Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales.

In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion.

A wearable cardiac ultrasound imager.

Continuous imaging of cardiac functions is highly desirable for the assessment of long-term cardiovascular health, detection of acute cardiac dysfunction and clinical management of critically ill or surgical patients1-4. However, conventional non-invasive approaches to image the cardiac function cannot provide continuous measurements owing to device bulkiness5-11, and existing wearable cardiac devices can only capture signals on the skin12-16. Here we report a wearable ultrasonic device for continuous, real-time and direct cardiac function assessment. We introduce innovations in device design and material fabrication that improve the mechanical coupling between the device and human skin, allowing the left ventricle to be examined from different views during motion. We also develop a deep learning model that automatically extracts the left ventricular volume from the continuous image recording, yielding waveforms of key cardiac performance indices such as stroke volume, cardiac output and ejection fraction. This technology enables dynamic wearable monitoring of cardiac performance with substantially improved accuracy in various environments.

Large DNA Methylation Nadirs Anchor Chromatin Loops Maintaining Hematopoietic Stem Cell Identity.

Higher-order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we find that large (>7.3 kb) DNA methylation nadirs (termed "grand canyons") can form long loops connecting anchor loci that may be dozens of megabases (Mb) apart, as well as inter-chromosomal links. The interacting loci cover a total of ∼3.5 Mb of the human genome. The strongest interactions are associated with repressive marks made by the Polycomb complex and are diminished upon EZH2 inhibitor treatment. The data are suggestive of the formation of these loops by interactions between repressive elements in the loci, forming a genomic subcompartment, rather than by cohesion/CTCF-mediated extrusion. Interestingly, unlike previously characterized subcompartments, these interactions are present only in particular cell types, such as stem and progenitor cells. Our work reveals that H3K27me3-marked large DNA methylation grand canyons represent a set of very-long-range loops associated with cellular identity.

De novo protein design with a denoising diffusion network independent of pretrained structure prediction models.

The recent success of RFdiffusion, a method for protein structure design with a denoising diffusion probabilistic model, has relied on fine-tuning the RoseTTAFold structure prediction network for protein backbone denoising. Here, we introduce SCUBA-diffusion (SCUBA-D), a protein backbone denoising diffusion probabilistic model freshly trained by considering co-diffusion of sequence representation to enhance model regularization and adversarial losses to minimize data-out-of-distribution errors. While matching the performance of the pretrained RoseTTAFold-based RFdiffusion in generating experimentally realizable protein structures, SCUBA-D readily generates protein structures with not-yet-observed overall folds that are different from those predictable with RoseTTAFold. The accuracy of SCUBA-D was confirmed by the X-ray structures of 16 designed proteins and a protein complex, and by experiments validating designed heme-binding proteins and Ras-binding proteins. Our work shows that deep generative models of images or texts can be fruitfully extended to complex physical objects like protein structures by addressing outstanding issues such as the data-out-of-distribution errors.

Topology-driven collective dynamics of nematic colloidal entanglement.

Entanglement in a soft condensed matter system is enabled in the form of entangled disclination lines by using colloidal particles in nematic liquid crystals. These topological excitations are manifested as colloidal entanglement at equilibrium. How to further utilize nonequilibrium disclination lines to manipulate colloidal entanglement remains a nontrivial and challenging task. In this work, we use experiments and simulations to demonstrate the reconfigurations of nematic colloidal entanglement in light-driven spatiotemporal evolutions of disclination lines. Colloidal entanglement can sense subtle changes in the topological structures of disclination lines and realize chirality conversion. This conversion is manifested as the "domino effect" of the collective rotation of colloids in the disclination lines. By programming the topological patterns and the geometry of the disclination lines, colloidal entanglement can be assembled and split. More remarkably, a double-helix entangled structure can be formed by controlling the changes in the morphology of the disclination lines. Thus, this work will provide opportunities to program colloidal composites for smart materials and micromachines.

LRG1 promotes atherosclerosis by inducing macrophage M1-like polarization.

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the accumulation of cholesterol-rich lipoproteins in macrophages. How macrophages commit to proinflammatory polarization under atherosclerosis conditions is not clear. Report here that the level of a circulating protein, leucine-rich alpha-2 glycoprotein 1 (LRG1), is elevated in the atherosclerotic tissue and serum samples from patients with coronary artery disease (CAD). LRG1 stimulated macrophages to proinflammatory M1-like polarization through the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways. The LRG1 knockout mice showed significantly delayed atherogenesis progression and reduced levels of macrophage-related proinflammatory cytokines in a high-fat diet-induced Apoe-/- mouse atherosclerosis model. An anti-LRG1 neutralizing antibody also effectively blocked LRG1-induced macrophage M1-like polarization in vitro and conferred therapeutic benefits to animals with ApoE deficiency-induced atherosclerosis. LRG1 may therefore serve as an additional biomarker for CAD and targeting LRG1 could offer a potential therapeutic strategy for CAD patients by mitigating the proinflammatory response of macrophages.

Revisiting Solar Energy Flow in Nanomaterial-Microorganism Hybrid Systems.

Nanomaterial-microorganism hybrid systems (NMHSs), integrating semiconductor nanomaterials with microorganisms, present a promising platform for broadband solar energy harvesting, high-efficiency carbon reduction, and sustainable chemical production. While studies underscore its potential in diverse solar-to-chemical energy conversions, prevailing NMHSs grapple with suboptimal energy conversion efficiency. Such limitations stem predominantly from an insufficient systematic exploration of the mechanisms dictating solar energy flow. This review provides a systematic overview of the notable advancements in this nascent field, with a particular focus on the discussion of three pivotal steps of energy flow: solar energy capture, cross-membrane energy transport, and energy conversion into chemicals. While key challenges faced in each stage are independently identified and discussed, viable solutions are correspondingly postulated. In view of the interplay of the three steps in affecting the overall efficiency of solar-to-chemical energy conversion, subsequent discussions thus take an integrative and systematic viewpoint to comprehend, analyze and improve the solar energy flow in the current NMHSs of different configurations, and highlighting the contemporary techniques that can be employed to investigate various aspects of energy flow within NMHSs. Finally, a concluding section summarizes opportunities for future research, providing a roadmap for the continued development and optimization of NMHSs.

A jingmenvirus RNA-dependent RNA polymerase structurally resembles the flavivirus counterpart but with different features at the initiation phase.

Jingmenviruses are a category of emerging segmented viruses that have garnered global attention in recent years, and are close relatives of the flaviviruses in the Flaviviridae family. One of their genome segments encodes NSP1 homologous to flavivirus NS5. NSP1 comprises both the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRP) modules playing essential roles in viral genome replication and capping. Here we solved a 1.8-Å resolution crystal structure of the NSP1 RdRP module from Jingmen tick virus (JMTV), the type species of jingmenviruses. The structure highly resembles flavivirus NS5 RdRP despite a sequence identity less than 30%. NSP1 RdRP enzymatic properties were dissected in a comparative setting with several representative Flaviviridae RdRPs included. Our data indicate that JMTV NSP1 produces characteristic 3-mer abortive products similar to the hepatitis C virus RdRP, and exhibits the highest preference of terminal initiation and shorter-primer usage. Unlike flavivirus NS5, JMTV RdRP may require the MTase for optimal transition from initiation to elongation, as an MTase-less NSP1 construct produced more 4-5-mer intermediate products than the full-length protein. Taken together, this work consolidates the evolutionary relationship between the jingmenvirus group and the Flaviviridae family, providing a basis to the further understanding of their viral replication/transcription process.

Structural basis of transition from initiation to elongation in de novo viral RNA-dependent RNA polymerases.

De novo viral RNA-dependent RNA polymerases (RdRPs) utilize their priming element (PE) to facilitate accurate initiation. Upon transition to elongation, the PE has to retreat from the active site to give room to the template-product RNA duplex. However, PE conformational change upon this transition and the role of PE at elongation both remain elusive. Here, we report crystal structures of RdRP elongation complex (EC) from dengue virus serotype 2 (DENV2), demonstrating a dramatic refolding of PE that allows establishment of interactions with the RNA duplex backbone approved to be essential for EC stability. Enzymology data from both DENV2 and hepatitis C virus (HCV) RdRPs suggest that critical transition of the refolding likely occurs after synthesis of a 4- to 5-nucleotide (nt) product together providing a key basis in understanding viral RdRP transition from initiation to elongation.

Collective transport and reconfigurable assembly of nematic colloids by light-driven cooperative molecular reorientations.

Nanomotors in nature have inspired scientists to design synthetic molecular motors to drive the motion of microscale objects by cooperative action. Light-driven molecular motors have been synthesized, but using their cooperative reorganization to control the collective transport of colloids and to realize the reconfiguration of colloidal assembly remains a challenge. In this work, topological vortices are imprinted in the monolayers of azobenzene molecules which further interface with nematic liquid crystals (LCs). The light-driven cooperative reorientations of the azobenzene molecules induce the collective motion of LC molecules and thus the spatiotemporal evolutions of the nematic disclination networks which are defined by the controlled patterns of vortices. Continuum simulations provide physical insight into the morphology change of the disclination networks. When microcolloids are dispersed in the LC medium, the colloidal assembly is not only transported and reconfigured by the collective change of the disclination lines but also controlled by the elastic energy landscape defined by the predesigned orientational patterns. The collective transport and reconfiguration of colloidal assemblies can also be programmed by manipulating the irradiated polarization. This work opens opportunities to design programmable colloidal machines and smart composite materials.

Alternative splicing of CsbHLH133 regulates geraniol biosynthesis in tea plants.

Geraniol is one of the most abundant aromatic compounds in fresh tea leaves and contributes to the pleasant odor of tea products. Additionally, it functions as an airborne signal that interacts with other members of the ecosystem. To date, the regulation of the geraniol biosynthesis in tea plants remains to be investigated. In this study, a correlation test of the content of geraniol and its glycosides with gene expression data revealed that nudix hydrolase, CsNudix26, and its transcription factor, CsbHLH133 are involved in geraniol biosynthesis. In vitro enzyme assays and metabolic analyses of genetically modified tea plants confirmed that CsNudix26 is responsible for the formation of geraniol. Yeast one-hybrid, dual-luciferase reporter, and EMSA assays were used to verify the binding of CsbHLH133 to the CsNudix26 promoter. Overexpression of CsbHLH133 in tea leaves enhanced CsNudix26 expression and geraniol accumulation, whereas CsbHLH133 silencing reduced CsNudix26 transcript levels and geraniol content. Interestingly, CsbHLH133-AS, produced by alternative splicing, was discovered and proved to be the primary transcript expressed in response to various environmental stresses. Furthermore, geraniol release was found to be affected by various factors that alter the expression patterns of CsbHLH133 and CsbHLH133-AS. Our findings indicate that distinct transcript splicing patterns of CsbHLH133 regulate geraniol biosynthesis in tea plants in response to different regulatory factors.

Maize lipid droplet-associated protein 2 is recruited by a virus to enhance viral multiplication and infection through regulating cellular fatty acid metabolism.

Pathogen infection induces massive reprogramming of host primary metabolism. Lipid and fatty acid (FA) metabolism is generally disrupted by pathogens and co-opted for their proliferation. Lipid droplets (LDs) that play important roles in regulating cellular lipid metabolism are utilized by a variety of pathogens in mammalian cells. However, the function of LDs during pathogenic infection in plants remains unknown. We show here that infection by rice black streaked dwarf virus (RBSDV) affects the lipid metabolism of maize, which causes elevated accumulation of C18 polyunsaturated fatty acids (PUFAs) leading to viral proliferation and symptom development. The overexpression of one of the two novel LD-associated proteins (LDAPs) of maize (ZmLDAP1 and ZmLDAP2) induces LD clustering. The core capsid protein P8 of RBSDV interacts with ZmLDAP2 and prevents its degradation through the ubiquitin-proteasome system mediated by a UBX domain-containing protein, PUX10. In addition, silencing of ZmLDAP2 downregulates the expression of FA desaturase genes in maize, leading to a decrease in C18 PUFAs levels and suppression of RBSDV accumulation. Our findings reveal that plant virus may recruit LDAP to regulate cellular FA metabolism to promote viral multiplication and infection. These results expand the knowledge of LD functions and viral infection mechanisms in plants.

Liquid-liquid phase separation underpins the formation of replication factories in rotaviruses.

RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.

A microRNA396b-growth regulating factor module controls castor seed size by mediating auxin synthesis.

Castor (Ricinus communis L.) is an importance crop cultivated for its oil and economic value. Seed size is a crucial factor that determines crop yield. Gaining insight into the molecular regulatory processes of seed development is essential for the genetic enhancement and molecular breeding of castor. Here, we successfully fine-mapped a major QTL related to seed size, qSS3, to a 180 kb interval on chromosome 03 using F2 populations (DL01×WH11). A 17.6-kb structural variation (SV) was detected through genomic comparison between DL01 and WH11. Analysis of haplotypes showed that the existence of the complete 17.6 kb structural variant may lead to the small seed characteristic in castor. In addition, we found that qSS3 contains the microRNA396b (miR396b) sequence, which is situated within the 17.6 kb SV. The results of our experiment offer additional evidence that miR396-Growth Regulating Factor 4 (GRF4) controls seed size by impacting the growth and multiplication of seed coat and endosperm cells. Furthermore, we found that RcGRF4 activates the expression of YUCCA6 (YUC6), facilitating the production of IAA in seeds and thereby impacting the growth of castor seeds. Our research has discovered a crucial functional module that controls seed size, offering a fresh understanding of the mechanism underlying seed size regulation in castor.

Exercise-induced ß-hydroxybutyrate promotes Treg cell differentiation to ameliorate colitis in mice.

Increasing attention is being paid to the mechanistic investigation of exercise-associated chronic inflammatory disease improvement. Ulcerative colitis (UC) is one type of chronic inflammatory bowel disease with increasing incidence and prevalence worldwide. It is known that regular moderate aerobic exercise (RMAE) reduces the incidence or risk of UC, and attenuates disease progression in UC patients. However, the mechanisms of this RMAE's benefit are still under investigation. Here, we revealed that ß-hydroxybutyrate (ß-HB), a metabolite upon prolonged aerobic exercise, could contribute to RMAE preconditioning in retarding dextran sulfate sodium (DSS)-induced mouse colitis. When blocking ß-HB production, RMAE preconditioning-induced colitis amelioration was compromised, whereas supplementation of ß-HB significantly rescued impaired ß-HB production-associated defects. Meanwhile, we found that RMAE preconditioning significantly caused decreased colonic Th17/Treg ratio, which is considered to be important for colitis mitigation; and the downregulated Th17/Treg ratio was associated with ß-HB. We further demonstrated that ß-HB can directly promote the differentiation of Treg cell rather than inhibit Th17 cell generation. Furthermore, ß-HB increased forkhead box protein P3 (Foxp3) expression, the core transcriptional factor for Treg cell, by enhancing histone H3 acetylation in the promoter and conserved noncoding sequences of the Foxp3 locus. In addition, fatty acid oxidation, the key metabolic pathway required for Treg cell differentiation, was enhanced by ß-HB treatment. Lastly, administration of ß-HB without exercise significantly boosted colonic Treg cell and alleviated colitis in mice. Together, we unveiled a previously unappreciated role for exercise metabolite ß-HB in the promotion of Treg cell generation and RMAE preconditioning-associated colitis attenuation.

Engineered Living Materials For Sustainability.

Recent advances in synthetic biology and materials science have given rise to a new form of materials, namely engineered living materials (ELMs), which are composed of living matter or cell communities embedded in self-regenerating matrices of their own or artificial scaffolds. Like natural materials such as bone, wood, and skin, ELMs, which possess the functional capabilities of living organisms, can grow, self-organize, and self-repair when needed. They also spontaneously perform programmed biological functions upon sensing external cues. Currently, ELMs show promise for green energy production, bioremediation, disease treatment, and fabricating advanced smart materials. This review first introduces the dynamic features of natural living systems and their potential for developing novel materials. We then summarize the recent research progress on living materials and emerging design strategies from both synthetic biology and materials science perspectives. Finally, we discuss the positive impacts of living materials on promoting sustainability and key future research directions.

Effect of M2-macrophage treated lymphatic endothelial cells on angiogenesis that promoted osteointegration.

Early vascularization plays an essential role during the whole process in bone regeneration because of the function of secreting cytokines, transporting nutrients and metabolic wastes. As the preliminary basis of bone repair, angiogenesis is regulated by immune cells represented by macrophages to a great extent. However, with the discovery of the endolymphatic circulation system inside bone tissue, the role of vascularization became complicated and confusing. Herein, we developed a macrophage/lymphatic endothelial cells (LECs)/human umbilical vein endothelial cells (HUVECs) co-culture system to evaluate the effect of macrophage treated lymphatic endothelial cells on angiogenesis in vitro and in vivo. In this study, we collected the medium from macrophage (CM) for LECs culture. We found that CM2 could promote the expression of LECs markers and migration ability, which indicated the enhanced lymphogenesis. In addition, the medium from LECs was collected for culturing HUVECs. The CM2-treated LECs showed superior angiogenesis property including the migration capacity and expression of angiogenetic markers, which suggested the superior vascularization. Rat femoral condyle defect model was applied to confirm the hypothesis in vivo. Generally, M2-macrophage treated LECs showed prominent angiogenetic potential coupling with osteogenesis.

Active transformations of topological structures in light-driven nematic disclination networks.

SignificanceTopological defects are marvels of nature. Understanding their structures is important for their applications in, for example, directed self-assembly, sensing, and photonic devices. There is recent interest in active motion and transformation of topological defects in active nematics. In these nonequilibrium systems, however, the motion and transformation of disclinations are difficult to control, thereby hindering their applications. Here, we propose a surface-patterned system engendering periodic three-dimensional disclinations, which can be excited by light irradiation and undergo a programmable transformation between different topological states. Continuum simulations recapitulating these topological structures characterize the bending, breaking, and relinking events of the disclinations during the nonequilibrium process. Our work provides an alternative dynamic system in which active transformation of topological defects can be engineered.

The impact of glucose on mitochondria and life span is determined by the integrity of proline catabolism in Caenorhabditis elegans.

Mutations in genes involved in mitochondrial proline catabolism lead to the rare genetic disorder hyperprolinemia in humans. We have previously reported that mutations of proline catabolic genes in Caenorhabditis elegans impair mitochondrial homeostasis and shorten life span, and that these effects surprisingly occur in a diet type-dependent manner. Therefore, we speculated that a specific dietary component may mitigate the adverse effects of defective proline catabolism. Here, we discovered that high dietary glucose, which is generally detrimental to health, actually improves mitochondrial homeostasis and life span in C. elegans with faulty proline catabolism. Mechanistically, defective proline catabolism results in a shift of glucose catabolism toward the pentose phosphate pathway, which is crucial for cellular redox balance. This shift helps to maintain mitochondrial reactive oxygen species homeostasis and to extend life span, as suppression of the pentose phosphate pathway enzyme GSPD-1 prevents the favorable effects of high glucose. In addition, we demonstrate that this crosstalk between proline and glucose catabolism is mediated by the transcription factor DAF-16. Altogether, these findings suggest that a glucose-rich diet may be advantageous in certain situations and might represent a potentially viable treatment strategy for disorders involving impaired proline catabolism.

Cotton peroxisome-localized lysophospholipase counteracts the toxic effects of Verticillium dahliae NLP1 and confers wilt resistance.

Plasma membrane represents a critical battleground between plants and attacking microbes. Necrosis-and-ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), cytolytic toxins produced by some bacterial, fungal and oomycete species, are able to target on lipid membranes by binding eudicot plant-specific sphingolipids (glycosylinositol phosphorylceramide) and form transient small pores, causing membrane leakage and subsequent cell death. NLP-producing phytopathogens are a big threat to agriculture worldwide. However, whether there are R proteins/enzymes that counteract the toxicity of NLPs in plants remains largely unknown. Here we show that cotton produces a peroxisome-localized enzyme lysophospholipase, GhLPL2. Upon Verticillium dahliae attack, GhLPL2 accumulates on the membrane and binds to V. dahliae secreted NLP, VdNLP1, to block its contribution to virulence. A higher level of lysophospholipase in cells is required to neutralize VdNLP1 toxicity and induce immunity-related genes expression, meanwhile maintaining normal growth of cotton plants, revealing the role of GhLPL2 protein in balancing resistance to V. dahliae and growth. Intriguingly, GhLPL2 silencing cotton plants also display high resistance to V. dahliae, but show severe dwarfing phenotype and developmental defects, suggesting GhLPL2 is an essential gene in cotton. GhLPL2 silencing results in lysophosphatidylinositol over-accumulation and decreased glycometabolism, leading to a lack of carbon sources required for plants and pathogens to survive. Furthermore, lysophospholipases from several other crops also interact with VdNLP1, implying that blocking NLP virulence by lysophospholipase may be a common strategy in plants. Our work demonstrates that overexpressing lysophospholipase encoding genes have great potential for breeding crops with high resistance against NLP-producing microbial pathogens.