Assembly Pathway Research Articles

Transcriptome Analysis of Intermittent Light Induced Early Bolting in Flowering Chinese Cabbage.

In flowering Chinese cabbage, early booting is one of the most important characteristics that is linked with quality and production. Through fixed light intensity (280 μmol·m-2·s-1) and fixed intermittent lighting in flowering Chinese cabbage, there was early bolting, bud emergence, and flowering. Moreover, the aboveground fresh weight, blade area, dry weight of blade, and quantification of the leaves in flowering Chinese cabbage were significantly reduced, while the thickness of tillers, tillers height, dry weight of tillers, and tillers weight were significantly increased. The chlorophyll contents and soil-plant analysis and development (SPAD) value decreased in the early stage and increased in the later stage. The nitrate content decreased, while the photosynthetic rate, vitamin C content, soluble sugar content, soluble protein content, phenolic content, and flavonoid content increased, and mineral elements also accumulated. In order to explore the mechanism of intermittent light promoting the early bolting and flowering of '49d' flowering Chinese cabbage, this study analyzed the transcriptional regulation from a global perspective using RNA sequencing. A total of 17,086 differentially expressed genes (DEGs) were obtained and 396 DEGs were selected that were closely related to early bolting. These DEGs were mainly involved in pollen wall assembly and plant circadian rhythm pathways, light action (34 DEGs), hormone biosynthesis and regulation (26 DEGs), development (21 DEGs), and carbohydrate synthesis and transport (6 DEGs). Three hub genes with the highest connectivity were identified through weighted gene co-expression network analysis (WGCNA): BrRVE, BrLHY, and BrRVE1. It is speculated that they may be involved in the intermittent light regulation of early bolting in flowering Chinese cabbage. In conclusion, intermittent light can be used as a useful tool to regulate plant growth structure, increase planting density, enhance photosynthesis, increase mineral accumulation, accelerate growth, and shorten the breeding cycle.

C. elegans spermatocyte divisions show a weak spindle checkpoint response.

Male meiotic division exhibits two consecutive chromosome separation events without apparent pausing. Several studies have shown that spermatocyte divisions are not stringently regulated as in mitotic cells. In this study, we investigated the role of the canonical spindle assembly (SAC) pathway in Caenorhabditis elegans spermatogenesis. We found the intensity of chromosome-associated outer kinetochore protein BUB-1 and SAC effector MDF-1 oscillates between the two divisions. However, the SAC target securin is degraded during the first division and remains undetectable for the second division. Inhibition of proteasome-dependent protein degradation did not affect the progression of the second division but stopped the first division at metaphase. Perturbation of spindle integrity did not affect the duration of meiosis II, and only slightly lengthened meiosis I. Our results demonstrate that male meiosis II is independent of SAC regulation, and male meiosis I exhibits only weak checkpoint response.

Time and Space Dual-Blockade Strategy for Highly Invasive Nature of Triple-Negative Breast Cancer in Enhanced Sonodynamic Therapy Based on Fe-MOF Nanoplatforms.

Triple-negative breast cancer (TNBC), due to its high malignant degree and strong invasion ability, leads to poor prognosis and easy recurrence, so effectively curbing the invasion of TNBC is the key to obtaining the ideal therapeutic effect. Herein, a therapeutic strategy is developed that curbs high invasions of TNBC by inhibiting cell physiological activity and disrupting tumor cell structural function to achieve the time and space dual-blockade. The time blockade is caused by the breakthrough of the tumor-reducing blockade based on the ferroptosis process and the oxidation-toxic free radicals generated by enhanced sonodynamic therapy (SDT). Meanwhile, alkyl radicals from 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) and 1O2 attacked the organelles of tumor cells under ultrasound (US), reducing the physiological activity of the cells. The attack of free radicals on the cytoskeleton, especially on the proteins of F-actin and its assembly pathway, achieves precise space blockade of TNBC. The damage to the cytoskeleton and the suppression of the repair process leads to a significant decline in the ability of tumor cells to metastasize and invade other organs. In summary, the FTM@AM nanoplatforms have a highly effective killing and invasion inhibition effect on invasive TNBC mediated by ultrasound, showcasing promising clinical transformation potential.

HSP90β controls NLRP3 autoactivation.

Gain-of-function mutations in NLRP3 are linked to cryopyrin-associated periodic syndromes (CAPS). Although NLRP3 autoinflammasome assembly triggers inflammatory cytokine release, its activation mechanisms are not fully understood. Our study used a functional genetic approach to identify regulators of NLRP3 inflammasome formation. We identified the HSP90β-SGT1 chaperone complex as crucial for autoinflammasome activation in CAPS. A deficiency in HSP90β, but not in HSP90α, impaired the formation of ASC specks without affecting the priming and expression of inflammasome components. Conversely, activating NLRP3 with stimuli such as nigericin or alum bypassed the need for SGT1 and HSP90β, suggesting the existence of alternative inflammasome assembly pathways. The role of HSP90β was further demonstrated in PBMCs derived from CAPS patients. In these samples, the pathological constitutive secretion of IL-1β could be suppressed using a pharmacological inhibitor of HSP90β. This finding underscores the potential of SGT1-HSP90β modulation as a therapeutic strategy in CAPS while preserving NLRP3's physiological functions.

Targeting mitochondrial bioenergetics by combination treatment with imatinib and dichloroacetate in human erythroleukemic K‑562 and colorectal HCT‑116 cancer cells.

Tumor malignant cells are characterized by dysregulation of mitochondrial bioenergetics due to the 'Warburg effect'. In the present study, this metabolic imbalance was explored as a potential target for novel cancer chemotherapy. Imatinib (IM) downregulates the expression levels of SCΟ2 and FRATAXIN (FXN) genes involved in the heme‑dependent cytochrome c oxidase biosynthesis and assembly pathway in human erythroleukemic IM‑sensitive K‑562 chronic myeloid leukemia cells (K‑562). In the present study, it was investigated whether the treatment of cancer cells with IM (an inhibitor of oxidative phosphorylation) separately, or together with dichloroacetate (DCA) (an inhibitor of glycolysis), can inhibit cell proliferation or cause death. Human K‑562 and IM‑chemoresistant K‑562 chronic myeloid leukemia cells (K‑562R), as well as human colorectal carcinoma cells HCT‑116 (+/+p53) and (‑/‑p53, with double TP53 knock-in disruptions), were employed. Treatments of these cells with either IM (1 or 2µM) and/or DCA (4mΜ) were also assessed for the levels of several process biomarkers including SCO2, FXN, lactate dehydrogenase A, glyceraldehyde‑3‑phosphate dehydrogenase, pyruvate kinase M2, hypoxia inducing factor‑1a, heme oxygenase‑1, NF‑κB, stem cell factor and vascular endothelial growth factor via western blot analysis. Computational network biology models were also applied to reveal the connections between the ten proteins examined. Combination treatment of IM with DCA caused extensive cell death (>75%) in K‑562 and considerable (>45%) in HCT‑116 (+/+p53) cultures, but less in K‑562R and HCT‑116 (‑/‑p53), with the latter deficient in full length p53 protein. Such treatment, markedly reduced reactive oxygen species levels, as measured by flow‑cytometry, in K‑562 cells and affected the oxidative phosphorylation and glycolytic biomarkers in all lines examined. These findings indicated, that targeting of cancer mitochondrial bioenergetics with such a combination treatment was very effective, although chemoresistance to IM in leukemia and the absence of a full length p53 in colorectal cells affected its impact.

Anti-phage defence through inhibition of virion assembly

Bacteria have evolved diverse antiviral defence mechanisms to protect themselves against phage infection. Phages integrated into bacterial chromosomes, known as prophages, also encode defences that protect the bacterial hosts in which they reside. Here, we identify a type of anti-phage defence that interferes with the virion assembly pathway of invading phages. The protein that mediates this defence, which we call Tab (for ‘Tail assembly blocker’), is constitutively expressed from a Pseudomonas aeruginosa prophage. Tab allows the invading phage replication cycle to proceed, but blocks assembly of the phage tail, thus preventing formation of infectious virions. While the infected cell dies through the activity of the replicating phage lysis proteins, there is no release of infectious phage progeny, and the bacterial community is thereby protected from a phage epidemic. Prophages expressing Tab are not inhibited during their own lytic cycle because they express a counter-defence protein that interferes with Tab function. Thus, our work reveals an anti-phage defence that operates by blocking virion assembly, thereby both preventing formation of phage progeny and allowing destruction of the infected cell due to expression of phage lysis genes.

Unique high-temperature tolerance mechanisms of zoochlorellae Symbiochlorum hainanensis derived from scleractinian coral Porites lutea.

The increasing ocean temperature above 31°C-32°C might trigger a breakdown of the coral-Symbiodiniaceae symbioses or coral bleaching because of the thermosensitivity of Symbiodiniaceae; therefore, the exploration of alternative coral symbiotic algae with high-temperature tolerance is important for the corals' protection under warming oceans. This study proves that zoochlorellae Symbiochlorum hainanensis can tolerate 38°C, which is the highest temperature tolerance known for coral symbiotic algae to date, with unique high-temperature tolerance mechanisms. Particularly, for the first time, an internal selenium antioxidant mechanism of coral symbiotic S. hainanensis to high temperature was suggested.

Conditional Cooperativity in RAS Assembly Pathways on Nanodiscs and Altered GTPase Cycling.

Self-assemblies (i.e., nanoclusters) of the RAS GTPase on the membrane act as scaffolds that activate downstream RAF kinases and drive MAPK signaling for cell proliferation and tumorigenesis. However, the mechanistic details of nanoclustering remain largely unknown. Here, size-tunable nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses revealed the structural basis of the cooperative assembly processes of fully processed KRAS, mutated in a quarter of human cancers. The cooperativity is modulated by the mutation and nucleotide states of KRAS and the lipid composition of the membrane. Notably, the oncogenic mutants assemble in nonsequential pathways with two mutually cooperative 'α/α' and 'α/β' interfaces, while α/α dimerization of wild-type KRAS promotes the secondary α/β interaction sequentially. Mutation-based interface engineering was used to selectively trap the oligomeric intermediates of KRAS and probe their favorable interface interactions. Transiently exposed interfaces were available for the assembly. Real-time NMR demonstrated that higher-order oligomers retain higher numbers of active GTP-bound protomers in KRAS GTPase cycling. These data provide a deeper understanding of the nanocluster-enhanced signaling in response to the environment. Furthermore, our methodology is applicable to assemblies of many other membrane GTPases and lipid nanoparticle-based formulations of stable protein oligomers with enhanced cooperativity.

Conditional Cooperativity in RAS Assembly Pathways on Nanodiscs and Altered GTPase Cycling

AbstractSelf‐assemblies (i.e., nanoclusters) of the RAS GTPase on the membrane act as scaffolds that activate downstream RAF kinases and drive MAPK signaling for cell proliferation and tumorigenesis. However, the mechanistic details of nanoclustering remain largely unknown. Here, size‐tunable nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses revealed the structural basis of the cooperative assembly processes of fully processed KRAS, mutated in a quarter of human cancers. The cooperativity is modulated by the mutation and nucleotide states of KRAS and the lipid composition of the membrane. Notably, the oncogenic mutants assemble in nonsequential pathways with two mutually cooperative ‘α/α’ and ‘α/β’ interfaces, while α/α dimerization of wild‐type KRAS promotes the secondary α/β interaction sequentially. Mutation‐based interface engineering was used to selectively trap the oligomeric intermediates of KRAS and probe their favorable interface interactions. Transiently exposed interfaces were available for the assembly. Real‐time NMR demonstrated that higher‐order oligomers retain higher numbers of active GTP‐bound protomers in KRAS GTPase cycling. These data provide a deeper understanding of the nanocluster‐enhanced signaling in response to the environment. Furthermore, our methodology is applicable to assemblies of many other membrane GTPases and lipid nanoparticle‐based formulations of stable protein oligomers with enhanced cooperativity.

Using cryo-EM to understand the assembly pathway of respiratory complex I.

Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the first component of the mitochondrial respiratory chain. In recent years, high-resolution cryo-EM studies of complex I from various species have greatly enhanced the understanding of the structure and function of this important membrane-protein complex. Less well studied is the structural basis of complex I biogenesis. The assembly of this complex of more than 40 subunits, encoded by nuclear or mitochondrial DNA, is an intricate process that requires at least 20 different assembly factors in humans. These are proteins that are transiently associated with building blocks of the complex and are involved in the assembly process, but are not part of mature complex I. Although the assembly pathways have been studied extensively, there is limited information on the structure and molecular function of the assembly factors. Here, the insights that have been gained into the assembly process using cryo-EM are reviewed.

Cerium Nanophases from Cerium Ammonium Nitrate.

Ceria nanomaterials with facile CeIII/IV redox behavior are used in sensing, catalytic, and therapeutic applications, where inclusion of CeIII has been correlated with reactivity. Understanding assembly pathways of CeO2 nanoparticles (NC-CeO2) in water has been challenged by "blind" synthesis, including rapid assembly/precipitation promoted by heat or strong base. Here, we identify a layered phase denoted Ce-I with a proposed formula CeIV(OH)3(NO3)·xH2O (x ≈ 2.5), obtained by adding electrolytes to aqueous cerium ammonium nitrate (CAN) to force precipitation. Ce-I represents intermediate hydrolysis species between dissolved CAN and NC-CeO2, where CAN is a commonly used CeIV compound that exhibits unusual aqueous and organic solubility. Ce-I features Ce-(OH)2-Ce units, representing the first step of hydrolysis toward NC-CeO2 formation, challenging prior assertions about CeIV hydrolysis. Structure/composition of poorly crystalline Ce-I was corroborated by a pair distribution function, Ce-L3 XAS (X-ray absorption spectroscopy), compositional analysis, and 17O nuclear magnetic resonance spectroscopy. Formation of Ce-I and its transformation to NC-CeO2 is documented in solution by small-angle X-ray scattering (SAXS) and in the solid-state by transmission electron microscopy (TEM) and powder X-ray diffraction. Morphologies identified by TEM support form factor models for SAXS analysis, evidencing the incipient assembly of Ce-I. Finally, two morphologies of NC-CeO2 are identified. Sequentially, spherical NC-CeO2 particles coexist with Ce-I, and asymmetric NC-CeO2 with up to 35% CeIII forms at the expense of Ce-I, suggesting direct replacement.

Modeling bacterial microcompartment architectures for enhanced cyanobacterial carbon fixation.

The carboxysome is a bacterial microcompartment (BMC) which plays a central role in the cyanobacterial CO2-concentrating mechanism. These proteinaceous structures consist of an outer protein shell that partitions Rubisco and carbonic anhydrase from the rest of the cytosol, thereby providing a favorable microenvironment that enhances carbon fixation. The modular nature of carboxysomal architectures makes them attractive for a variety of biotechnological applications such as carbon capture and utilization. In silico approaches, such as molecular dynamics (MD) simulations, can support future carboxysome redesign efforts by providing new spatio-temporal insights on their structure and function beyond in vivo experimental limitations. However, specific computational studies on carboxysomes are limited. Fortunately, all BMC (including the carboxysome) are highly structurally conserved which allows for practical inferences to be made between classes. Here, we review simulations on BMC architectures which shed light on (1) permeation events through the shell and (2) assembly pathways. These models predict the biophysical properties surrounding the central pore in BMC-H shell subunits, which in turn dictate the efficiency of substrate diffusion. Meanwhile, simulations on BMC assembly demonstrate that assembly pathway is largely dictated kinetically by cargo interactions while final morphology is dependent on shell factors. Overall, these findings are contextualized within the wider experimental BMC literature and framed within the opportunities for carboxysome redesign for biomanufacturing and enhanced carbon fixation.

Optimisation of surfactin yield in Bacillus using data-efficient active learning and high-throughput mass spectrometry

Integration of machine learning and high throughput measurements are essential to drive the next generation of the design-build-test-learn (DBTL) cycle in synthetic biology. Here, we report the use of active learning in combination with metabolomics for optimising production of surfactin, a complex lipopeptide resulting from a non-ribosomal assembly pathway. We designed a media optimisation algorithm that iteratively learns the yield landscape and steers the media composition toward maximal production. The algorithm led to a 160 % yield increase after three DBTL runs as compared to an M9 baseline. Metabolomics data helped to elucidate the underpinning biochemistry for yield improvement and revealed Pareto-like trade-offs in production of other lipopeptides from related pathways. We found positive associations between organic acids and surfactin, suggesting a key role of central carbon metabolism, as well as system-wide anisotropies in how metabolism reacts to shifts in carbon and nitrogen levels. Our framework offers a novel data-driven approach to improve yield of biological products with complex synthesis pathways that are not amenable to traditional yield optimisation strategies.

Hierarchical assembly is more robust than egalitarian assembly in synthetic capsids

Self-assembly of complex and functional materials remains a grand challenge in soft material science. Efficient assembly depends on a delicate balance between thermodynamic and kinetic effects, requiring fine-tuning affinities and concentrations of subunits. By contrast, we introduce an assembly paradigm that allows large error-tolerance in the subunit affinity and helps avoid kinetic traps. Our combined experimental and computational approach uses a model system of triangular subunits programmed to assemble into T = 3 icosahedral capsids comprising 60 units. The experimental platform uses DNA origami to create monodisperse colloids whose three-dimensional geometry is controlled to nanometer precision, with two distinct bonds whose affinities are controlled to kBT precision, quantified in situ by static light scattering. The computational model uses a coarse-grained representation of subunits, short-ranged potentials, and Langevin dynamics. Experimental observations and modeling reveal that when the bond affinities are unequal, two distinct hierarchical assembly pathways occur, in which the subunits first form dimers in one case and pentamers in another. These hierarchical pathways produce complete capsids faster and are more robust against affinity variation than egalitarian pathways, in which all binding sites have equal strengths. This finding suggests that hierarchical assembly may be a general engineering principle for optimizing self-assembly of complex target structures.

Revealing pathways of ribosome assembly and viral RNA processing with time-resolved fluorescence

Revealing pathways of ribosome assembly and viral RNA processing with time-resolved fluorescence

Structural investigations of the cytosolic iron-sulfur cluster assembly pathway late acting proteins

Structural investigations of the cytosolic iron-sulfur cluster assembly pathway late acting proteins

Multiple Cations Nanoconfinement in Ultrathin V2O5 Nanosheets Enables Ultrafast Ion Diffusion Kinetics Toward High-performance Zinc Ion Battery.

Nanoconfinement of cations in layered oxide cathode is an important approach to realize advanced zinc ion storage performance. However, thus far, the conventional hydrothermal/solvothermal route for this nanoconfinement has been restricted to its uncontrollable phase structure and the difficulty on the multiple cation co-confinement simultaneously. Herein, this work reports a general, supramolecular self-assembly of ultrathin V2O5 nanosheets using various unitary cations including Na+, K+, Mg2+, Ca2+, Zn2+, Al3+, NH4 +, and multiple cations (NH4 + + Na+, NH4 + + Na+ + Ca2+, NH4 + + Na+ + Ca2+ +Mg2+). The unitary cation confinement results in a remarkable increase in the specific capacity and Zn-ion diffusion kinetics, and the multiple cation confinement gives rise to superior structural and cycling stability by multiple cation synergetic pillaring effect. The optimized diffusion coefficient of Zn-ion (7.5 × 10-8 cm2 s-1) in this assembly series surpasses most of the V-based cathodes reported up to date. The work develops a novel multiple-cations nanoconfinement strategy toward high-performance cathode for aqueous battery. It also provides new insights into the guest cation regulation of zinc-ion diffusion kinetics through a general, supramolecular assembly pathway.

Dynamic Study of Kinetically Trapped Byproducts during DNA Assembly: Case Study on a Pathway-Dependent Assembly.

Despite 40 years of development of DNA nanotechnology, the fundamental knowledge of the process of DNA strand assembly into targeted nanostructures remains unclear. Study of the dynamic process, especially the competing hybridizations in kinetic traps, provides insight into DNA assembly. In this study, a system of middle-domain first assembly (MDFA) was proposed to enable oligonucleotides to assemble into a 2D DNA monolayer in a pathway-dependent approach. This system was an ideal case to study the dynamic interactions between competing hybridizations during oligonucleotide assembly. Dynamic study revealed the coexistence of the kinetically trapped dead-end byproduct and target product at the early stage of annealing, followed by transformation of the byproduct into the target product by reverse disassembly, due to the equilibrium of the competing hybridizations increasingly favoring the target product pathway. This study offered a better understanding of the assembly pathway of DNA nanostructures for future design.

Methods to monitor mitochondrial disulfide bonds.

Mitochondria contain numerous proteins that utilize the chemistry of cysteine residues, which can be reversibly oxidized. These proteins are involved in mitochondrial biogenesis, protection against oxidative stress, metabolism, energy transduction to adenosine triphosphate, signaling and cell death among other functions. Many proteins located in the mitochondrial intermembrane space are imported by the mitochondrial import and assembly pathway the activity of which is based on the reversible oxidation of cysteine residues and oxidative trapping of substrates. Oxidative modifications of cysteine residues are particularly difficult to study because of their labile character. Here we present techniques that allow for monitoring the oxidative state of mitochondrial proteins as well as to investigate the mitochondrial import and assembly pathway. This chapter conveys basic concepts on sample preparation and techniques to monitor the redox state of cysteine residues in mitochondrial proteins as well as the strategies to study mitochondrial import and assembly pathway.

Structures and Efflux Mechanisms of the AcrAB-TolC Pump.

The global emergence of multidrug resistance (MDR) in gram-negative bacteria has become a matter of worldwide concern. MDR in these pathogens is closely linked to the overexpression of certain efflux pumps, particularly the resistance-nodulation-cell division (RND) efflux pumps. Inhibition of these pumps presents an attractive and promising strategy to combat antibiotic resistance, as the efflux pump inhibitors can effectively restore the potency of existing antibiotics. AcrAB-TolC is one well-studied RND efflux pump, which transports a variety of substrates, therefore providing resistance to a broad spectrum of antibiotics. To develop effective pump inhibitors, a comprehensive understanding of the structural aspect of the AcrAB-TolC efflux pump is imperative. Previous studies on this pump's structure have been limited to individual components or in vitro determination of fully assembled pumps. Recent advancements in cellular cryo-electron tomography (cryo-ET) have provided novel insights into this pump's assembly and functional mechanism within its native cell membrane environment. Here, we present a summary of the structural data regarding the AcrAB-TolC efflux pump, shedding light on its assembly pathway and operational mechanism.