Design Glutamate Dehydrogenase for Nonaqueous System by Motifs Reassembly and Interaction Network Analysis.

Glutamate dehydrogenases (GDH) serve as the key regulated enzyme that links protein and carbohydrate metabolism. Combined with motif reassembly and mutation, novel GDHs were designed. Motif reassembly of thermophilic GDH and malate dehydrogenase aims to overcome stability and activity tradeoff in nonaqueous systems. Structural compatibility and dynamic cooperation of the designed AaDHs were studied by molecular dynamics simulation. Furthermore, multipoint mutations improved its catalytic activity for unnatural substrates. Amino acid interaction network analysis indicated that the high density of hydrogen-bonded salt bridges is beneficial to the stability. Finally, the experimental verification determines the kinetics of AaDHs in a nonaqueous system. The activity of Aa05 was increased by 1.78-fold with ionic liquid [EMIM]BF4. This study presents the strategy of a combination of rigid motif assembly and mutations of active sites for robust dehydrogenases with high activity in the nonaqueous system, which overcomes the activity-stability tradeoff effect.

Feature reassembly and L1 preemption: Acquiring CLLD in L2 Italian and L2 Romanian.

This study investigates feature acquisition and feature reassembly associated with Clitic Left Dislocation (CLLD). The article compares the acquisition of CLLD in second language (L2) Italian to L2 Romanian to examine effects of first language (L1) transfer, construction frequency and the type of interface involved (external vs. internal interface) within the same syntactic construction. The results from an acceptability judgment task and a written elicitation task show that while English near-native speakers of Italian/Romanian acquired the L2 constraints on CLLD, which is [+anaphor] for Italian and [+specific] for Romanian, data from both Romanian L2 learners of Italian and Italian L2 learners of Romanian showed persistent L1 transfer effects. Target-like acquisition for these groups requires both grammatical expansion and retraction; Romanian CLLD requires the addition of an L1-unavailable [+specific] feature and the loss of a [+anaphor] feature, while Italian CLLD requires the addition of an L1-unavailable [+anaphor] and the loss of a [+specific] feature. The reported findings extend evidence in favour of the Feature Reassembly Hypothesis to the syntax-discourse interface, as reassembly of interpretational features associated with CLLD proved more difficult than feature acquisition. While learners at the near-native levels were able to broaden the contexts that allow a clitic in the L2 (grammatical expansion), L1 preemption difficulties were attested as well. This was the case regardless of the frequency of the relevant construction in the input and the type of L2 feature that needed to be added/removed.

Standardized Parts for Activation of Small GTPase Signaling in Living Cells.

Small GTPases comprise a superfamily of over 167 proteins in the human genome and are critical regulators of a variety of pathways including cell migration and proliferation. Despite the importance of these proteins in cell signaling, a standardized approach for controlling small GTPase activation within living cells is lacking. Herein, we report a split-protein-based approach to directly activate small GTPase signaling in living cells. Importantly, our fragmentation site can be applied across the small GTPase superfamily. We highlight the utility of these standardized parts by demonstrating the ability to directly modulate the activity of four different small GTPases with user-defined inputs, providing the first plug and play system for direct activation of small GTPases in living cells.

Reconstitution of nuclear envelope subdomain formation on mitotic chromosomes in semi-intact cells.

In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called "core" region and "noncore" region on telophase chromosomes, which corresponded to the "pore-free" region and the "pore-rich" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.

Crosstalk between mitotic reassembly and repair of the nuclear envelope.

In eukaryotic cells, the nuclear envelope (NE) is a membrane partition between the nucleus and the cytoplasm to compartmentalize nuclear contents. It plays an important role in facilitating nuclear functions including transcription, DNA replication and repair. In mammalian cells, the NE breaks down and then reforms during cell division, and in interphase it is restored shortly after the NE rupture induced by mechanical force. In this way, the partitioning effect is regulated through dynamic processes throughout the cell cycle. A failure in rebuilding the NE structure triggers the mixing of nuclear and cytoplasmic contents, leading to catastrophic consequences for the nuclear functions. Whereas the precise details of molecular mechanisms for NE reformation during cell division and NE restoration in interphase are still being investigated, here, we mostly focus on mammalian cells to describe key aspects that have been identified and to discuss the crosstalk between them.

Rapid one-pot isothermal amplification reassembled of fluorescent RNA aptamer for SARS-CoV-2 detection.

The outbreak of SARS-CoV-2 poses a serious threat to human life and health. A rapid nucleic acid tests can effectively curb the spread of the disease. With the advantages of fluorescent RNA aptamers, low background and high sensitivity. A variety of fluorescent RNA aptamer sensors have been developed for the detection of nucleic acid. Here, we report a hypersensitive detection platform in which SARS-CoV-2 initiates RTF-EXPAR to amplify trigger fragments. This activation leads to the reassembled of the SRB2 fluorescent RNA aptamer, restoring its secondary structure for SR-DN binding and turn-on fluorescence. The platform completes the assay in 30 min and all reactions occur in one tube. The detection limit is as low as 116 aM. Significantly, the platform's quantitative analyses were almost identical to qPCR results in simulated tests of positive samples. In conclusion, the platform is sensitive, accurate and provides a new protocol for point-of-care testing of viruses.

Long-term combined blockade of CXCR4 and PD-L1 with in vivo reassembly for intensive tumor interference.

Negative immunoregulatory signal (PD-L1, CXCR4, et al.) and weak immunogenicity elicited immune system failing to detect and destroy cancerous cells. CXCR4 blockade promoted T cell tumor infiltration and increased tumor sensitivity to anti-PD-L1 therapy. Here, pH-responsive reassembled nanomaterials were constructed with anti-PD-L1 peptide and CXCR4 antagonists grafting (APAB), synergized with photothermal therapy for melanoma and breast tumor interference. The self-assembled APAB nanoparticles accumulated in the tumor and rapidly transformed into nanofibers in response to the acidic tumor microenvironment, leading to the exposure of grafted therapeutic agents. APAB enabling to reassemble around tumor cells and remained stable for over 96 h due to the aggregation induced retention (AIR) effect, led to long-term efficiently combined PD-L1 and CXCR4 blockade. Photothermal efficiency (ICG) induced immunogenic cell death (ICD) of tumor cells so as to effectively improve the immunogenicity. The combined therapy (ICG@APAB) could effectively inhibit the growth of primary tumor (∼83.52%) and distant tumor (∼76.24%) in melanoma-bearing mice, and significantly (p < 0.05) prolong the survival time over 42 days. The inhibition assay on tumor metastasis in 4 T1 model mice exhibited ICG@APAB almostly suppressed the occurrence of lung metastases and the expression levels of CD31, MMP-9 and VEGF in tumor decreased by 82.26%, 90.45% and 41.54%, respectively. The in vivo reassembly strategy will offer novel perspectives benefical future immunotherapies and push development of combined therapeutics into clinical settings.

A Semiquantitative Protein-Fragment Complementation Assay to Study Protein-Protein Interactions of the Polymerase Complex in Cellula.

Protein-fragment complementation assays (PCAs) are powerful tools to investigate protein-protein interactions in a cellular context. These are especially useful to study unstable proteins and weak interactions that may not resist protein isolation or purification. The PCA based on the reconstitution of the Gaussia princeps luciferase (split-luc) is a sensitive approach allowing the mapping of protein-protein interactions and the semiquantitative measurement of binding affinity. Here, we describe the split-luc protocol we used to map the viral interactome of measles virus polymerase complex.

Split Proteins and Reassembly Modules for Biological Applications.

Split systems, modular entities enabling controlled biological processes, have become instrumental in biological research. This review highlights their utility across applications like gene regulation, protein interaction identification, and biosensor development. Covering significant progress over the last decade, it revisits traditional split proteins such as GFP, luciferase, and inteins, and explores advancements in technologies like Cas proteins and base editors. We also examine reassembly modules and their applications in diverse fields, from gene regulation to therapeutic innovation. This review offers a comprehensive perspective on the recent evolution of split systems in biological research.

Development of Novel Nanocarriers by Ultrasound and Ethanol-Assisted Soy Protein Isolate: Enhancing the Resistance of Lutein to Environmental Stress.

The aim of this work was to investigate the effects of the processing sequence of ultrasound and ethanol on the physicochemical properties of soy protein isolate (SPI), which were further evaluated for the morphology and stability of SPI-lutein coassembled nanoparticles. The results showed that the sequence of ultrasound followed by ethanol treatment was the optimal one. The samples were subjected to ultrasonication followed by subunit disassembly and reassembly induced by 40% (v/v) ethanol, with the resulting molecular unfolding and subsequent aggregation being attributed to intramolecular hydrogen bonds. The recombined nanoparticles had smaller particle size (142.43 ± 2.91 nm) and turbidity (0.16 ± 0.01), and the exposure of more hydrophobic groups (H0 = 6221.00 ± 130.20) induced a shift of SPI structure toward a more ordered direction. The homogeneous and stable particle provided excellent stability for the loading of lutein. The bioaccessibility (from 25.48 ± 2.35 to 65.85 ± 1.78%) and release rate of lutein were modulated in gastrointestinal digestion experiments. Our discoveries provide a new perspective for the development of combined physicochemical modification of proteins as nanocarriers in functional foods.

Reassembly of the vesicular structure of niosomes after their destruction in a mechanical field.

This paper presents, for the first time, evidence for vesicle destruction and payload loss at the stage of purification of niosome dispersions by centrifugation, an important operation in the assembly of vesicular materials. The ability of niosomes of different compositions to reassemble, i.e., to restore the vesicular structure after destruction in the field of centrifugal forces, was demonstrated by dynamic light scattering and fluorescence spectroscopy. The kinetics of reassembly of vesicular structures is determined by the strength of the centrifugal field and the composition of niosomes. In contrast to ternary compositions, where particle size and modality are essentially unchanged after redispersion of the precipitate resulting from centrifugation, niosome dispersions containing anionic dicetyl phosphate includes micron-sized particles after redispersion, which vary in size over a wide range throughout the observation period. The reassembly process is complicated by the presence of charge on the surface of the niosomes. Elastic niosomes - ethosomes have been synthesised which, due to the high deformability of the shells, are less susceptible to destruction in the centrifugal field and retain the contents of the aqueous core. Using the "energy landscape" approximation, it is shown that vesicular structures assembled during hydration and reassembled after their centrifugation occupy different positions in the energetic pathway of their preparation. The results obtained should also be taken into account when determining the entrapment efficiency, since this procedure uses centrifugation to separate the load. It is important to note that the physical stability of niosomes, which is usually considered in terms of the functional activity of particles, is manifest and should be considered at the material preparation stage.

Myristoylated GRASP55 dimerizes in the presence of model membranes.

The Golgi Reassembly and Stacking Proteins (GRASPs) are engaged in various functions within the cell, both in unconventional secretion mechanisms and structuring and organizing the Golgi apparatus. Understanding their specific role in each situation still requires more structural and functional data at the molecular level. GRASP55 is one of the GRASP members in mammals, anchored to the membrane via the myristoylation of a Gly residue at its N-terminus. Therefore, co-translational modifications, such as myristoylation, are fundamental when considering a strategy to obtain detailed information on the interactions between GRASP55 and membranes. Despite its functional relevance, the N-terminal myristoylation has been underappreciated in the studies reported to date, compromising the previously proposed models for GRASP-membrane interactions. Here, we investigated the synergy between the presence of the membrane and the formation of oligomeric structures of myristoylated GRASP55, using a series of biophysical techniques to perform the structural characterization of the lipidated GRASP55 and its interaction with biological lipid model membranes. Our data fulfill an unexplored gap: the adequate evaluation of the presence of lipidations and lipid membranes on the structure-function dyad of GRASPs.Communicated by Ramaswamy H. Sarma.

Inverse priority effects: A role for historical contingency during species losses.

Communities worldwide are losing multiple species at an unprecedented rate, but how communities reassemble after these losses is often an open question. It is well established that the order and timing of species arrival during community assembly shapes forthcoming community composition and function. Yet, whether the order and timing of species losses can lead to divergent community trajectories remains largely unexplored. Here, we propose a novel framework that sets testable hypotheses on the effects of the order and timing of species losses-inverse priority effects-and suggests its integration into the study of community assembly. We propose that the order and timing of species losses within a community can generate alternative reassembly trajectories, and suggest mechanisms that may underlie these inverse priority effects. To formalize these concepts quantitatively, we used a three-species Lotka-Volterra competition model, enabling to investigate conditions in which the order of species losses can lead to divergent reassembly trajectories. The inverse priority effects framework proposed here promotes the systematic study of the dynamics of species losses from ecological communities, ultimately aimed to better understand community reassembly and guide management decisions in light of rapid global change.

Disassembly and reassembly of the non-conventional thermophilic C-phycocyanin.

C-phycocyanin (CPC), which contains open-chain tetrapyrroles, is a major light-harvesting red-fluorescent protein with an important role in aquatic photosynthesis. Recently, we reported a non-conventional CPC from Thermoleptolyngbya sp. O-77 (CPCO77) that contains two different structures, i.e., a hexameric structure and a non-conventional octameric structure. However, the assembly and disassembly mechanisms of the non-conventional octameric form of CPC remain unclear. To understand this assembly mechanism, we performed an in vitro experiment to study the disassembly and reassembly behaviors of CPC using isolated CPC subunits. The dissociation of the CPCO77 subunit was performed using a Phenyl-Sepharose column in 20 mM potassium phosphate buffer (pH 6.0) containing 7.0 M urea. For the first time, crystals of isolated CPC subunits were obtained and analyzed after separation. After the removal of urea from the purified α and ß subunits, we performed an in vitro reassembly experiment for CPC and analyzed the reconstructed CPC using spectrophotometric and X-ray crystal structure analyses. The crystal structure of the reassembled CPC was nearly identical to that of the original CPCO77. The findings of this study indicate that the octameric CPCO77 is a naturally occurring form in the thermophilic cyanobacterium Thermoleptolyngbya sp. O-77.

Transformation of SBUs and Synergy of MOF Host-Guest in Single Crystalline State: Ingenious Strategies for Modulating Third-Order NLO Signals.

Central metal exchange can innovatively open the cavity of metal-organic frameworks (MOFs) by alternating the framework topology. Here, the single-crystal-to-single-crystal (SC-SC) transformation is reported from a Co-based MOF {[Co1.25 (HL)0.5 (Pz-NH2 )0.25 (µ3 -O)0.25 (µ2 -OH)0.25 (H2 O)]·0.125 Co·0.125 L·10.25H2 O}n (Co-MOF, L = 5,5'-(1H-2,3,5-triazole-1,4-diyl)diisophthalic acid) into two novel MOF materials, {[Cu1.75 L0.75 (Pz-NH2 )0.125 (µ3 -O)0.125 (µ2 -OH)0.25 (H2 O)0.375 ]•3CH3 CN}n (Cu-MOF) and {[Zn1.75 L0.625 (Pz-NH2 )0.25 (µ3 -O)0.25 (µ2 -O)0.25 (H2 O)1.25 ]•4CH3 CN}n (Zn-MOF), through exchanging the Co2+ in the MOF into Cu2+ or Zn2+ , respectively. The free Co2+ and L4- in the Co-MOF channels fuse with the skeleton during the Co→Cu and Co→Zn exchange processes, leading to the expansion of the channel space and the transformation of the secondary building units (SBUs) to form an adjustable skeleton. The nonlinear optical response results show that the MOFs generated by the exchange of the central metal exhibit different saturable absorption and the self-focusing effect. In addition, loading polypyrrole (PPy) into the MOFs can not only improve the stability of the MOFs but also further optimize the nonlinear optical behavior. This work suggests that SC-SC central metal exchange and the introduction of polymer molecules can tune the nonlinear optical response, which provides a new perspective for the future study of nonlinear optical materials.

Better soon than never: climate change induces strong phenological reassembly in the flowering of a Mediterranean shrub community.

● Flowering is a key process in the life cycle of a plant. Climate change is shifting flowering phenologies in the Northern Hemisphere, but studies with long data series at community level are scarce, and even more so those regarding the consequences of phenological changes for emerging ecological interactions. In the Mediterranean region, the effects of climate change are stronger than the global average and there is an urgent need to understand how biodiversity will be affected in this area. ● In this study we investigated how the entire flowering phenology of a community comprising 51 perennial species from the south of the Iberian Peninsula changed from the decade of the 1980s to the 2020s. Furthermore, we have analysed the consequences of these changes for flowering order and co-flowering patterns. ● We have found that the flowering phenology of the community has advanced by about 20 days, and was coherent with the increasing temperatures related to climate change. Individual species have generally advanced their entire flowering phenology (start and end) and increased their flowering duration. The early flowering has resulted in a re-organisation of the flowering order of the community and generated a new co-flowering assemblages of species, with a slight trend towards an increase of shared flowering time among species. ● The advanced flowering phenology and changes on flower duration reported here were of unprecedented magnitude, showcasing the extreme effects of climate change on Mediterranean ecosystems. Furthermore, the effects were not similar among species, which could be attributed to differences in sensitivities of environmental cues for flowering. One consequence of these changes in flowering times is the ecological mismatches indicated by the changes in flower order and co-flowering between decades. The novel scenario may lead to new competition or facilitation interactions and to the loss or gain of pollinators.

Rapid Reassembly, Biomass-Derived Adhesive Based on Soybean Oil and Diels-Alder Bonds.

Synthetic adhesives play a crucial role in holding together solid materials through interfacial interactions. Thermoplastic and thermosetting adhesives are important types of synthetic adhesives, with thermoplastic adhesives being reassemblable and thermosetting adhesives exhibiting high adhesive strength and creep resistance. However, there is a need to combine the advantages of both types and develop high bonding strength, reassemblable adhesives. Here, epoxidized soybean oil (ESO) was used to prepare adhesive networks and Diels-Alder bonds were incorporated to enhance reassembly ability. The ESO was functionalized with furyl groups and cross-linked via the reaction between furyl and imide groups to involve the Diels-Alder bonds. The resulting adhesive exhibited good solvent resistance and mechanical properties, which could be regulated by adjusting the quantity of cross-linker. The prepared adhesives also demonstrated self-healing capabilities, as the scratch on the surface gradually diminished with heating. Additionally, the adhesives showed the ability to undergo recycling without significant changes in properties. The prepared adhesives exhibited hydrophilicity and the flow characteristics during reassembly were characterized by a decrease in torque. This study provides a promising approach for the development of synthetic adhesives with reassembly ability, which has important implications for the field of bonding.

Metabolism of glucose activates TORC1 through multiple mechanisms in Saccharomyces cerevisiae.

Target of Rapamycin Complex 1 (TORC1) is a conserved eukaryotic protein complex that links the presence of nutrients with cell growth. In Saccharomyces cerevisiae, TORC1 activity is positively regulated by the presence of amino acids and glucose in the medium. However, the mechanisms underlying nutrient-induced TORC1 activation remain poorly understood. By utilizing an in vivo TORC1 activation assay, we demonstrate that differential metabolism of glucose activates TORC1 through three distinct pathways in yeast. The first "canonical Rag guanosine triphosphatase (GTPase)-dependent pathway" requires conversion of glucose to fructose 1,6-bisphosphate, which activates TORC1 via the Rag GTPase heterodimer Gtr1GTP-Gtr2GDP. The second "non-canonical Rag GTPase-dependent pathway" requires conversion of glucose to glucose 6-phosphate, which activates TORC1 via a process that involves Gtr1GTP-Gtr2GTP and mitochondrial function. The third "Rag GTPase-independent pathway" requires complete glycolysis and vacuolar ATPase reassembly for TORC1 activation. We have established a roadmap to deconstruct the link between glucose metabolism and TORC1 activation.

Microbiome re-assembly boosts anaerobic digestion under volatile fatty acid inhibition: focusing on reactive oxygen species metabolism.

The accumulation of volatile fatty acids (VFAs) in anaerobic digestion (AD) systems resulting from food waste overload poses a risk of system collapse. However, limited understanding exists regarding the inhibitory mechanisms and effective strategies to address VFAs-induced stress. This study found that accumulated VFAs exert reactive oxygen species (ROS) stress on indigenous microbiota, particularly impacting methanogens due to their lower antioxidant capability compared to bacteria, which is supposed to be the primary reason for methanogenesis failure. To enhance the VFAs-stressed AD process, microbiome re-assembly using customized propionate-degrading consortia and bioaugmentation with concentrated digestate were implemented. Microbiome re-assembly demonstrated superior efficiency, yielding an average methane yield of 563.6±159.8 mL/L·d and reducing VFAs to undetectable levels for a minimum of 80 days. This strategy improved the abundance of Syntrophomonas, Syntrophobacter and Methanothrix, alleviating ROS stress. Conversely, microbial community in reactor with other strategy experienced an escalating intracellular damage, as indicated by the increase of ROS generation-related genes. This study fills knowledge gaps in stress-related metabolic mechanisms of anaerobic microbiomes exposed to VFAs and microbiome re-assembly to boost methanogenesis process.

Dynamics and ecological reassembly of the human gut microbiome and the host metabolome in response to prolonged fasting.

Introduction: Prolonged fasting is an intervention approach with potential benefits for individuals with obesity or metabolic disorders. Changes in gut microbiota during and after fasting may also have significant effects on the human body. Methods: Here we conducted a 7-days medically supervised water-only fasting for 46 obese volunteers and characterized their gut microbiota based on whole-metagenome sequencing of feces at five timepoints. Results: Substantial changes in the gut microbial diversity and composition were observed during fasting, with rapid restoration after fasting. The ecological pattern of the microbiota was also reassembled during fasting, reflecting the reduced metabolic capacity of diet-derived carbohydrates, while other metabolic abilities such as degradation of glycoproteins, amino acids, lipids, and organic acid metabolism, were enhanced. We identified a group of species that responded significantly to fasting, including 130 fasting-resistant (consisting of a variety of members of Bacteroidetes, Proteobacteria, and Fusobacteria) and 140 fasting-sensitive bacteria (mainly consisting of Firmicutes members). Functional comparison of the fasting-responded bacteria untangled the associations of taxon-specific functions (e.g., pentose phosphate pathway modules, glycosaminoglycan degradation, and folate biosynthesis) with fasting. Furthermore, we found that the serum and urine metabolomes of individuals were also substantially changed across the fasting procedure, and particularly, these changes were largely affected by the fasting-responded bacteria in the gut microbiota. Discussion: Overall, our findings delineated the patterns of gut microbiota alterations under prolonged fasting, which will boost future mechanistic and clinical intervention studies.