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Abstract The male reproductive system has a standard immune response regulatory mechanism, However, a variety of external stimuli, including viruses, bacteria, heat, and medications can damage the testicles and cause orchitis and epididymitis. It has been shown that various RNA viruses are more likely to infect the testis than DNA viruses, inducing orchitis and impairing testicular function. It was found that local injection of the viral RNA analog poly(I:C) into the testes markedly disrupted the structure of the seminiferous tubules, accompanied by apoptosis and inflammation. Poly(I:C) mainly inhibited the expression of testosterone synthesis-associated proteins, STAR and MGARP, and affected the synthesis and metabolism of amino acids and lipids in the testis. This led to the disruption of the metabolite levels in the testis of mice, thus affecting the normal spermatogenesis process. The present study analyzed the acute inflammatory response of the testis to viral infection using a multi-omics approach. It provides insights into how RNA virus infection impairs testicular function and offers a theoretical basis for future studies on immune homeostasis and responses under stress conditions in male reproduction.

ABSTRACTAcetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded β-barrel of the partially resolved BCCP domain. Disruption of β-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTβ-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTβ to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the “swing domain model” where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.

Methanol–gasoline blends have emerged as a promising and environmentally friendly bio-fuel option, garnering widespread attention and promotion globally. The methanol content within these blends significantly influences their quality and combustion performance. This study explores the qualitative and qualitative analysis of methanol–gasoline blends using Raman spectroscopy coupled with machine learning methods. Experimentally, methanol–gasoline blends with varying methanol concentrations were artificially configured, commencing with initial market samples. For qualitative analysis, the partial least squares discriminant analysis (PLS-DA) model was employed to classify the categories of blends, demonstrating high prediction performance with an accuracy of nearly 100% classification. For the quantitative analysis, a consensus model was proposed to accurately predict the methanol content. It integrates member models developed on clustered variables, using the unsupervised clustering method of the self-organizing mapping neural network (SOM) to accomplish the regression prediction. The performance of this consensus model was systemically compared to that of the PLS model and uninformative variable elimination (UVE)–PLS model. Results revealed that the unsupervised consensus model outperformed other models in predicting the methanol content across various types of methanol gasoline blends. The correlation coefficients for prediction sets consistently exceeded 0.98. Consequently, Raman spectroscopy emerges as a suitable choice for both qualitative and quantitative analysis of methanol–gasoline blend quality. This study anticipates an increasing role for Raman spectroscopy in analysis of fuel composition.

ABSTRACT Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, respectively. It catalyzes the two-step reduction of malonyl-CoA to 3-hydroxypropionate (3-HP), a key reaction in the autotrophic CO2 fixation cycles of Chloroflexaceae green non-sulfur bacteria and the archaea Crenarchaeota. However, the structural basis underlying substrate selection, coordination, and the subsequent catalytic reactions of full-length MCR is largely unknown. For the first time, we here determined the structure of full-length MCR from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii (RfxMCR) at 3.35 Å resolution. Furthermore, we determined the crystal structures of the N- and C-terminal fragments bound with reaction intermediates NADP+ and malonate semialdehyde (MSA) at 2.0 Å and 2.3 Å, respectively, and elucidated the catalytic mechanisms using a combination of molecular dynamics simulations and enzymatic analyses. Full-length RfxMCR was a homodimer of two cross-interlocked subunits, each containing four tandemly arranged short-chain dehydrogenase/reductase (SDR) domains. Only the catalytic domains SDR1 and SDR3 incorporated additional secondary structures that changed with NADP+–MSA binding. The substrate, malonyl-CoA, was immobilized in the substrate-binding pocket of SDR3 through coordination with Arg1164 and Arg799 of SDR4 and the extra domain, respectively. Malonyl-CoA was successively reduced through protonation by the Tyr743–Arg746 pair in SDR3 and the catalytic triad (Thr165–Tyr178–Lys182) in SDR1 after nucleophilic attack from NADPH hydrides. IMPORTANCE The bi-functional MCR catalyzes NADPH-dependent reduction of malonyl-CoA to 3-HP, an important metabolic intermediate and platform chemical, from biomass. The individual MCR-N and MCR-C fragments, which contain the alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities, respectively, have previously been structurally investigated and reconstructed into a malonyl-CoA pathway for the biosynthetic production of 3-HP. However, no structural information for full-length MCR has been available to illustrate the catalytic mechanism of this enzyme, which greatly limits our capacity to increase the 3-HP yield of recombinant strains. Here, we report the cryo-electron microscopy structure of full-length MCR for the first time and elucidate the mechanisms underlying substrate selection, coordination, and catalysis in the bi-functional MCR. These findings provide a structural and mechanistic basis for enzyme engineering and biosynthetic applications of the 3-HP carbon fixation pathways.

Astragalus species have a certain capacity to enrich selenium (Se) and are the strongest Se hyperaccumulator legumes known globally at present. The biochar application to medicinal plants has been reported to affect plant metabolites. In this study, we aimed to employ hyperaccumulating Astragalus species in the plant growth of selenium-lacked soil, while also investigating the impact of varying selenium doses and biochar application on legumes growth, selenium content, and secondary metabolite production. Applying biochar to soil, along with a Se concentration of 6 mg/kg, significantly enhanced the growth, Se content, total polysaccharide content, and calycosin-7-glucoside content of Astragalus species (p < 0.05). Importantly, the Se and biochar application also led to a significant improvement in Se content in ABH roots (p < 0.05). Meanwhile, the content of total flavonoids in ABH roots could be promoted by a Se concentration of 3 mg/kg and biochar application in soil. Additionally, the Se enrichment coefficients of Astragalus species under Se treatments were significantly higher than those under control treatment, with a marked difference observed across all treatments, whether roots or above-ground (p < 0.05). Remarkably, the Se transport coefficients of Astragalus species were observed to be lower than one, except for the transport coefficient of AB in the Se concentration of the control treatment (0 mg/kg). This result showed that a medium concentration treatment of Se and biochar application in soil not only promotes the growth of Astragalus species and the uptake of exogenous Se but also increases the active component content, meanwhile enhancing the Se enrichment and transport capacity. Taken as a whole, the present findings offer a more comprehensive understanding of the interplay between distinct Se levels, as well as the addition of biochar in soil, providing valuable insight for the cultivation of Se-rich Astragalus in Se-deficient soil-plant systems.

Coenzyme A (CoA) transferases catalyze reversible transfer of CoA groups from CoA-thioesters to free acids, playing important roles in the metabolism of carboxylic acids in all organisms. An intramolecular CoA transferase, Mesaconyl-CoA C1-C4 CoA transferase (MCT) was identified in the autotrophic CO2 fixation pathway, 3-hydroxypropionic acid cycle of filamentous anoxygenic phototrophs (FAPs). Different from the well-known CoA transferases that catalyze CoA transfer between two distinct substrates, MCT specifically catalyzes the reversible transformation of mesaconyl-C1-CoA to mesaconyl-C4-CoA, a key reaction intermediate for carbon fixation. However, the molecular mechanism of MCT in employing one substrate is enigmatic. Here we determined the crystal structure of MCT from a chlorosome-less FAP Roseiflexus castenholzii at 2.5 Å resolution, and characterized the catalytic mechanisms through structural analyses and molecular dynamic simulations. The structure of R. castenholzii MCT consists of a Rossmann fold larger domain and a small domain that are connected by two linkers. Two MCT subunits are cross interlocked at the linker regions to form a functional dimer in solution, in which the substrate binding pockets are located at the interface of the Rossmann fold larger domain from one subunit and the small domain from the other subunit. In the simulated binding structures, both the substrate mesaconyl-C1-CoA and product mesaconyl-C4-CoA form extensive electrostatic and hydrogen bonding interactions with MCT. But some differences exist in the binding mode of these two CoA analogs, Arg314’ from the second subunit of the dimer presenting dramatic conformational changes in binding with mesaconyl-C4-CoA. Together with Arg47 and one water molecule, a strictly conserved residue Asp165 are essential for catalyzing the reversible intramolecular CoA transfer reaction, through the electrostatic and hydrogen bonding interactions with the mesaconic tail of both the substrate and product. This study revealed a previously unrecognized mechanism for the uncommon intramolecular CoA transfer reaction, which will not only broaden the knowledge on the catalytic mechanisms of CoA transferases, but also contribute to enzyme engineering or biosynthetic applications of the 3-HP cycle for synthesis of fine chemicals and important metabolites.

At present, the research of robot door opening method is basically realized by identifying the door handle through the synchronous sensing system on the premise that the bio-inspired mobile manipulator is located in front of the door. An adaptive door opening strategy of a bio-inspired mobile manipulator based on a synchronous sensing system is proposed. Firstly, the random delay distribution in clock synchronization technology is analyzed in detail, and its distribution is verified on the experimental platform of adjacent nodes. Based on the Gaussian distribution of random delay, the relative frequency offset and relative phase offset of adjacent nodes are calculated. The clock synchronization of network cable sensor nodes is realized. Secondly, based on the information data of synchronous sensing system, this article realizes target detection and tracking based on depth network. In addition, based on the sliding mode control theory, the dynamic model of the nonholonomic bio-inspired mobile manipulator is applied. Finally, a robust adaptive sliding mode control method for nonlinear systems with input gain uncertainty and unmatched uncertainty is proposed by combining adaptive backstepping with sliding mode control. By adding sliding mode control in the last step of adaptive backstepping, the uncertainty of the system is compensated, and the system trajectory is maintained on the specified sliding mode manifold. The tracking control and stability control of the nonholonomic bio-inspired mobile manipulator are simulated. The experimental and simulation results show that the control method proposed in this article is effective and feasible.

Summary: The mitochondrial uniporter is a Ca2+-selective ion-conducting channel in the inner mitochondrial membrane that is involved in various cellular processes. The components of this uniporter, including the pore-forming membrane subunit MCU and the modulatory subunits MCUb, EMRE, MICU1, and MICU2, have been identified in recent years. Previously, extensive studies revealed various aspects of uniporter activities and proposed multiple regulatory models of mitochondrial Ca2+ uptake. Recently, the individual auxiliary components of the uniporter and its holocomplex have been structurally characterized, providing the first insight into the component structures and their spatial relationship within the context of the uniporter. Here, we review recent uniporter structural studies in an attempt to establish an architectural framework, elucidating the mechanism that governs mitochondrial Ca2+ uptake and regulation, and to address some apparent controversies. This information could facilitate further characterization of mitochondrial Ca2+ permeation and a better understanding of uniporter-related disease conditions.

Based on the active interference suppression algorithm, this study combines the radar working mode and the interference type and realizes the effective detection of the flaw detection signal by successively processing the radar receiving signal and the filtering processing. Firstly, this article builds a simulation platform similar to the actual situation to verify the existing conventional active interference suppression algorithms. Secondly, for the detection of chirp active deception jamming signals entering from the main lobe, a radar active deception jamming detection method based on the characteristic parameter matching of the harmonic components of active deception jamming signals is proposed. After that, the spectral characteristics of the harmonic components of the deception interference signal are analyzed, and the center frequency and the tuning frequency of the real target echo are obtained. Finally, by establishing a frequency modulation parameter library for possible interference harmonic signal components, the acquisition phase of the radar gate by the jammer matched analysis with the preestablished frequency modulation parameter library is implemented to achieve active deception interference detection. This method can effectively detect active deception jamming signals in a complex tunnel environment. The interference suppression algorithms verified by simulation include noise FM interference suppression algorithm based on cancellation and distance false target interference suppression algorithm based on LFM radar summary processing. Through actual measurement data processing and analysis, the effectiveness of the method is verified and the idea of interference suppression is expanded. The construction of the simulation platform is obtained by appropriately modifying the actual parameters, a certain type of suppression jammer, and a certain type of deception jammer used in a certain countermeasure field test at a radar station.

Phosphoenolpyruvate carboxykinase (PEPCK1) is ubiquitinated by E3 ubiquitin ligase UBR5, which was thought to be facilitated by the acetylation of Lys70, Lys71 and Lys594 in PEPCK1. Here, we made a series of UBR5 HECT domain truncation variants and, through pull-down assay, showed that the N-terminal lobe of the UBR5 HECT domain is largely responsible for interacting with PEPCK1. We mutated all three lysine residues thought to be acetylated in PEPCK1 but were surprised to observe no loss of binding to UBR5 HECT domain. Furthermore, two PEPCK1 truncation variants (74-622 aa and 10-560 aa) lacking these lysine residues were still able to bind with UBR5 and ubiquitinated in HEK293T cells. To discover the ubiquitination site(s) of PEPCK1, which is currently unknown, the Lys residues of PEPCK1 were mutated to Ala and the ubiquitination level of the PEPCK1 mutants was assessed. Results revealed at least two ubiquitination sites (Lys243 and Lys342), which represent the first time that ubiquitination sites of PEPCK1 have been identified. Our pull-down experiments further show that the lack of ubiquitination of PEPCK1 Lys243Ala and Lys342Ala mutants is not due to their binding to UBR5, which remained unchanged. Taken together, our work has provided new insights into UBR5 mediated ubiquitination of PEPCK1.

Mrakia psychrophila is an obligate psychrophilic fungus. The cold adaptation mechanism of psychrophilic fungi remains unknown. Comparative genomics analysis indicated that M. psychrophila had a specific codon usage preference, especially for codons of Gly and Arg and its major facilitator superfamily (MFS) transporter gene family was expanded. Transcriptomic analysis revealed that genes involved in ribosome and energy metabolism were upregulated at 4°, while genes involved in unfolded protein binding, protein processing in the endoplasmic reticulum, proteasome, spliceosome, and mRNA surveillance were upregulated at 20°. In addition, genes related to unfolded protein binding were alternatively spliced. Consistent with other psychrophiles, desaturase and glycerol 3-phosphate dehydrogenase, which are involved in biosynthesis of unsaturated fatty acid and glycerol respectively, were upregulated at 4°. Cold adaptation of M. psychrophila is mediated by synthesizing unsaturated fatty acids to maintain membrane fluidity and accumulating glycerol as a cryoprotectant. The proteomic analysis indicated that the correlations between the dynamic patterns between transcript level changes and protein level changes for some pathways were positive at 4°, but negative at 20°. The death of M. psychrophila above 20° might be caused by an unfolded protein response.

MICU2 has been reported to interact with MICU1 and participate in the regulation of mitochondrial Ca2+ uptake, although the molecular determinants underlying the function of MICU2 is unknown. In order to characterize MICU2 we screened a series of N-terminal and C-terminal truncations and obtained constructs which can be expressed in abundance, giving rise to soluble samples to enable subsequent characterizations. Size exclusion chromatography (SEC) and multi-angle laser light scattering (MALLS) revealed that MICU2 exists as a monomer in Ca2+-free conditions but forms a dimer in Ca2+-bound conditions. Unlike MICU1, the C-helix domain of MICU2 exhibits no influence on protein conformation in both Ca2+-free and Ca2+-bound forms. Furthermore, mutation of the first EF-hand abolishes the ability of MICU2 to switch to a dimer in the presence of Ca2+, indicating that the first EF-hand is not only involved in Ca2+ binding but also in conformational change. Our pull-down and co-immunoprecipitation assays suggest that, in addition to disulfide bonds, salt bridges also contribute to MICU1-MICU2 heterodimer formation.

To calculate the stress of cement-soil pile (flexible pile) in composite foundation accurately and overcome the defective preposition that the pile and the soil are elastic, the nonlinear analysis and calculation of pile-soil stress by adopting the energy method are performed. First, on the basis of the universally adopted load transfer method, it is assumed that the friction and the relative displacement of the pile shaft conform with the elastic-plastic relationship. Then, the modified Cam-clay model is introduced to calculate the compressive amount of soil around the pile. Further, the stress and strain characteristics curve of the flexible pile is used to calculate the compression of the pile, deducing the energy equation of the pile and soil around the pile, establishing the total potential energy expression of the flexible pile composite foundation, and the load sharing value and the corresponding stress value of the pile-soil are obtained based on the minimum potential energy principle. Finally, this method is verified by indoor laboratory model test and the reported case. The results show that the calculated solutions are in agreement with the measurements; therefore, the calculation method is reasonable.

The silos are widely used in bulk material in many fields such as agriculture, mining, chemical, electric power storage, etc. Thin metal cylindrical silo shells are vulnerable to buckling failure caused by the compressive wall friction force. In this paper, the structural analysis of two types of steel silo with cylindrical-wall bearing and profile-steel bearing is implemented by Abaqus finite element analysis. The results indicate that under the same loading conditions, steel silos with profile-steel bearing and cylindrical-Wall bearing have similar values in Mises stress, but the steel silo with profile-steel bearing has a smaller radial displacement and a better capability of buckling resistance. Meanwhile, the total steel volumes reduced 8.0% comparing to the steel silo with cylindrical-wall bearing. Therefore, steel soil with profile-steel bearing not only has a less steel volumes but also a good stability.

Load-depth curves of an unpoled Lead Zirconate Titanate (PZT) film composite as a function of temperature were measured by nanoindentation technique. Its reduce modulus and hardness were calculated by the typical Oliver-Pharr method. Then the true modulus and hardness of the PZT film were assessed by decoupling the influence of substrate using methods proposed by Zhou et al. and Korsunsky et al., respectively. Results show that the indentation depth and modulus increase, but the hardness decreases at elevated temperature. The increasing of indentation depth and the decreasing of hardness are thought to be caused by the decreasing of the critical stress needed to excite dislocation initiation at high temperature. The increasing of true modulus is attributed to the reducing of recoverable indentation depth induced by back-switched domains. The influence of residual stress on the indentation behavior of PZT film composite was also investigated by measuring its load-depth curves with pre-load strains.

BACKGROUND: An aptamer based biosensor (aptasensor) was developed and evaluated for rapid colorimetric detection of Escherichia coli (E. coli) O157:H7. METHODOLOGY/PRINCIPAL FINDINGS: The aptasensor was assembled by modifying the truncated lipopolysaccharides (LPS)-binding aptamer on the surface of nanoscale polydiacetylene (PDA) vesicle using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between E. coli O157:H7 and aptamer at the interface of the vesicle lead to blue-red transition of PDA which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR). Confocal laser scanning microscope (CLSM) and transmission electron microscopy (TEM) was used to confirm the specific interactions between the truncated aptamer and E. coli O157:H7. The aptasensor could detect cellular concentrations in a range of 10(4)~ 10(8) colony-forming units (CFU)/ml within 2 hours and its specificity was 100% for detection of E. coli O157:H7. Compared with the standard culture method, the correspondent rate was 98.5% for the detection of E. coli O157:H7 on 203 clinical fecal specimens with our aptasensor. CONCLUSIONS: The new aptasensor represents a significant advancement in detection capabilities based on the combination of nucleic acid aptamer with PDA vesicle, and offers a specific and convenient screening method for the detection of pathogenic bacteria. This technic could also be applied in areas from clinical analysis to biological terrorism defense, especially in low-resource settings.

The study of the constitutive relationship of lunar soil is the key to a deep understanding of the mechanical properties of lunar soil. Previous models mostly focused on the strengthening behavior, while rarely reflected the post-peak softening and residual deformation stages during shear deformation. A new elastoplastic constitutive relation is derived with combining kinematic hardening model and initial shear stress, which effectively compensates for the shortcomings of existing constitutive models, and the validity of the model is verified by comparing with existed laboratory test results. The developed constitutive model not only effectively captures the shear dilatancy and softening characteristics of lunar soil simulant, but also only requires fewer parameters to be easily determined by simple initial loading curves from direct shear tests, Furthermore, the influences of some key parameters on shear strength and softening behavior of lunar soil simulant can be easily obtained based on this constitutive model. [ABSTRACT FROM AUTHOR]