Types Of Domains Research Articles

Functional analysis and modification of anti-lipopolysaccharide factor (ALF) from the freshwater crab Sinopotamon henanense and preparation of a novel ShALF6-2 K-AgNPs complex.

Overuse of antibiotics has led to the emergence of drug-resistant bacteria and environmental problems. Antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) can potentially replace antibiotics. Therefore, it is possible to create composite nanostructures with synergistic bactericidal properties by combining AgNPs and AMPs. In this study, a novel anti-lipopolysaccharide factor 6, named ShALF6, was identified in the freshwater crab Sinopotamon henanense. Full-length ShALF6 is 654 bp long and contains a typical lipopolysaccharide-binding domain spanning from Cys51 to Lys72. ShALF6 is highly expressed in hemocytes and responds to infection by the gram-negative bacterium Aeromonas hydrophila. ShALF6 inhibited the growth of gram-negative bacteria by binding to them and disrupting their cell membranes. To alter the charge of ShALF6, the negatively charged glutamic acid (E) in the sequence was replaced with a positively charged lysine (K) and the modified protein was named ShALF6-2 K. The bacteriostatic activity of ShALF6-2 K was significantly enhanced by an increase in the protein's cations. ShALF6-2 K showed high binding efficiency after 36 h of co-incubation with AgNPs and modifying the surface potential of the AgNPs. ShALF6-2 K-AgNPs exhibited synergistic inhibition with enhanced effectiveness against gram-negative bacteria. Finally, the cytotoxicity of ShALF6-2 K-AgNPs was investigated. The combination of ShALF6-2 K and AgNPs significantly reduced the toxic effects of AgNPs on the cells. This study provides theoretical and experimental bases for the development of novel bioactive AMP-coated composite AgNPs.

Localization and Molecular Cloning of the ASMT Gene for Melatonin Synthesis in Pigs.

Melatonin is synthesized in multiple tissues and organs of pigs, and existing studies have shown the presence of the melatonin-synthesizing enzyme ASMT protein. However, the genomic information for the ASMT gene has been lacking. The aim of this study was to locate the genomic information of the ASMT gene in pigs using comparative genomics analysis and then obtain the coding region information through molecular cloning. First, using the NCBI Genome Data Viewer, we found that in most animals, the AKAP17A gene is often located next to the ASMT gene, with both genes arranged in the same direction. Similarly, the P2RY8 gene is commonly adjacent to the ASMTL gene, also in the same orientation. We also discovered that the ASMTL gene is frequently adjacent to the ASMT gene and arranged in the opposite direction. Using the "three-point localization" principle, we inferred the position of the ASMT gene based on the coordinates of AKAP17A and ASMTL in pigs. Our results revealed that on the pig X chromosome, a gene called LOC110258194 is located next to the AKAP17A and ASMTL genes, and its arrangement aligns with the ASMT gene in other species. Additionally, Ensembl contains a gene, ENSSSCG00000032659, at the same position, with completely overlapping exons, though it is not annotated as ASMT. Further analysis using the TreeFam tool from EMBL-EBI and the CDD tool from NCBI revealed that LOC110258194 and ENSSSCG00000032659 do not contain the typical Maf domain of ASMTL and, thus, should not be annotated as ASMTL, but rather as the ASMT gene. Using a slow-down PCR method for high-GC content genes, we successfully cloned the full CDS region of the pig ASMT gene and identified a new transcript missing Exon 6 and Exon 7. This transcript was submitted to NCBI and assigned the GenBank accession number MW847601. Our results represent the first successful localization of the ASMT gene in pigs, the first cloning of the ASMT gene's coding region, and the first discovery of a new transcript of the pig ASMT gene.

Genome-wide identification of CAMTA gene family in teak (Tectona grandis) and functional characterization of TgCAMTA1 and TgCAMTA3 in cold tolerance

BackgroundCalmodulin-binding transcription activator (CAMTA) proteins play significant roles in signal transduction, growth and development, as well as abiotic stress responses, in plants. Understanding their involvement in the low-temperature stress response of teak is vital for revealing cold resistance mechanisms.ResultsThrough bioinformatics analysis, the CAMTA gene family in teak was examined, and six CAMTA genes were identified in teak. The encoded proteins were predicted to be located in the nucleus and exhibited hydrophilic properties, with molecular weights ranging from 103.4 to 123.3 kDa and isoelectric points ranging from 5.49 to 7.55. On the basis of protein sequence homology, the CAMTA family could be divided into three subgroups. Domain and 3D structure analyses demonstrated that all the TgCAMTA proteins contained the typical CAMTA domain with the CaMBD binding domain, which was exposed on the surface. Expression analysis of different tissues revealed the expression of TgCAMTA genes in teak roots, stems, leaves, flowers, fruits, and branches. Furthermore, the promoter region contained various cis-acting elements related to light, hormone, and abiotic stress responses. After low-temperature stress treatment, different expression patterns of TgCAMTAs were observed in teak roots, stems, and leaves, with TgCAMTA1 showing the highest expression level in leaves compared with stems. Transgenic lines carrying the TgCAMTA1/3 promoter::GUS construct cold stress induction of TgCAMTA1/3 genes revealed the presence of multiple low-temperature responsive cis-acting elements in the TgCAMTA1/3 promoter region. Subcellular localization analysis indicated that these genes were functional predominantly in the nucleus. Compared with wild-type Arabidopsis, TgCAMTA1/3-overexpressing Arabidopsis presented increased tolerance to freezing stress, with increased expression of AtCOR genes. Moreover, under low-temperature conditions, TgCAMTA3-overexpressing Arabidopsis presented significantly elevated expression levels of genes related to the CBF signaling pathway, including AtCBF1/2/3.ConclusionsOur findings add significantly to the existing knowledge regarding cold stress tolerance and help elucidate cold response mechanisms in teak.

Transcriptome-wide identification and characterization of Toll pathway genes in Riptortus pedestris (Hemiptera: Alydidae)

The Toll pathway was first identified in Drosophila and plays an essential role in defense against infection by various pathogens. To date, various noncoding RNAs (ncRNAs) have been demonstrated to maintain immune homeostasis by regulating several target genes in the insect Toll pathway. However, the characterization and function of Toll pathway genes involved in the response to environmental changes at the posttranscriptional level associated with gut bacterial changes in Riptortus pedestris, which is a significant pest of soybeans, remain unclear. In this study, we identified and classified six Toll genes into three subtypes with typical Toll domain arrangements, including a Toll/interleukin receptor (TIR) domain, a transmembrane domain, and multiple leucine-rich repeat (LRR) domains; in addition, only one positive selection site was found in hemipteran sPP-Tolls, and a total of five downstream members in the Toll signaling pathway were selected and characterized. The expression patterns revealed that all these genes were widely expressed at all developmental stages of R. pedestris, and they presented variable expression levels among the different feeding treatments in the R. pedestris gut. Our comprehensive prediction analysis revealed that there are sixty miRNA‒mRNA interaction pairs, including fifty-six miRNA and six Toll pathway genes (P‒Toll1, sP‒Toll, Myd88, Pelle, Tube, and Cactus), and a ceRNA network comprising two lncRNA‒miRNA‒Toll pairs was constructed in response to environmental changes. Finally, the expression of some above genes and ncRNAs from the ceRNA network exhibited positive or negative association with the most changes in gut bacterial genera via Pearson correlation analysis. These findings provide valuable insights into how the Toll pathway of R. pedestris is involved in environmental adaptation at the posttranscriptional level and identifies new avenues for developing more effective methods for pest control through integration with gut bacteria.

Identification of ZmSNAC06, a Maize NAC Family Transcription Factor with Multiple Transcripts Conferring Drought Tolerance in Arabidopsis

Drought is one of the most serious environmental stresses affecting crop production. NAC transcription factors play a crucial role in responding to various abiotic stresses in plants. Here, we identified a maize NAC transcription factor, ZmSNAC06, between drought-tolerant and drought-sensitive inbred lines through RNA-seq analysis and characterized its function in Arabidopsis. ZmSNAC06 had five transcripts, of which ZmSNAC06-T02 had a typical NAC domain, while ZmSNAC06-P02 was localized in the nucleus of maize protoplasts and had transactivation activity in yeasts. The expression of ZmSNAC06 in maize was induced by drought. The overexpression of ZmSNAC06-T02 in Arabidopsis resulted in hypersensitivity to abscisic acid (ABA) at the germination stage, and overexpression lines exhibited higher survival rates and higher antioxidant enzyme activities compared with the wild-type under drought stress. These results suggest that ZmSNAC06 acts as a positive regulator in drought tolerance and may be used to improve drought tolerance in crops.

Identification of 3-hydroxy-3-methylglutaryl monoacyl-coenzyme A reductase (HMGR) associated with the synthesis of terpenoids in Santalum album L.

Santalum album is an economically important plant in the craft, spices and medicine industries. The main chemical constituents found in sandalwood essential oils are sesquiterpenes. 3-Hydroxy-3-methylglutaryl monoacyl-coenzyme A reductase (HMGR) is one of the rate-limiting enzymes required for the synthesis of sandal sesquiterpenes, but there are no studies on the HMGR gene in S. album. In this study, the full-length ORFs of the upper rate-limiting enzyme genes SaHMGR1 and SaHMGR2, which lie upstream of the MVA metabolic pathway of sandal sesquiterpenes, were cloned for the first time. Bioinformatics and phylogenetic analyses were conducted. The results showed that SaHMGR1 and SaHMGR2 had typical domains of HMGR class I enzymes in the HMGR superfamily, including four catalytic sites, six NADPH-binding sites, five substrate binding regions, four inhibitor binding sites, and several dimer interface regions. A phylogenetic analysis showed that SaHMGR1 and SaHMGR2 were highly conserved relative to corresponding genes in other plants. An analysis of subcellular localization showed that these SaHMGR genes were located in the endoplasmic reticulum. SaHMGR1 and SaHMGR2 were detected by real-time PCR in roots, sapwood, heartwood, young leaves, mature leaves and twigs. Highest expression was in roots. SaHMGR1 expression was higher in mature leaves than in heartwood while SaHMGR2 expression was lower in mature leaves than in heartwood. Expression in Escherichia coli strain DH5α with plasmid pET-32a (+) was also used to verify the functionality of both HMGR proteins, which catalyzed the formation of MVA from HMG-CoA. In E. coli, the enzymatic activity of SaHMGR1 was higher than that of SaHMGR2. These findings provide a basis for further studies on the function of SaHMGR genes and the regulation of sesquiterpene biosynthesis in S. album.

Characterization of a maltononaose-producing amylopullulanase from Bacillus aryabhattai W310.

The recombinated amylopullulanase of PulW310B, pullulanase from Bacillus aryabhattai W310, was characterized. Sequence analysis of PulW310B showed that PulW310B has type I pullulanase structures including its typical region and the conserved regions of glycoside hydrolase family 13. Moreover, PulW310B was predicted to has typical domains of pullulanase and SSF51445 belonging to tansglycosidase. While the substrate specificity of PulW310B presented that it is not a type I pullulanase. PulW310B could hydrolyze pullulan into maltotriose, maltohexaose, and maltononaose as main products with the proportions of 73.6 %, 16.7 %, and 6.7 %, which indicated that PulW310B has a certain potential on maltooligosaccharides production due to its 4-α-transglycosylation activity that was consistent with its domain analysis. Based on substrate specificity and hydrolytic properties, PulW310B is an amylopullulanase. This is the first report of maltononaose producing activity of amylopullulanase, which will be very useful in food industry. Moreover, PulW310B is a moderate pullulanase with optimal temperature of 40 °C and pH of 7.0. 50 % of the maximum activity was retained at room temperature (25 °C), which indicated that it is an environmentally friendly enzyme in maltooligosaccharides production.

The Drosophila toothrin Gene Related to the d4 Family Genes: An Evolutionary View on Origin and Function.

D. melanogaster has two paralogs, tth and dd4, related to the evolutionarily conserved d4 family genes. In mammals, the family consists of Dpf1-3, encoding transcription co-factors involved in the regulation of development and cell fate determination. The function of tth and dd4 in Drosophila remains unclear. The typical domain structure of the proteins encoded by the d4 family consists of an N-terminal 2/3 domain (Requiem_N), a central Kruppel-type zinc finger, and a C-terminal D4 domain of paired PHD zinc fingers (DPFs). In Drosophila, both paralogs lack the Kruppel-type ZF, and tth encodes a protein that contains only Requiem_N. In contrast, vertebrate Dpf1-3 paralogs encode all the domains, but some paralogs have specific splice isoforms. For example, the DPF3a isoform lacks the D4 domain necessary for histone reading. The occurrence of proteins without the D4 domain in mammals and flies implies functional significance and analogous roles across animal taxa. In this study, we reconstructed the evolutionary events that led to the emergence of Drosophila tth by analyzing the divergence of d4 paralogs across different evolutionary lineages. Our genomic and transcriptomic data analysis revealed duplications and gene copy loss events. Among insects, gene duplication was only observed in Diptera. In other lineages, we found the specialization of paralogs for producing isoforms and further specialization for coding proteins with specific domain organizations. We hypothesize that this pathway is a common mechanism for the emergence of paralogues lacking the D4 domain across different evolutionary lineages. We, thus, postulate that TTH may function as a splice isoform of the ancestral single-copy gene, possibly a DPF3a-like isoform characteristic of related insect species. Our analysis provides insights into the possible impact of paralogue divergence, emphasizing the functional significance of the 2/3 domain and the potential roles of isoforms lacking the D4 domain.

MYB transcription factors in Peucedanum Praeruptorum Dunn: the diverse roles of the R2R3-MYB subfamily in mediating coumarin biosynthesis

BackgroundThe MYB superfamily (v-myb avian myeloblastosis viral oncogene homolog) plays a role in plant growth and development, environmental stress defense, and synthesis of secondary metabolites. Little is known about the regulatory function of MYB genes in Peucedanum praeruptorum Dunn, although many MYB family members, especially R2R3-MYB genes, have been extensively studied in model plants.ResultsA total of 157 R2R3-MYB transcription factors from P. praeruptorum were identified using bioinformatics analysis. Comprehensive analyses including chromosome location, microsynteny, gene structure, conserved motif, phylogenetic tree, and conserved domain were further performed. The length of the 157 transcription factors ranged from 120 to 1,688 amino acids (molecular weight between 14.21 and 182.69 kDa). All proteins were hydrophilic. Subcellular localization predictions showed that 155 PpMYB proteins were localized in the nucleus, with PpMYB12 and PpMYB157 localized in the chloroplasts and mitochondria, respectively. Ten conserved motifs were identified in the PpMYBs, all of which contained typical MYB domains. Transcriptome analysis identified 47,902 unigenes. Kyoto Encyclopedia of Genes and Genomes analysis revealed 136 pathways, of which 524 genes were associated with the phenylpropanoid pathway. Differential expressed genes (DEGs) before and after bolting showed that 11 genes were enriched in the phenylpropanoid pathway. Moreover, the expression patterns of transcription genes were further verified by qRT-PCR. With high-performance liquid chromatography (HPLC), 8 coumarins were quantified from the root, stem, and leaf tissue samples of P. praeruptorum at different stages. Praeruptorin A was found in both roots and leaves before bolting, whereas praeruptorin B was mainly concentrated in the roots, and the content of both decreased in the roots and stems after bolting. Praeruptorin E content was highest in the leaves and increased with plant growth. The correlation analysis between transcription factors and coumarin content showed that the expression patterns of PpMYB3 and PpMYB103 in roots align with the accumulation trends of praeruptorin A, praeruptorin B, praeruptorin E, scopoletin, and isoscopoletin, which declined in content after bolting, suggesting that these genes may positively regulate the biosynthesis of coumarins. Eleven distinct metabolites and 48 DEGs were identified. Correlation analysis revealed that the expression of all DEGs were significantly related to the accumulation of coumarin metabolites, indicating that these genes are involved in the regulation of coumarin biosynthesis.ConclusionsR2R3-MYB transcription factors may be involved in the synthesis of coumarin. Our findings provide basic data and a rationale for future an in-depth studies on the role of R2R3-MYB transcription factors in the growth and regulation of coumarin synthesis.

Gibberellin promotes theanine synthesis by relieving the inhibition of CsWRKY71 on CsTSI in tea plant (Camellia sinensis)

Abstract Theanine is a crucial indicator of tea quality, its significance is closely tied to the economic value of tea. There were many reports on the regulation mechanism of theanine synthesis and accumulation, but the mechanism of gibberellin regulates theanine synthesis in tea plants is poorly understood. Previous studies have shown that the content of theanine experiences significant changed in the growth stages of tea shoots, displayed a strong correlation with gibberellin. This study confirmed that gibberellin significantly promoted the expression of the major gene of theanine synthesis, known as CsTSI. Additionally, the study identified CsWRKY71, a transcription factor mediated the regulation of gibberellin on theanine synthesis in tea plants. CsWRKY71 was localized in the nucleus and had a typical WRKY domain. It was a member of class IIC sub- and its expression was significantly suppressed following exogenous GA3 treatment. Further assay such as EMSA, dual luciferase and asODN interfering demonstrated that CsWRKY71 significantly interacted with the promoter of CsTSI, which inhibited theanine synthesis by binding to the cis-acting element (C/T)TGAC(T/C) of CsTSI promoter. Overall, the addition of exogenous gibberellin alleviated the inhibition of CsTSI by down-regulating the expression of CsWRKY71, ultimately facilitated the rapid biosynthesis of theanine. This study elucidated the molecular mechanism of CsWRKY71 mediated gibberellin regulation of theanine synthesis in tea plant. The findings not only enhance our understanding of the regulatory processes involved in theanine synthesis in tea plants, but also provide important references for maintaining the characteristics of high theanine in tea plant.

The JNK signalling pathway gene BmJun is involved in the regulation of egg quality and production in the silkworm, Bombyx mori.

The Jun N-terminal kinase (JNK) signalling pathway has a key role in tissue remodelling during insect metamorphosis by regulating programmed cell death. However, multiple members of the JNK pathway in Lepidoptera remain uncharacterized. In this study, two key genes of the JNK pathway, BmJun and BmFos, were cloned from the silkworm Bombyx mori, a lepidopteran model insect, and their effects on reproductive development were investigated. BmJun and BmFos encode 239 and 380 amino acids, respectively. Both proteins have typical basic leucine zipper domains and form a BmJUN-BmFOS dimer activator protein to exert transcriptional regulation. During the wandering stage of silkworm development, interference in BmJun expression had no effect on pupation, whereas B. mori vitellogenin (BmVg) expression, which is essential for egg development, was suppressed in the fat body and egg laying was significantly reduced. Additionally, numerous eggs appeared shrivelled and deformed, suggesting that they were nutritionally stunted. Inhibition of the JNK pathway caused abnormal pupal metamorphosis, an increase in shrivelled, unfertilized eggs, a decrease in fat body synthesis, and accumulation of BmVg in the ovaries of female B. mori. The results indicated that BmJUN and BmFOS can form an AP-1 dimer. Interfering with BmJun or inhibiting the phosphorylation of BmJUN leads to a reduction in the synthesis of BmVg in the fat body and its accumulation in the ovaries, thereby affecting the quality and production of the progeny eggs. These findings suggest that regulating Jun in the JNK pathway could be a potential way to inhibit female reproduction in Lepidoptera.

Screening and computational characterization of novel antimicrobial cathelicidins from amphibian transcriptomic data

As cold-blooded organisms living in damp and dark environments, amphibians have evolved robust defense mechanisms to protect themselves from predators and infections. Among the wide repertoire of bioactive compounds they produce are antimicrobial peptides (AMPs), which are required as part of innate immunity. One important class of AMPs is cathelicidins, known for their broad-spectrum activity against pathogens and their immunoregulatory roles. However, despite their promising biomedical potential and the increasing availability of omics data, few cathelicidins have been studied in amphibians, mostly through conventional experimental techniques. Here, we present 210 novel cathelicidin sequences from amphibian transcriptomes, identified through a comprehensive computational pipeline, which employed HMMER and BLAST tools to screen cathelicidin domains. These sequences reveal a typical tripartite domain architecture that was confirmed by SignalP and InterProScan analysis. Phylogenetic inference with IQ-TREE classified the sequences into six categories based on evolutionary relationships. Compared to cathelicidins from other vertebrates, amphibian mature peptides exhibit longer average lengths (around 50 amino acids), fewer aromatic and hydrophobic residues, and reduced thermal stability. Furthermore, these amphibian cathelicidins were characterized for their physicochemical and biological properties, revealing significant antimicrobial potential with lower hemolytic capability, especially in anurans, which suggests a balance between their antimicrobial and hemolytic activities predicted through AMPlify, ampir, AmpGram, and HemoPI. Secondary structure estimations, including three-dimensional modeling using AlphaFold2, indicate that amphibian cathelicidins predominantly feature α-helices and coils. Some representative models also display a high α-helix composition with amphipathic topology, facilitating interactions with simulated bacterial membranes as assessed by the PPM approach. Thus, these findings highlight the functional role of cathelicidins in amphibian immunity and their promising biomedical applicability, emphasizing the importance of applying computational methods to expand the scope and reveal the diverse landscape of cathelicidins across vertebrates.

Correlative light and soft X-ray tomography of in situ mesoscale heterochromatin structure in intact cells

Heterochromatin organization is critical to many genome-related programs including transcriptional silencing and DNA repair. While super-resolution imaging, electron microscopy, and multiomics methods have provided indirect insights into the heterochromatin organization, a direct measurement of mesoscale heterochromatin ultrastructure is still missing. We use a combination of correlative light microscopy and cryo-soft X-ray tomography (CLXT) to analyze heterochromatin organization in the intact hydrated state of human mammary fibroblast cells. Our analysis reveals that the heterochromatin ultra-structure has a typical mean domain size of approximately 80 nm and a mean separation of approximately 120 nm between domains. Functional perturbations yield further insights into the molecular density and alterations in the mesoscale organization of the heterochromatin regions. Furthermore, our polymer simulations provide a mechanistic basis for the experimentally observed size and separation distributions of the mesoscale chromatin domains. Collectively, our results provide direct, label-free observation of heterochromatin organization in the intact hydrated state of cells.

Genome-Wide Identification of Basic Helix-Loop-Helix (bHLH) Family in Peanut: Potential Regulatory Roles in Iron Homeostasis.

The basic helix-loop-helix (bHLH) superfamily is the second-largest transcription factor family that participates in a wide range of biological processes in plants, including iron homeostasis. Although the family has been studied in several plant species, a comprehensive investigation is still needed for peanut (Arachis hypogaea). Here, a genome-wide analysis identified 373 AhbHLH genes in peanut, which were divided into 14 groups or subfamilies according to phylogenetic analysis. Clustered members generally share similar gene/protein structures, supporting the evolutionary relationships among AhbHLH proteins. Most AhbHLHs experienced whole-genome or segmental duplication. The majority of AhbHLH proteins had a typical bHLH domain, while several phylogenetic groups, including Group VI, X, XIII, and XIV, had the HLH domain. The expression of several AhbHLH genes, including AhbHLH001.3, AhbHLH029.1/.2, AhbHLH047.1/.2, AhbHLH115.1/.2, AhbHLH097.1/.2, AhbHLH109.4, and AhbHLH135.1, was induced by Fe deficiency for both cultivars, or at least in Silihong, suggesting an important role in the Fe deficiency response in peanut. Nine genes (AhbHLH001.3, AhbHLH029.1/.2, AhbHLH047.1/.2, AhbHLH097.1/.2, and AhbHLH115.1/.2) were specifically induced by Fe deficiency in Silihong, and their expression was higher in Silihong than that in Fenghua 1. These genes might be responsible for higher tolerance to Fe deficiency in Silihong. Our findings provide comprehensive information for further elucidating the regulatory mechanism of Fe homeostasis in peanut.

Rab5 regulates the antimicrobial phagocytosis of hemocytes through class B scavenger receptor in Procambarus clarkii

The crucial involvement of Rab5 in regulating phagocytosis through mediating receptors, such as the Class B scavenger receptor (SRB), has been documented in higher animal species. However, little is known about the molecular mechanisms of Rab5-mediated phagocytosis in invertebrates. The present study aims to investigate whether the phagocytic function of hemocytes in Procambarus clarkii can be regulated by Rab5 through SRB. We first cloned the open reading frame of SRB from P. clarkii (defined as PcSRB), encoding a putative protein containing a typical CD36 domain. PcSRB was widely expressed in all tested tissues of P. clarkii and induced by viral and bacterial stimulation. The extracellular segment of PcSRB (PcSRB-EX) can agglutinate as well as bind with both Gram-positive (G+) and Gram-negative (G−) bacteria. Knockdown of PcSRB resulted in down-regulation of the phagocytic capacity and bactericidal activity against Aeromonas hydrophila, along with impaired expression of several phagocytosis-related genes and antimicrobial peptide (AMP) genes. PcSRB interference also affected the mRNA expression level of Tolls, indicating that PcSRB might function as a recognition receptor or co-receptor in the Toll signaling pathway. Furthermore, Rab5 interference significantly impaired the expression of PcSRB at both gene and protein levels, which confirmed its significant regulatory effect on PcSRB. Subsequently, we observed a significant decrease in the phagocytic activity and bacteria clearance of hemocytes when Rab5 and PcSRB were simultaneously knocked down. Additionally, there was a notable down-regulation in the expression levels of genes related to phagocytosis and AMPs. Overall, these findings provide preliminary evidence that Rab5 mediates PcSRB to influence the phagocytosis of P. clarkii hemocytes against A. hydrophila.

CDC42 enhances phagocytic activity and exhibits antimicrobial functions through the recruitment of IQGAP1 in Apostichopus japonicus

Cell Division Cycle 42 protein (CDC42) is a pivotal member of the Rho family of small GTPases, integral to cell signaling and the regulation of cell polarity. CDC42 is extensively documented to participate in host immunity, particularly in processes such as phagocytosis and cell migration. Nevertheless, the precise mechanisms by which CDC42 affects host-bacteria interactions remain unclear in Echinoderms. In this study, we have isolated and characterized the CDC42 gene from Apostichopus japonicus, referred to as AjCDC42. The complete cDNA sequence of AjCDC42 extends 1282 base pairs, including a typical classical RHO domain, spanning amino acids from 6 to 179. Spatial expression analysis demonstrated that AjCDC42 is ubiquitously expressed across all examined tissues. Following infection with Vibrio splendidus and stimulation with LPS, the protein and mRNA levels of AjCDC42 were significantly upregulated in coelomocytes. Moreover, the knockdown of AjCDC42 significantly decreased V. splendidus-induced phagocytic activity in coelomocytes, leading to a reduced intracellular load of V. splendidus, as evidenced by flow cytometry and bacterial plate counting assays. Mechanistically, AjCDC42 was identified as an interacting partner of IQ Motif Containing GTPase Activating Protein 1 (AjIQGAP1) through the use of co-immunoprecipitation, immunofluorescence, and GST pull-down assays combined with mass spectrometry techniques. Additional functional analyses revealed that the knockdown of AjIQGAP1 similarly decreased the phagocytic activity and the intracellular load of V. splendidus in coelomocytes. Furthermore, we detected that decreased levels of AjCDC42 and AjIQGAP1 impaired cytoskeletal rearrangement and delayed lysosomal clearance of V. splendidus. In summary, our findings underscore the pivotal role of the AjCDC42-AjIQGAP1 axis in regulating coelomocytes phagocytosis in A. japonicus during V. splendidus infection.

MaEng1, an endo-1,3-glucanase, contributes to the conidiation pattern shift through changing the cell wall structure in Metarhizium acridum

Microcycle conidiation has displayed the greater potential than normal conidiation in large-scale production of mycopesticides. Fungi require partial hydrolysis of the cell wall to achieve the necessary plasticity during their morphological changes. Therefore, various cell wall-associated hydrolases are crucial for fungal morphogenesis. Eng1, as an endo-β-1,3-glucanase, is involved in the cell separation of fungi, but its role in morphological changes of entomopathogenic fungi is not yet clear. Here, the endo-β-1,3-glucanase gene MaEng1 was characterized in the model entomopathogenic fungi M. acridum. MaEng1 possesses a typical carbohydrate hydrolase domain and belongs to the GH81 family. The functions of MaEng1 in fungal growth, stress tolerance, pathogenicity, and conidiation capacity were analyzed using targeted gene disruption. The results displayed that the absence of MaEng1 does not affect the fungal growth, stress tolerances, and pathogenicity in M. acridum. However, the knockout of MaEng1 led to the normal conidiation of M. acridum on the SYA medium, which can induce the microcycle conidiation. Moreover, the content of β-1,3-glucan in the cell wall of the MaEng1-disruption strain were significantly reduced and the exposures of β-1,3-glucan on the surface of the mature conidia and mycelia in ΔMaEng1 were declined, indicating that MaEng1 contributes to the conversion of conidiation mode in M. acridum by affecting the cell wall structure.

Interferon Regulatory Factors (IRF1, IRF4, IRF5, IRF7 and IRF9) in Sichuan taimen (Hucho bleekeri): Identification and Functional Characterization.

Background/Objectives: Interferon regulatory factors (IRFs) are multifunctional transcription factors that play important roles in the transcriptional regulation of interferons and in the immune response to pathogens. Therefore, studying the interferon system in fish is highly relevant in the prevention and treatment of viral diseases. Methods: In this study, five IRF genes (IRF1, IRF4, IRF5, IRF7 and IRF9) were identified and characterized in Hucho bleekeri, and their expression profiles were determined after LPS and Poly(I:C) treatment. Results: These IRFs have typical DNA-binding domains and IRF-association domains. Amino acid sequence comparison revealed high homology between these IRFs and those of other vertebrates, with the highest homology being with other salmonid fish. Phylogenetic analysis revealed that these IRFs are divided into four subfamilies (IRF1, IRF3, IRF4 and IRF5), with both IRF4 and IRF9 belonging to the IRF4 subfamily. IRF genes were widely expressed in all of the tested tissues, with IRF1, IRF4 and IRF9 being highly expressed in the spleen and kidney and IRF5 and IRF7 highly expressed in the gonads. IRF1, IRF4 and IRF5 expression was induced at different time points post-LPS challenge. IRF7 and IRF9 expression in the spleen and head kidney was not significantly altered by LPS induction. Poly(I:C) treatment altered IRF expression more significantly than LPS treatment. Poly(I:C) significantly altered the spleen and head kidney expression of all five IRFs. Conclusions: These findings reveal the potential role of IRFs in the antiviral response of H. bleekeri and provide a reference for examining signal transduction pathways in the interferon system in fish.

Substrate binding and catalytic mechanism of UDP-α-D-galactofuranose: β-galactofuranoside β-(1→5)-galactofuranosyltransferase GfsA.

UDP-α-D-galactofuranose (UDP-Galf): β-galactofuranoside β-(1→5)-galactofuranosyltransferase, known as GfsA, is essential in synthesizing β-(1→5)-galactofuranosyl oligosaccharides that are incorporated into the cell wall of pathogenic fungi. This study analyzed the structure and function of GfsA from Aspergillus fumigatus. To provide crucial insights into the catalytic mechanism and substrate recognition, the complex structure was elucidated with manganese (Mn2+), a donor substrate product (UDP), and an acceptor sugar molecule (β-galactofuranose). In addition to the typical GT-A fold domain, GfsA has a unique domain formed by the N and C termini. The former interacts with the GT-A of another GfsA, forming a dimer. The active center that contains Mn2+, UDP, and galactofuranose forms a groove structure that is highly conserved in the GfsA of Pezizomycotina fungi. Enzymatic assays using site-directed mutants were conducted to determine the roles of specific active-site residues in the enzymatic activity of GfsA. The predicted enzyme-substrate complex model containing UDP-Galf characterized a specific β-galactofuranosyltransfer mechanism to the 5'-OH of β-galactofuranose. Overall, the structure of GfsA in pathogenic fungi provides insights into the complex glycan biosynthetic processes of fungal pathogenesis and may inform the development of novel antifungal therapies.

A DSMC-CFD coupling method using surrogate modelling for low-speed rarefied gas flows

A new Micro-Macro-Surrogate (MMS) hybrid method is presented that couples the Direct Simulation Monte Carlo (DSMC) method with Computational Fluid Dynamics (CFD) to simulate low-speed rarefied gas flows. The proposed MMS method incorporates surrogate modelling instead of direct coupling of DSMC data with the CFD, addressing the limitations CFD has in accurately modelling rarefied gas flows, the computational cost of DSMC for low-speed and multiscale flows, as well as the pitfalls of noise in conventional direct coupling approaches. The surrogate models, trained on the DSMC data using Bayesian inference, provide noise-free and accurate corrections to the CFD simulation enabling it to capture the non-continuum physics. The MMS hybrid approach is validated by simulating low-speed, steady-state, force-driven rarefied gas flows in a canonical 1D parallel-plate system, where corrections to the boundary conditions and stress tensor are considered and shows excellent agreement with DSMC benchmark results. A comparison with the typical domain decomposition DSMC-CFD hybrid method is also presented, to demonstrate the advantages of noise-avoidance in the proposed approach. The method also inherently captures the uncertainty arising from micro-model fluctuations, allowing for the quantification of noise-related uncertainty in the predictions. The proposed MMS method demonstrates the potential to enable multiscale simulations where CFD is inaccurate and DSMC is prohibitively expensive.