MicroRNA-27a-5p Downregulates Expression of Proinflammatory Cytokines in Lipopolysaccharide-Stimulated Human Dental Pulp Cells via the NF-κB Signaling Pathway.

MicroRNA-27a-5p (miR-27a-5p) was significantly upregulated in dental pulp inflammation, yet its underlying mechanisms remain unclear. This study investigated the effect of miR-27a-5p on the expression of proinflammatory cytokines in human dental pulp cells (hDPCs) stimulated by lipopolysaccharide (LPS). LPS-stimulated hDPCs showed concurrent increases in the expression of miR-27a-5p and proinflammatory cytokines (IL-6, IL-8, and MCP1), and the increased expression was suppressed by NF-κB inhibitor BAY 11-0785. Transfection of the miR-27a-5p mimic downregulated the expression of proinflammatory cytokines, NF-κB activity, and the expression of NF-κB signaling activators (TAB1, IRAK4, RELA, and FSTL1) in LPS-stimulated hDPCs. Luciferase reporter assays revealed that miR-27a-5p bound directly to the 3'-UTR of TAB1. siTAB1 downregulated NF-κB activity and proinflammatory cytokine expression. Downregulation of proinflammatory cytokine expression, NF-κB activity, and NF-κB signaling activator expression (TAB1, IRAK4, and RELA) was also found in LPS-stimulated rat incisor pulp tissue explants following transfection with the miR-27a-5p mimic ex vivo. MiR-27a-5p, whose expression was induced by NF-κB signaling, negatively regulated the synthesis of proinflammatory cytokines via targeting NF-κB signaling. In particular, TAB1, a potent NF-κB activator, was targeted by miR-27a-5p. These results provide insights into the negative regulatory effects of miR-27a-5p, particularly those targeting the TAB1-NF-κB signaling pathway, on pulp inflammation.

Machine Learning-Driven Discovery and Evaluation of Antimicrobial Peptides from Crassostrea gigas Mucus Proteome.

Marine antimicrobial peptides (AMPs) represent a promising source for combating infections, especially against antibiotic-resistant pathogens and traditionally challenging infections. However, traditional drug discovery methods face challenges such as time-consuming processes and high costs. Therefore, leveraging machine learning techniques to expedite the discovery of marine AMPs holds significant promise. Our study applies machine learning to develop marine AMPs, focusing on Crassostrea gigas mucus rich in antimicrobial components. We conducted proteome sequencing of C. gigas mucous proteins, used the iAMPCN model for peptide activity prediction, and evaluated the antimicrobial, hemolytic, and cytotoxic capabilities of six peptides. Proteomic analysis identified 4490 proteins, yielding about 43,000 peptides (8-50 amino acids). Peptide ranking based on length, hydrophobicity, and charge assessed antimicrobial potential, predicting 23 biological activities. Six peptides, distinguished by their high relative scores and promising biological activities, were chosen for bactericidal assay. Peptides P1 to P4 showed antimicrobial activity against E. coli, with P2 and P4 being particularly effective. All peptides inhibited S. aureus growth. P2 and P4 also exhibited significant anti-V. parahaemolyticus effects, while P1 and P3 were non-cytotoxic to HEK293T cells at detectable concentrations. Minimal hemolytic activity was observed for all peptides even at high concentrations. This study highlights the potent antimicrobial properties of naturally occurring oyster mucus peptides, emphasizing their low cytotoxicity and lack of hemolytic effects. Machine learning accurately predicted biological activity, showcasing its potential in peptide drug discovery.

Correction of thermal airflow distortion in warpage measurements of microelectronic packaging structures via deep learning-based digital image correlation.

The projected speckle-based three-dimensional digital image correlation method (3D-DIC) is being increasingly used in the reliability measurement of microelectronic packaging structures because of its noninvasive nature, high precision, and low cost. However, during the measurement of the thermal reliability of packaging structures, the thermal airflow generated by heating introduces distortions in the images captured by the DIC measurement system, impacting the accuracy and reliability of noncontact measurements. To address this challenge, a thermal airflow distortion correction model based on the transformer attention mechanism is proposed specifically for the measurement of thermal warpage in microelectronic packaging structures. This model avoids the oversmoothing issue associated with convolutional neural networks and the lack of physical constraints in generative adversarial networks, ensuring the precision of grayscale gradient changes in speckle patterns and minimizing adverse effects on DIC calculation accuracy. By inputting the distorted images captured by the DIC measurement system into the network, corrected images are obtained for 3D-DIC calculations, thus allowing the thermal warpage measurement results of the sample to be acquired. Through experiments measuring topography with customized step block specimens, the effectiveness of the proposed method in improving warpage measurement accuracy is confirmed; this is particularly true when captured images are affected by thermal airflow at 140 °C and 160 °C, temperatures commonly encountered in thermal reliability testing of packaging structures. The method successfully reduces the standard deviation from 9.829 to 5.943 µm and from 12.318 to 6.418 µm, respectively. The results demonstrate the substantial practical value of this method for measuring thermal warpage in microelectronic packaging structures.

ChRIPK1 caused necroptosis signaling pathway deficiency in Crassostrea hongkongensis.

RIPK1/TAK1 are important for programmed cell death, including liver death, necroptosis and apoptosis. However, there have been few published reports on the functions of RIPK1/TAK1 in invertebrates. In this study, full-length ChRIPK1 and ChTAK1 were cloned from C. hongkongensis through the rapid amplification of cDNA ends (RACE) technology. ChRIPK1 has almost no homology with human RIPK1 and lacks a kinase domain at the N-terminus but has a DD and RHIM domain. ChTAK1 is conserved throughout evolution. qRT‒PCR was used to analyze the mRNA expression patterns of ChRIPK1 in different tissues, developmental stages, and V. coralliilyticus-infected individuals, and both were highly expressed in the mantle and gills, while ChRIPK1 was upregulated in hemocytes and gills after V. coralliilyticus or S. aureus infection, which indicates that ChRIPK1 is involved in immune regulation. Fluorescence assays revealed that ChRIPK1 localized to the cytoplasm of HEK293T cells in a punctiform manner, but the colocalization of ChRIPK1 with ChTAK1 abolished the punctiform morphology. In the dual-luciferase reporter assay, both ChRIPK1 and ChRIPK1-RIHM activated the NF-κB signaling pathway in HEK293T cells, and ChTAK1 activated ChRIPK1 in the NF-κB signaling pathway. The apoptosis rate of the hemocytes was not affected by the necroptosis inhibitor Nec-1 but was significantly decreased, and ChRIPK1 expression was knocked down in the hemocytes of C. hongkongensis. These findings indicated that ChRIPK1 induces apoptosis but not necroptosis in oysters. This study provides a theoretical basis for further research on the molecular mechanism by which invertebrates regulate the programmed cell death of hemocytes in oysters.

Stimulating the biofilm formation of Bacillus populations to mitigate soil antibiotic resistome during insect fertilizer application.

Antibiotic resistance in soil introduced by organic fertilizer application pose a globally recognized threat to human health. Insect organic fertilizer may be a promising alternative due to its low antibiotic resistance. However, it is not yet clear how to regulate soil microbes to reduce antibiotic resistance in organic fertilizer agricultural application. In this study, we investigated soil microbes and antibiotic resistome under black soldier fly organic fertilizer (BOF) application in pot and field systems. Our study shows that BOF could stimulate ARB (antibiotic resistant - bacteria) - suppressive Bacillaceae in the soil microbiome and reduce antibiotic resistome. The carbohydrate transport and metabolism pathway of soil Bacillaceae was strengthened, which accelerated the synthesis and transport of polysaccharides to form biofilm to antagonistic soil ARB, and thus reduced the antibiotic resistance. We further tested the ARB - suppressive Bacillus spp. in a microcosm assay, which resulted in a significant decrease in the presence of ARGs and ARB together with higher abundance in key biofilm formation gene (epsA). This knowledge might help to the development of more efficient bio-fertilizers aimed at mitigating soil antibiotic resistance and enhancing soil health, in particular, under the requirements of global "One Health".

A Testis-Specific DMRT1 (Double Sex and Mab-3-Related Transcription Factor 1) Plays a Role in Spermatogenesis and Gonadal Development in the Hermaphrodite Boring Giant Clam Tridacna crocea.

The testis-specific double sex and mab-3-related transcription factor 1 (DMRT1) has long been recognized as a crucial player in sex determination across vertebrates, and its essential role in gonadal development and the regulation of spermatogenesis is well established. Here, we report the cloning of the key spermatogenesis-related DMRT1 cDNA, named Tc-DMRT1, from the gonads of Tridacna crocea (T. crocea), with a molecular weight of 41.93 kDa and an isoelectric point of 7.83 (pI). Our hypothesis is that DMRT1 machinery governs spermatogenesis and regulates gonadogenesis. RNAi-mediated Tc-DMRT1 knockdown revealed its critical role in hindering spermatogenesis and reducing expression levels in boring giant clams. A histological analysis showed structural changes, with normal sperm cell counts in the control group (ds-EGFP) but significantly lower concentrations of sperm cells in the experimental group (ds-DMRT1). DMRT1 transcripts during embryogenesis exhibited a significantly high expression pattern (p < 0.05) during the early zygote stage, and whole-embryo in-situ hybridization confirmed its expression pattern throughout embryogenesis. A qRT-PCR analysis of various reproductive stages revealed an abundant expression of Tc-DMRT1 in the gonads during the male reproductive stage. In-situ hybridization showed tissue-specific expression of DMRT1, with a positive signal detected in male-stage gonadal tissues comprising sperm cells, while no signal was detected in other stages. Our study findings provide an initial understanding of the DMRT1 molecular machinery controlling spermatogenesis and its specificity in male-stage gonads of the key bivalve species, Tridacna crocea, and suggest that DMRT1 predominantly functions as a key regulator of spermatogenesis in giant clams.

Aquaculture Performance and Genetic Diversity of a New [(Crassostrea hongkongensis ♀ × C. gigas ♂) ♂ × C. hongkongensis ♀] Variety of the Oyster "South China No. 1" in Beibu Gulf, China.

Crassostrea hongkongensis is an economically important bivalve found in various parts of the South China Sea. A new interspecific backcross ([(Crassostrea hongkongensis ♀ × C. gigas ♂) ♂ × C. hongkongensis ♀]) variety was bred by the South China Sea Institute of Oceanology which named "South China No. 1". This study aims to explore the effects of stocking density on the growth performance of "South China No. 1", compared their growth performance and genetic diversity to C. hongkongensis, and found the best place breeding site for "South China No. 1" in Beibu Gulf. The results showed that stocking a density of 20 oysters/substrate can significantly increase the shell height, shell width, total weight, survival rate, daily shell height gain and daily body mass gain. It was found that the shell height and total weight of "South China No. 1" cultured in Fangchenggang were significantly higher than that of those in Beihai and Qinzhou from September 2018 to November 2018. Similarly, the shell width of oysters in Fangchenggang and Qinzhou was also significantly higher in September 2018, and the interaction between site and stocking density had significant effects on the shell width in March 2018 and November 2018. In addition, the shell height and shell width of "South China No. 1" were significantly higher than that of C. hongkongensis in all three sites. At all three sites, the phytoplankton community structure was mostly dominated by Bacillariophyta. In the Hardy-Weinberg equilibrium test, for the seven populations and ten microsatellites, in 10 of the 70 groups, the segregation distortion was significant. These results suggest that a stocking density of 20 oysters/substrate can promote the shell height, shell width and total weight of "South China No. 1" in Beibu Gulf, China. "South China No. 1" has better growth performance compared with C. hongkongensis. Fangchenggang is a suitable place to cultivate the "South China No. 1" breed according to the total weight and sum of all algal genus abundances. The results of this study can be used as a reference to further understand the stocking density and genetic diversity of the "South China No. 1" breed in Beibu Gulf, China.

Larval biomass production from the co-digestion of mushroom root waste and soybean curd residues by black soldier fly larvae (Hermetia illucens L.).

People are increasingly using black soldier fly larvae (BSFL) as a sustainable waste management solution. They are high in protein and other essential nutrients, making them an ideal food source for livestock, poultry, and fish. Prior laboratory studies with BSFL developed on pure mushroom root waste (MRW) showed poor conversion efficiency compared to a regular artificial diet. Therefore, we mixed the nutrient-rich soybean curd residues (SCR) with MRW in different ratios (M2-M5). Pure mushroom root waste (M1, MRW 100%) had the lowest survival rate (86.2%), but it increased up to 96.9% with the SCR percentage increasing. M1 had the longest developmental period (31.1 days) and the lowest BSFL weight (7.4 g). However, the addition of SCR reduced the development time to 22.0 and 21.5 days in M4 (MRW 40%, SCR 60%) and M5 (MRW 20%, SCR 80%), respectively, and improved the larval weight to 10.9 g in M4 and 11.8 g in M5. Other groups did not have as much feed conversion ratio (FCR) (8.4 for M4 and M5), bioconversion (M4 5.4%; M5 5.9%), or lipid content (M4 25.2%; M5 24.3%). These mixtures did. Compare this to M1. We observed better results, with no significant differences between the M4 and M5 groups and their parameters. In the present study, our main target was to utilize more MRW. Therefore, we preferred the M4 group in our nutritional and safety investigation and further compared it with the artificial diet (M7). The heavy metals and essential amino acids (histidine 3.6%, methionine 2.7%, and threonine 3.8%) required for human consumption compared to WHO/FAO levels showed satisfactory levels. Furthermore, fatty acids (capric acid 1.9%, palmitic acid 15.3%, oleic acid 17.3%, and arachidonic acid 0.3%) also showed higher levels in M4 than M7. The SEM images and FT-IR spectra from the residues showed that the BSFL in group M4 changed the structure of the compact fiber to crack and remove fibers, which made the co-conversion mixture better.

Resistance of an intertidal oyster(Saccostrea mordax)to marine heatwaves and the implication for reef building.

Marine heatwaves (MHWs) have a significant impact on intertidal bivalves and the ecosystems they sustain, causing the destruction of organisms' original habitats. Saccostrea mordax mainly inhabits the intertidal zone around the equator, exhibiting potential tolerance to high temperatures and maybe a species suitable for habitat restoration. However, an understanding about the tolerance mechanism of S. mordax to high temperatures is unclear. It is also unknown the extent to which S. mordax can tolerate repeated heatwaves of increasing intensity and frequency. Here, we simulated the effects of two scenarios of MHWs and measured the physiological and biochemical responses and gene expression spectrum of S. mordax. The predicted responses varied greatly across heatwaves, and no heatwave had a significant impact on the survival of S. mordax. Specifically, there were no statistically significant changes apparent in the standard metabolic rate and the activities of enzymes of the oyster during repeated heatwaves. S. mordax exposed to high-intensity heatwaves enhanced their standard metabolic rate to fuel essential physiological maintenance and increasing activity of SOD and expression of HSP70/90. These strategies are presumably at the expense of functions related to immunity and growth, as best exemplified by significant depressions in activities of enzymes (NaK, CaMg, T-ATP, and AKP) and expression levels of genes (Rab, eEF-2, HMGR, Rac1, SGK, Rab8, etc.). The performance status of S. mordax tends to improve by implementing a suite of less energy-costly compensatory mechanisms at various levels of biological organization when re-exposed to heatwaves. The adaptive abilities shown by S. mordax indicate that they can play a crucial role in the restoration of oyster reefs in tropical seas.

Potential Function of 3,5-Dihydroxy-4-Methoxybenzyl Alcohol from Pacific Oyster (Crassostrea gigas) in Brain of Old Mice.

SCOPE: 3,5-Dihydroxy-4-methoxybenzyl alcohol (DHMBA) is found in oyster extracts in recent years and is reported to have antioxidant activity. Although it has been reported to be protective in various models of oxidative stress, the therapeutic effect of DHMBA on neurological damage caused by aging remains to be demonstrated. METHODS AND RESULTS: The present study investigates the potential functions of DHMBA in brain of old C57BL/6J mice and aging cell model. Administration of DHMBA improves working memory, reduces anxiety behavior, decreases the expression levels of cell cycle proteins, cycin-dependent kinase inhibitor 1(P21) and peptidase inhibitor 16(P16)  and inhibits neuronal loss in old mice. The data obtained from the aging cell model are consistent with those from the old mice. The interaction between DHMBA and Kelch-like ECH-associated protein 1 (Keap1) is predicted by molecular docking assay, and then it is verified by co-immunopricipitation (CoIP) that factor red lineage 2-related factor 2 (Nrf2)-Keap1 protein-protein interaction is inhibited by DHMBA. Protein levels of Nrf2 and its target genes, such as glutathione peroxidase 4(GPX4) and heme oxygenase 1 (HO-1), are detected in old mice and aging cell model. CONCLUSION: This study provides new evidence that explores the antioxidant mechanism of DHMBA and implies a potential role of DHMBA on antiaging in brain.

Identification and involvement of DAX1 gene in spermatogenesis of boring giant clam Tridacna crocea.

DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on X chromosome gene 1), a key sex determinant in various species, plays a vital role in gonad differentiation and development and controls spermatogenesis. However, the identity and function of DAX1 are still unclear in bivalves. In the present study, we identified a DAX1 (designed as Tc-DAX1) gene from the boring giant clam Tridacna crocea, a tropical marine bivalve. The full length of Tc-DAX1 was 1877 bp, encoding 462 amino acids, with a Molecular weight of 51.81 kDa and a theoretical Isoelectric point of 5.87 (pI). Multiple sequence alignments and phylogenetic analysis indicated a putative ligand binding domain (LBD) conserved regions clustered with molluscans DAX1 homologs. The tissue distributions in different reproductive stages revealed a dimorphic pattern, with the highest expression trend in the male reproductive stage, indicating its role in spermatogenesis. The DAX1 expression data from embryonic stages shows its highest expression profile (P < 0.05) in the zygote stage, followed by decreasing trends in the larvae stages (P > 0.05). The localization of DAX1 transcripts has also been confirmed by whole mount in situ hybridization, showing high positive signals in the fertilized egg, 2, and 4-cell stage, and gastrula. Moreover, RNAi knockdown of the Tc-DAX1 transcripts shows a significantly lower expression profile in the ds-DAX1 group compared to the ds-EGFP group. Subsequent histological analysis of gonads revealed that spermatogenesis was affected in a ds-DAX1 group compared to the ds-EGFP group. All these results indicate that Tc-DAX1 is involved in the spermatogenesis and early embryonic development of T. crocea, providing valuable information for the breeding and aquaculture of giant clams.

Correction: Han et al. MicroRNA-146b-5p Suppresses Pro-Inflammatory Mediator Synthesis via Targeting TRAF6, IRAK1, and RELA in Lipopolysaccharide-Stimulated Human Dental Pulp Cells. Int. J. Mol. Sci. 2023, 24, 7433.

In the original publication, there was a mistake in Figure 4J,K as published [...].

Chromosome-level genome assembly and annotation of rare and endangered tropical bivalve, Tridacna crocea.

Tridacna crocea is an ecologically important marine bivalve inhabiting tropical coral reef waters. High quality and available genomic resources will help us understand the population structure and genetic diversity of giant clams. This study reports a high-quality chromosome-scale T. crocea genome sequence of 1.30 Gb, with a scaffold N50 and contig N50 of 56.38 Mb and 1.29 Mb, respectively, which was assembled by combining PacBio long reads and Hi-C sequencing data. Repetitive sequences cover 71.60% of the total length, and a total of 25,440 protein-coding genes were annotated. A total of 1,963 non-coding RNA (ncRNA) were determined in the T. crocea genome, including 62 micro RNA (miRNA), 58 small nuclear RNA (snRNA), 83 ribosomal RNA (rRNA), and 1,760 transfer RNA (tRNA). Phylogenetic analysis revealed that giant clams diverged from oyster about 505.7 Mya during the evolution of bivalves. The genome assembly presented here provides valuable genomic resources to enhance our understanding of the genetic diversity and population structure of giant clams.

Cellulose-degrading bacteria improve conversion efficiency in the co-digestion of dairy and chicken manure by black soldier fly larvae.

Black soldier fly larvae (BSFL) have potential utility in converting livestock manure into larval biomass as a protein source for livestock feed. However, BSFL have limited ability to convert dairy manure (DM) rich in lignocellulose. Our previous research demonstrated that feeding BSFL with mixtures of 40% dairy manure and 60% chicken manure (DM40) provides a novel strategy for significantly improving their efficiency in converting DM. However, the mechanisms underlying the efficient conversion of DM40 by BSFL are unclear. In this study, we conducted a holistic study on the taxonomic stucture and potential functions of microbiota in the larval gut and manure during the DM and DM40 conversion by BSFL, as well as the effects of BSFL on cellulosic biodegradation and biomass production. Results showed that BSFL can consume cellulose and other nutrients more effectively and harvest more biomass in a shorter conversion cycle in the DM40 system. The larval gut in the DM40 system yielded a higher microbiota complexity. Bacillus and Amphibacillus in the BSFL gut were strongly correlated with the larval cellulose degradation capacity. Furthermore, in vitro screening results for culturable cellulolytic microbes from the larval guts showed that the DM40 system isolated more cellulolytic microbes. A key bacterial strain (DM40L-LB110; Bacillus subtilis) with high cellulase activity from the larval gut of DM40 was validated for potential industrial applications. Therefore, mixing an appropriate proportion of chicken manure into DM increased the abundance of intestinal bacteria (Bacillus and Amphibacillus) producing cellulase and improved the digestion ability (particularly cellulose degradation) of BSFL to cellulose-rich manure through changes in microbial communities composition in intestine. This study reveals the microecological mechanisms underlying the high-efficiency conversion of cellulose-rich manure by BSFL and provide potential applications for the large-scale cellulose-rich wastes conversion by intestinal microbes combined with BSFL.

Insights into the reduction of antibiotic-resistant bacteria and mobile antibiotic resistance genes by black soldier fly larvae in chicken manure.

The increasing prevalence of antibiotic-resistant bacteria (ARB) from animal manure has raised concerns about the potential threats to public health. The bioconversion of animal manure with insect larvae, such as the black soldier fly larvae (BSFL, Hermetia illucens [L.]), is a promising technology for quickly attenuating ARB while also recycling waste. In this study, we investigated BSFL conversion systems for chicken manure. Using metagenomic analysis, we tracked ARB and evaluated the resistome dissemination risk by investigating the co-occurrence of antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and bacterial taxa in a genetic context. Our results indicated that BSFL treatment effectively mitigated the relative abundance of ARB, ARGs, and MGEs by 34.9%, 53.3%, and 37.9%, respectively, within 28 days. Notably, the transferable ARGs decreased by 30.9%, indicating that BSFL treatment could mitigate the likelihood of ARG horizontal transfer and thus reduce the risk of ARB occurrence. In addition, the significantly positive correlation links between antimicrobial concentration and relative abundance of ARB reduced by 44.4%. Moreover, using variance partition analysis (VPA), we identified other bacteria as the most important factor influencing ARB, explaining 20.6% of the ARB patterns. Further analysis suggested that antagonism of other bacteria on ARB increased by 1.4 times, while nutrient competition on both total nitrogen and crude fat increased by 2.8 times. Overall, these findings provide insight into the mechanistic understanding of ARB reduction during BSFL treatment of chicken manure and provide a strategy for rapidly mitigating ARB in animal manure.

A novel Fas ligand plays an important role in cell apoptosis of Crassostrea hongkongensis: molecular cloning, expression profiles and functional identification of ChFasL.

Background: Apoptosis regulates normal development, homeostasis, immune tolerance and response to environmental stress by eliminating unwanted or diseased cells, and plays a key role in non-specific immunity of invertebrates. The exogenous pathway mediated by death receptors and death ligands is a very important pathway for cell apoptosis. Death ligands are mainly members of the tumour necrosis factor (TNF) family, of which FasL is an important member. The deep involvement of FasL in vertebrates cell apoptosis and immunity has been reported many times, but there is limited research on the FasL gene in shellfish, and its functional importance in oyster cell apoptosis and immunity remains unclear. Methods: The full length of ChFasL was identified and cloned based on the genome of Crassostrea hongkongensis. Quantitative PCR was used to detect the relative expression of ChFasL in different developmental stages and tissues, as well as the changes of relative expression in hemocytes after bacterial infection. The expression position of ChFasL in HEK293T cells was also located by subcellular localization, and the effect of increased recombinant protein content on the activity of reporter genes p53 and p21 was studied by dual-fluorescence reporter gene. Finally, the changes of apoptosis rate in hemocytes after ChFasL silencing was identified by RNA interference technology. Results: We identified a novel FasL gene from C. hongkongensis and named it ChFasL. We found that ChFasL has potential N-linked glycosylation site, a transmembrane domain and a TNF region, which was a typical characteristics of TNF family. ChFasL was expressed in all developmental stages of larvae and in all tissues of oysters. After stimulation by V. alginolyticus or S. haemolyticus, its relative expression in hemocytes increased significantly, suggesting that ChFasL was deeply engaged in the immune response process of C. hongkongensis to external microbial stimulation. The results of subcellular localization showed that ChFasL was mainly distributed in the cytoplasm of HEK293T cells. With the overexpression of the recombinant protein pcDNA3 1- ChFasL, the activity of p53 and p21 significantly increased, showing a positive regulatory effect. Moreover, after dsRNA successfully reduced the relative expression of ChFasL, the apoptosis rate of hemocytes was significantly lower than that the dsGFP group. Conclusion: These results comprehensively confirmed the important role of ChFasL in the apoptosis process of C. hongkongensis, which provided the basis and premise for the in-depth understanding of the immune function of apoptosis in molluscs, and also contributed to the research on the pathogenic death mechanism and disease resistance breeding of marine bivalves.

Hippo dictates signaling for cellular homeostasis and immune defense in Crassostrea hongkongensis hemocytes.

Introduction: The Hippo signaling pathway is an evolutionarily conserved signaling cascade that plays a crucial role in regulating cell proliferation, differentiation, and apoptosis. It has been shown to be a key regulator of cell fate and cellular homeostasis in various immune processes. Despite its well-established functions in vertebrate immunity, its roles in marine invertebrate immunity remain poorly understood. Therefore, our present work provides fresh mechanistic insights into how the Hippo pathway orchestrates hemocytic functions in Crassostrea hongkongensis, with implications for studies on its major forms and modifications in animal evolution. Method: The complete set of Hippo pathway genes, including SAV1, MOB1, LATS, YAP/TAZ, TEAD, and MST, were identified from the C. hongkongensis genome. Quantitative PCR assays were conducted to examine the mRNA expression levels of these genes in different tissues and the levels of these genes in hemocytes before and after bacterial challenges. The study also examined the crosstalk between the Hippo pathway and other immune pathways, such as the AP-1 and p53-dependent p21 signaling cascades. RNA interference was used to knock down MST and TEAD, and MST is a core orchestrator of non-canonical Hippo signaling, to investigate its impact on phagocytosis and bacterial clearance in hemocytes. Result: The results demonstrated that members of the Hippo pathway were highly expressed in hemocytes, with their expression levels significantly increasing following bacterial challenges. Crosstalk between the Hippo pathway and other immune pathways triggered hemocytic apoptosis, which functioned similarly to the canonical Mst-Lats-Yap signaling pathway in Drosophila and mammals. Knocking down MST resulted in increased phagocytosis and boosted the efficiency of bacterial clearance in hemocytes, presumably due to mobilized antioxidant transcription by Nrf for maintaining immune homeostasis. Discussion: This study provides novel insights into the regulatory mechanisms underlying the Hippo pathway in immune responses of C. hongkongensis hemocytes. The study highlights the importance of the Hippo pathway in maintaining immune homeostasis and orchestrating hemocytic functions in oysters. Moreover, this study demonstrates the divergence of the Hippo pathway's roles in marine invertebrate immunity from mammalian observations, indicating the need for further comparative studies across species. These findings have significant implications for future research aimed at elucidating the evolutionary trajectory and functional diversity of the Hippo signaling pathway in animal evolution.

MicroRNA-146b-5p Suppresses Pro-Inflammatory Mediator Synthesis via Targeting TRAF6, IRAK1, and RELA in Lipopolysaccharide-Stimulated Human Dental Pulp Cells.

MicroRNA-146b-5p (miR-146b-5p) is up-regulated during and to suppress the inflammation process, although mechanisms involved in the action of miR-146b-5p have not been fully elucidated. This study examined the anti-inflammation effects of miR-146b-5p in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). An increase in human miR-146b-5p (hsa-miR-146b-5p) expression following the mRNA expression of pro-inflammatory cytokines was observed in LPS-stimulated hDPCs. The expression of hsa-miR-146b-5p and pro-inflammatory cytokines was down-regulated by a nuclear factor-kappa B (NF-κB) inhibitor, and the expression of hsa-miR-146b-5p was also decreased by a JAK1/2 inhibitor. Enforced expression of hsa-miR-146b-5p abolished phosphorylation of NF-κB p65 and down-regulated the expression of pro-inflammatory cytokines and NF-κB signaling components, such as interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), and REL-associated protein involved in NF-κB (RELA). Expression of rat miR-146b-5p (rno-miR-146b-5p) and pro-inflammatory cytokine mRNA was also up-regulated in experimentally-induced rat pulpal inflammation in vivo, and rno-miR-146b-5p blocked the mRNA expression of pro-inflammatory mediators and NF-κB signaling components in LPS-stimulated ex vivo cultured rat incisor pulp tissues. These findings suggest that the synthesis of miR-146b-5p is controlled via an NF-κB/IL6/STAT3 signaling cascade, and in turn, miR-146b-5p down-regulates the expression of pro-inflammatory mediators by targeting TRAF6, IRAK1, and RELA in LPS-stimulated hDPCs.

Isolated and identified pathogenic bacteria from black soldier fly larvae with "soft rot" reared in mass production facilities and its incidence characteristics.

The black soldier fly larvae (BSFL) can transform organic waste into high-end proteins, lipids, chitin, biodiesel, and melanin at an industrial scale. But scaling up of its production capacity has also posed health risks to the insect itself. In this investigation, larval "soft rot" which is occurring in mass production facilities that cause larval developmental inhibition and a certain degree of death was reported. Responsible pathogen GX6 was isolated from BSFL with "soft rot" and identified to be Paenibacillus thiaminolyticus. No obvious impact on larval growth was observed when treated with GX6 spores, whereas mortality of 6-day-old BSFL increased up to 29.33% ± 2.05% when GX6 vegetative cells (1 × 106 cfu/g) were inoculated into the medium. Moreover, higher temperature further enhanced the BSFL mortality and suppressed larval development, but increasing substrate moisture showed the opposite effect. The middle intestine of infected larvae became swollen and transparent after dissection and examination. Transmission electron microscopy (TEM) observation indicated that GX6 had destroyed the peritrophic matrix and intestinal microvilli and damaged epithelial cells of larval gut. Furthermore, 16S rRNA gene sequencing analysis of intestinal samples revealed that gut microflora composition was significantly altered by GX6 infection as well. It can be noticed that Dysgonomonas, Morganella, Myroides, and Providencia bacteria became more numerous in the intestines of GX6-infected BSFL as compared to controls. This study will lay foundations for efficient control of "soft rot" and promote healthy development of the BSFL industry to contribute to organic waste management and circular economy.

Mitigation of antibiotic resistome in swine manure by black soldier fly larval conversion combined with composting.

The increasing prevalence of antibiotic resistance genes (ARGs) in animal manure has attracted considerable attention because of their potential contribution to the development of multidrug resistance worldwide. Insect technology may be a promising alternative for the rapid attenuation of ARGs in manure; however, the underlying mechanism remains unclear. This study aimed to evaluate the effects of black soldier fly (BSF, Hermetia illucens [L.]) larvae conversion combined with composting on ARGs dynamics in swine manure and to uncover the mechanisms through metagenomic analysis. Compared to natural composting (i.e. without BSF), BSFL conversion combined with composting reduced the absolute abundance of ARGs by 93.2 % within 28 days. The rapid degradation of antibiotics and nutrient reformulation during BSFL conversion combined with composting indirectly altered manure bacterial communities, resulting in a lower abundance and richness of ARGs. The number of main antibiotic-resistant bacteria (e.g., Prevotella, Ruminococcus) decreased by 74.9 %, while their potential antagonistic bacteria (e.g., Bacillus, Pseudomonas) increased by 128.7 %. The number of antibiotic-resistant pathogenic bacteria (e.g., Selenomonas, Paenalcaligenes) decreased by 88.3 %, and the average number of ARGs carried by each human pathogenic bacterial genus declined by 55.8 %. BSF larvae gut microbiota (e.g., Clostridium butyricum, C. bornimense) could help reduce the risk of multidrug-resistant pathogens. These results provide insight into a novel approach to mitigate multidrug resistance from the animal industry in the environment by using insect technology combined with composting, in particular in light of the global "One Health" requirements.