Corrigendum: Effects of scale worm parasitism on interactions between the symbiotic gill microbiome and gene regulation in deep sea mussel hosts.

[This corrects the article DOI: 10.3389/fmicb.2022.940766.].

Effects of scale worm parasitism on interactions between the symbiotic gill microbiome and gene regulation in deep sea mussel hosts.

Diverse adaptations to the challenging deep sea environment are expected to be found across all deep sea organisms. Scale worms Branchipolynoe pettiboneae are believed to adapt to the deep sea environment by parasitizing deep sea mussels; this biotic interaction is one of most known in the deep sea chemosynthetic ecosystem. However, the mechanisms underlying the effects of scale worm parasitism on hosts are unclear. Previous studies have revealed that the microbiota plays an important role in host adaptability. Here, we compared gill-microbiota, gene expression and host-microorganism interactions in a group of deep sea mussels (Gigantidas haimaensis) parasitized by scale worm (PA group) and a no parasitic control group (NPA group). The symbiotic microorganism diversity of the PA group significantly decreased than NPA group, while the relative abundance of chemoautotrophic symbiotic bacteria that provide the host with organic carbon compounds significantly increased in PA. Interestingly, RNA-seq revealed that G. haimaensis hosts responded to B. pettiboneaei parasitism through significant upregulation of protein and lipid anabolism related genes, and that this parasitism may enhance host mussel nutrient anabolism but inhibit the host's ability to absorb nutrients, thus potentially helping the parasite obtain nutrients from the host. In an integrated analysis of the interactions between changes in the microbiota and host gene dysregulation, we found an agreement between the microbiota and transcriptomic responses to B. pettiboneaei parasitism. Together, our findings provide new insights into the effects of parasite scale worms on changes in symbiotic bacteria and gene expression in deep sea mussel hosts. We explored the potential role of host-microorganism interactions between scale worms and deep sea mussels, and revealed the mechanisms through which scale worm parasitism affects hosts in deep sea chemosynthetic ecosystem.

Proteome and Transcriptome Analysis of Gonads Reveals Intersex in Gigantidas haimaensis.

Sex has proven to be one of the most intriguing areas of research across evolution, development, and ecology. Intersex or sex change occurs frequently in molluscs. The deep-sea mussel Gigantidas haimaensis often dominates within Haima cold seep ecosystems, but details of their reproduction remain unknown. Herein, we conducted a combined proteomic and transcriptomic analysis of G. haimaensis gonads to provide a systematic understanding of sexual development in deep-sea bivalves. A total of 2,452 out of 42,238 genes (5.81%) and 288 out of 7,089 proteins (4.06%) were significantly differentially expressed between ovaries and testes with a false discovery rate (FDR) <0.05. Candidate genes involved in sexual development were identified; among 12 differentially expressed genes between sexes, four ovary-biased genes (ß-catenin, fem-1, forkhead box L2 and membrane progestin receptor α) were expressed significantly higher in males than females. Combining histological characteristics, we speculate that the males maybe intersex undergoing sex change, and implied that these genes may be involved in the process of male testis converting into female gonads in G. haimaensis. The results suggest that this adaptation may be based on local environmental factors, sedentary lifestyles, and patchy distribution, and sex change may facilitate adaptation to a changing environment and expansion of the population. The findings provide a valuable genetic resource to better understand the mechanisms of sex change and survival strategies in deep-sea bivalves.

Molecular cloning and functional characterization of KCNQ1 in shell biomineralisation of pearl oyster Pinctada fucata martensii.

KCNQ1, a voltage-gated potassium ion channel, plays an important role in various physiological processes, including osteoblast differentiation in higher animals. However, its function in lower invertebrates such as marine shellfish remains poorly understood. Pearl oysters, such as P. fucata martensii, are ideal for studying biomineralisation. In this study, a full-length cDNA of KCNQ1 from P. fucata martensii (PfKCNQ1) was obtained, and its function in shell formation was investigated. The full-length 3945 bp cDNA of PfKCNQ1 included an open reading frame (ORF) of 1944 bp encoding a polypeptide of 647 amino acids. Multiple sequence alignment revealed high homology with KCNQ1 from other species, with six transmembrane domains (S1 - S6) and a pore (P) region. Expression pattern analysis showed that PfKCNQ1 was expressed in all tested tissues, with highest expression in mantle and heart, and shell notching induced PfKCNQ1 expression. Silencing PfKCNQ1 expression inhibited PfKCNQ1 expression and downregulated four biomineralisation-related genes (Shematrin, Pif80, N16 and MSI60). Disordered crystals or "hollows" were visible in the shell ultrastructure by scanning electron microscopy following PfKCNQ1 knockdown. The results suggested that PfKCNQ1 may participate in or regulate biomineralisation and shell formation in pearl oyster.

In vitro and in vivo evaluation of structurally-controlled silk fibroin coatings for orthopedic infection and in-situ osteogenesis.

Biomedical device-associated infections (BAI) and osteosynthesis are two main complications following the orthopedic implant surgery, especially while infecting bacteria form a mature biofilm, which can protect the organisms from the host immune system and antibiotic therapy. Comparing with the single antibiotics therapeutic method, the combination of silver nanoparticles (AgNPs) and conventional antibiotics exert a high level of antibacterial activity. Nevertheless, one major issue that extremely restricts the potential application of AgNP/antiviotics is the uncontrolled release. Moreover, the lack of osteogenic ability may cause the osteosynthesis. Thus, herein we fabricated a structure-controlled drug-loaded silk fibroin (SF) coating that can achieve the size and release control of AgNPs and high efficient osteogenesis. Three comparative SF-based coatings were fabricated: α-structured coating (α-helices 32.7%,), m-structured coating (ß-sheets 28.3%) and ß-structured coating (ß-sheets 41%). Owning to the high content of α-helices structure and small AgNPs (20 nm), α-structured coating displayed better protein adsorption and hydrophilicity, as well as pH-dependent and long-lasting antibacterial performance. In vitro studies demonstrated that α coating showed biocompatibility (cellular attachment, spreading and proliferation), high ALP expression, collagen secretion and calcium mineralization. Moreover, after one month subcutaneous implantation in vivo, α-structured coating elicited minimal, comparable inflammatory response. Additionally, in a rabbit femoral defect model, α-structured coating displayed a significant improvement on the generation of new-born bone and bonding between the new bone and the tissue, implying a rapid and durable osteointegration. Expectedly, this optimized structure-controlled SF-based coating can be an alternative and prospective solution for the current challenges in orthopedics. STATEMENT OF SIGNIFICANCE: In this study, an AgNPs/Gentamycin-loaded structured-controlled silk fibroin coatings were constructed on Ti implant's surface to guarantee the success of implantation even in the face of bacterial infection. In comparison, the α-structured coating had the lowest content of ß-sheets structure (19.0%) and the smallest particle size of AgNPs (~ 20 nm), and owned pH-responsive characteristic due to reversible α-helices structural. Thanks to pH-responsive release of Ag+, the α-structure coating could effectively inhibit adhesive bacteria and kill planktonic bacteria by releasing a large amount of reactive oxygen radicals. Through in vitro biological results (cell proliferation, differentiation and osteogenic gene expression) and in vivo rabbit femur implantation results, the α-structure coating had good biocompatible and osteogenic properties.

GKN2 promotes oxidative stress-induced gastric cancer cell apoptosis via the Hsc70 pathway.

BACKGROUND: The GKN2 is a secretory protein, whose levels decrease in gastric cancer. The present study aimed to investigate the expression, function and mechanism of action of GKN2 in gastric cancer. METHODS: Molecular biology assays were performed to elucidate the function and underlying mechanisms of GKN2 in gastric cancer under stress-induced condition in vivo and in vitro. Clinical specimens were used to assess the correlation of GKN2 and prognosis. RESULTS: We found that overexpression of GKN2 significantly enhanced apoptosis and growth arrest in vitro. GKN2 expression increased in gastric cancer cells exposed to hydrogen peroxide and promoted reactive oxygen species-induced mitochondrial dysfunction and resulted in increased cell apoptosis via inhibition of NF-κB signaling pathway and activation of JNK signaling pathway through the direct interaction of GKN2 with Hsc70. Trefoil factor 1 might contribute to the tumor suppressing effects of GKN2. MiR-216a downregulated GKN2 expression. GKN2 also inhibited xenograft tumor growth and was an independent and significant prognostic factor for patients with gastric cancer treated with oxaliplatin. CONCLUSIONS: Taken together, our data indicate that GKN2 may increase sensitivity of GC cells to the drugs which increase ROS levels in tumors. Inhibition of the interaction between GKN2 and Hsc70 could attenuate the effects induced by GKN2. GKN2 overexpression could be used to determine the subgroup of patients to obtain the more favorable outcome of oxaliplatin treatment and may be used as biomarker of the prognosis of this cancer.

H19/miR-148a/USP4 axis facilitates liver fibrosis by enhancing TGF-ß signaling in both hepatic stellate cells and hepatocytes.

Liver fibrosis is a wound-healing response represented by excessive extracellular matrix deposition. Activation of hepatic stellate cell (HSC) is the critical cellular basis for hepatic fibrogenesis, whereas hepatocyte undergoes epithelial-mesenchymal transition (EMT) which is also involved in chronic liver injury. Long noncoding RNA H19 has been found to be associated with cholestatic liver fibrosis lately. However, the role of H19 in liver fibrosis remains largely to be elucidated. In this study, we found that the expression of H19 was significantly upregulated in the liver tissue of CCl4 -induced mice, a toxicant-induced liver fibrogenesis model. Overexpression of H19 significantly aggravated activation of HSC and EMT of hepatocyte both by stimulating transforming growth factor-ß (TGF-ß) pathway. In terms of mechanism, H19 functioned as a competing endogenous RNA to sponge miR-148a and subsequently sustained the level of ubiquitin-specific protease 4 (USP4), which was an identified target of miR-148a and was able to stabilize TGF-ß receptor I. In conclusion, our findings revealed a novel H19/miR-148a/USP4 axis which promoted liver fibrosis via TGF-ß pathway in both HSC and hepatocyte, indicating that H19 could become a promising target for the treatment of liver fibrosis.

DNMT1 mediated promoter methylation of GNAO1 in hepatoma carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers worldwide and has recently become the second most common cause of cancer-related deaths in men of developing countries. Guanine nucleotide-binding protein (G protein) has been reported to be associated with the early process of HCC. In our previous study, GNAO1, one of members of G protein, was found to be down-regulated in HCC. Thus, the present study aimed to throw light upon the mechanism of the abnormal expression of GNAO1 in HCC. First, qPCR results from two HCC cell lines (SMMC-7721 and QGY-7703) confirmed the down-expression of GNAO1, followed by the validation of the methylation status of the promoter region by bisulfite sequence PCR (BSP). Moreover, 5-Aza-2'-deoxycytidine (DAC) with Trichostatin A (TSA) treatment made it much clear that GNAO1 transcription was inhibited by promoter hypermethylation, contributing to its low expression. It was further revealed that the silencing effect was regulated by methyltransferase 1 (DNMT1), and was further enhanced by transforming growth factor ß (TGF-ß). In addition, the up-regulation of GNAO1 with the help of recombinant plasmid was also found to accelerate cell apoptosis, confirmed by flow cytometry and western blotting analysis. All these results above indicated that the promoter hypermethylation of GNAO1 might play an important role in HCC, suggesting that it might be used as a promising biomarker for HCC diagnosis and targeted therapy.

Serum microRNA panel excavated by machine learning as a potential biomarker for the detection of gastric cancer.

Early detection of gastric cancer (GC) is crucial to improve the therapeutic effect and prolong the survival of patients. MicroRNAs (miRNAs) are a group of small non-protein-coding RNAs that function as repressors of diverse genes. We aimed to identify a microRNA panel in the serum of patients to predict GC non-invasively with high accuracy and sensitivity. Using six types of classifiers, we selected three markers (miR­21-5p, miR-22-3p and miR-29c-3p) from a published miRNA profiling study (GSE23739) which was treated as a training set. The values of the area under the receiver operating characteristic (ROC) curves (AUCs) were 0.9437, 0.9456 and 0.9563 in the three classifiers [Compound covariate classifier, Diagonal linear discriminant analysis (DLDA) classifier and Support vector machine classifier], respectively. Then the panel was validated further in another two miRNA profiles in GEO (Gene Expression Omnibus) databases (GSE26595, GSE28700) with high AUC values as well. Next, we found that the serum levels of miR-21 were significantly higher in GC patients than levels in healthy controls by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for confirmation, which was opposite to the serum levels of miR-22 and miR-29c (all P<0.0001). Finally, using bioinformatic tools, their biological mechanisms were elucidated by their predicted targets: Sp1 (miR-21) and PTEN (miR-22 and miR-29c). This miRNA panel is a non­invasive and potential biomarker for GC.

Neural precursor cell expressed, developmentally downregulated 8­activating enzyme inhibitor MLN4924 sensitizes colorectal cancer cells to oxaliplatin by inducing DNA damage, G2 cell cycle arrest and apoptosis.

Oxaliplatin-based chemotherapy is a primary treatment for patients with metastatic colorectal cancer (CRC); however, its efficacy is limited. Therefore, novel therapeutic agents are urgently required. MLN4924 is a first­in­class inhibitor of neural precursor cell expressed, developmentally downregulated 8 (NEDD8)­activating enzyme E1, and has entered various phase­I/II clinical trials for cancer therapy due to its significant anticancer efficacy. The aim of the present study was to examine the synergistic effect and underlying mechanisms of MLN4924 and oxaliplatin combined treatment for CRC. It was demonstrated that MLN4924 treatment induced the DNA damage response (DDR) by inactivating cullin­ring ubiquitin ligases, subsequently leading to cell cycle disturbance and apoptosis in CRC cells. MLN4924 treatment increased the oxaliplatin­induced DDR, G2 cell cycle arrest and apoptosis. Protein expression levels of phosphorylated checkpoint kinase 2 (p­CHK2), p21 and p53, which are well­known functional proteins involved in G2 cell cycle arrest, were assessed. p­CHK2 protein expression levels were increased following combined treatment with MLN4924 and oxaliplatin, whereas p21/p53 protein expression levels were not. In conclusion, MLN4924 treatment may sensitize CRC cells to oxaliplatin treatment by inducing the DDR and increasing protein expression levels of p­CHK2, leading to G2 cell cycle arrest and apoptosis. Therefore, combined MLN4924 and oxaliplatin­based chemotherapy may be a potential therapeutic strategy for the treatment of CRC.

Positive feedback loop of YB-1 interacting with Smad2 promotes liver fibrosis.

Y-box binding protein (YB-1), known as a multifunctional cellular protein in various biological processes, was recently reported to be associated with liver fibrosis. The critical role of TGF-ß/Smad signaling pathway in stimulating the transcription of fibrotic genes in fibroblasts have already been identified, however, whether and how YB-1 modulated liver fibrosis via TGF-ß/Smad signaling pathway remains largely unknown. In our previous study, we proved that ectopic TGF-ß was associated with YB-1 expression. Herein, by combining in vitro experiments in LX2 human hepatic stellate cells and in vivo studies by building CCl4 based mice liver fibrosis model, we showed that YB-1 and p-YB-1 were upregulated in liver fibrosis tissue, and YB-1 promoted the deposition of excess extracellular matrix. Mechanistically, Smad2, a key member in TGF-ß signaling pathway, acted as a transcription factor that triggered YB-1 promoter, while on the other hand, p-YB-1 stabilized Smad2 by attenuating its ubiquitination. Knockdown of Smad2 could reduce YB-1 expression, which in turn shorter the half time of Smad2. Furthermore, the serine102 residue of YB-1 both affected its binding and stabilizing activity to Smad2. These finding demonstrated that YB-1 and Smad2 played as a positive feedback loop in promoting liver fibrosis. In conclusion, TGF-ß signaling pathway may influence liver fibrosis by incorporating with YB-1, indicating that YB-1 could be a potential target for therapies against liver fibrosis.

Treatment of petrochemical wastewater by microaerobic hydrolysis and anoxic/oxic processes and analysis of bacterial diversity.

Microaerobic hydrolysis-acidification (MHA)-anoxic-oxic (A/O) processes were developed to treat actual petrochemical wastewater. The results showed that the overall COD removal efficiency was 72-79% at HRT=20h, and MHA accounted for 33-42% of COD removal, exhibiting good efficiency of acidogenic fermentation. Ammonium removal was more than 94%. The main pollutants in the influent were identified to be benzene, ketone, alcohols, amine, nitrile and phenols by GC-MS, and the majority of pollutants could be removed by MHA-A/O treatment. Proteobacteria was the most dominant bacteria in the system, accounting for more than 55% of the reads. The predominant genera in MHA, anoxic and oxic reactors were Anaerolineaceae and Sulfuritalea, Lactococcus and Blastocatella, and Saprospiraceae uncultured and Nitrosomonadaceae, respectively. This treatment system exhibited good performance in degrading the complex compounds in the petrochemical wastewater.