Prognostic biomarkers associated with immune checkpoint inhibitors in hepatocellular carcinoma.

The treatment of hepatocellular carcinoma (HCC), particularly advanced HCC, has been a serious challenge. Immune checkpoint inhibitors (ICIs) are landmark drugs in the field of cancer therapy in recent years, which have changed the landscape of cancer treatment. In the field of HCC treatment, this class of drugs has shown good therapeutic prospects. For example, atezolizumab in combination with bevacizumab has been approved as first-line treatment for advanced HCC due to significant efficacy. However, sensitivity to ICI therapy varies widely among HCC patients. Therefore, there is an urgent need to search for determinants of resistance/sensitivity to ICIs and to screen biomarkers that can predict the efficacy of ICIs. This manuscript reviews the research progress of prognostic biomarkers associated with ICIs in HCC in order to provide a scientific basis for the development of clinically individualised precision medication regimens.

The progress of research on immune checkpoint inhibitor resistance and reversal strategies for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in humans, which is prone to recurrence and metastasis and has a poor prognosis. The occurrence and progression of HCC are closely related to immune elimination, immune homeostasis, and immune escape of the immune system. In recent years, immunotherapy, represented by immune checkpoint inhibitors (ICIs), has shown powerful anti-tumor capabilities in HCC patients. However, there are still some HCC patients who cannot benefit from ICIs treatment due to their innate or acquired drug resistance. Therefore, it is of great practical significance to explore the possible mechanisms of resistance to ICIs in HCC and to use them as a target to design strategies to reverse resistance, to overcome drug resistance in HCC and to improve the prognosis of patients. This article summarizes the possible primary (tumor microenvironment alteration, and signaling pathways, etc.) and acquired (immune checkpoint upregulation) resistance mechanisms in patients with HCC treated with ICIs, and based on this, discusses the status and effectiveness of combination drug strategy to reverse drug resistance, to provide a reference for subsequent related studies and decisions.

Efficacy and safety of FDA-approved IDH inhibitors in the treatment of IDH mutated acute myeloid leukemia: a systematic review and meta-analysis.

OBJECTIVE: To systematically evaluate the efficacy and safety of FDA-approved isocitrate dehydrogenase (IDH) inhibitors in the treatment of IDH-mutated acute myeloid leukemia (AML). METHODS: We used R software to conduct a meta-analysis of prospective clinical trials of IDH inhibitors in the treatment of IDH-mutated AML published in PubMed, Embase, Clinical Trials, Cochrane Library and Web of Science from inception to November 15th, 2022. RESULTS: A total of 1109 IDH-mutated AML patients from 10 articles (11 cohorts) were included in our meta-analysis. The CR rate, ORR rate, 2-year survival (OS) rate and 2-year event-free survival (EFS) rate of newly diagnosed IDH-mutated AML (715 patients) were 47%, 65%, 45% and 29%, respectively. The CR rate, ORR rate, 2-year OS rate, median OS and median EFS of relapsed or refractory (R/R) IDH-mutated AML (394 patients) were 21%, 40%, 15%, 8.21 months and 4.73 months, respectively. Gastrointestinal adverse events were the most frequently occurring all-grade adverse events and hematologic adverse events were the most frequently occurring ≥ grade 3 adverse events. CONCLUSION: IDH inhibitor is a promising treatment for R/R AML patients with IDH mutations. For patients with newly diagnosed IDH-mutated AML, IDH inhibitors may not be optimal therapeutic agents due to low CR rates. The safety of IDH inhibitors is controllable, but physicians should always pay attention to and manage the differentiation syndrome adverse events caused by IDH inhibitors. The above conclusions need more large samples and high-quality RCTs in the future to verify.

The efficacy and safety of trastuzumab deruxtecan (T-DXd) in HER2-expressing solid tumours: a single-arm meta-analysis.

OBJECTIVE: We performed a meta-analysis to assess the efficacy and safety of T-DXd in the treatment of HER2-expressing solid tumours. METHODS: We systematically searched PubMed, Web of Science, Embase and the Cochrane Library and collected studies published before March 17, 2023, on T-DXd for HER2-expressing tumours for a meta-analysis. We performed a subgroup analysis based on the different cancer types and the doses used. RESULTS: There were 11 studies including 1349 HER2-expressing patients in this meta-analysis. The pooled ORR was 47.91%, and the pooled DCR was 87.01%. The mPFS and mOS combined were 9.63 and 10.71 months, respectively. The most common adverse reactions in grades 1-2 were decreased appetite (49.3%) and vomiting (43.0%). The netropemia (31.2%) and leukopenia (31.2%) were the most common grade 3 and higher adverse reactions. Subgroup analysis showed that breast cancer had the best ORR and DCR, with 66.96 and 96.52%, respectively. CONCLUSIONS: Overall, the efficacy of T-DXd in treating HER2-expressing solid tumours is encouraging, especially breast and non-small cell lung cancers, and has an acceptable safety profile. However, concerns remain about potentially serious treatment adverse events (e.g. interstitial lung disease/pneumonia). More well-designed, large-scale randomized controlled trials are needed to demonstrate our study.

MTHFD1 is critical for the negative regulation of retinoic acid receptor signalling in anencephaly.

Neural tube defects are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Retinoic acid, an active derivative of vitamin A, is critical for neural system development, and retinoic acid receptor (RAR) signalling malfunctions have been observed in human neural tube defects. However, retinoic acid-retinoic acid receptor signalling regulation and mechanisms in neural tube defects are not fully understood. The mRNA expression of RARs and retinoid X receptors in the different human neural tube defect phenotypes, including 11 pairs of anencephaly foetuses, 10 pairs of hydrocephalus foetuses and nine pairs of encephalocele foetuses, was investigated by NanoString nCounter technology. Immunoprecipitation-mass spectrometry was performed to screen the potential interacting targets of retinoic acid receptor γ. The interactions between proteins were confirmed by co-immunoprecipitation and immunofluorescence laser confocal microscopy. Luciferase and chromatin immunoprecipitation with quantitative real-time polymerase chain reaction assays were used to clarify the underlying mechanism. Moreover, a neural tube defect animal model, constructed using excess retinoic acid, was used for further analysis with established molecular biology technologies. We report that level of retinoic acid receptor γ (RARγ) mRNA was significantly upregulated in the brain tissues of human foetuses with anencephaly. To further understand the actions of retinoic acid receptor γ in neural tube defects, methylenetetrahydrofolate dehydrogenase 1 was identified as a specific retinoic acid receptor γ target from IP-MS screening. Additionally, methylenetetrahydrofolate dehydrogenase 1 negatively regulated retinoic acid receptor γ transcription factor activity. Furthermore, low expression of methylenetetrahydrofolate dehydrogenase 1 and activation of retinoic acid receptor signalling were further determined in human anencephaly and a retinoic acid-induced neural tube defect mouse model. This study reveals that methylenetetrahydrofolate dehydrogenase 1, the rate-determining enzyme in the one-carbon cycle, might be a specific regulator of retinoic acid receptors; these findings provide new insights into the functional linkage between nuclear folate metabolism and retinoic acid receptor signalling in neural tube defect pathology.

Incidence and risk of hypertension associated with PARP inhibitors in cancer patients: a systematic review and meta-analysis.

OBJECTIVE: To analyze the incidence and risk of hypertension associated with poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors in cancer patients and provide reference for clinicians. METHODS: We used R software to conduct a meta-analysis of phase II/III randomized controlled trials (RCT) on PARP inhibitors for cancer treatment published in PubMed, Embase, Clinical Trials, Cochrane Library and Web of Science from inception to July 29th, 2022. RESULTS: We included 32 RCTs with 10,654 participants for this meta-analysis. For total PARP inhibitors, the incidence and risk ratio of all-grade hypertension were 12% and 1.22 (95% CI: 0.91-1.65, P = 0.19, I2 = 81%), and the incidence and risk ratio of grade 3-4 hypertension were 4% and 1.24 (95% CI: 0.74-2.08, P = 0.42, I2 = 68%). Compared with the control group, the niraparib group, olaparib 800 mg/day group, and olaparib plus cediranib group increased the risk of any grade and grade 3-4 hypertension, while the veliparib group and rucaparib group did not increase the risk of any grade and grade 3-4 hypertension, and olaparib 200 mg-600 mg/day group (exclude olaparib plus cediranib regime) reduced the risk of any grade and grade 3-4 hypertension. CONCLUSION: Olaparib 200-600 mg/day (excluding olaparib plus cediranib regimen) may be the most suitable PARP inhibitor for cancer patients with high risk of hypertension, followed by veliparib and rucaparib. Niraparib, olaparib 800 mg/day and olaparib combined with cediranib may increase the risk of developing hypertension in cancer patients, clinicians should strengthen the monitoring of blood pressure in cancer patients and give medication in severe cases.

Adverse events of immune checkpoint inhibitors for patients with digestive system cancers: A systematic review and meta-analysis.

Objective: To study the incidence and distribution of adverse events in immune checkpoint inhibitors (ICI) for digestive system cancers and to provide a reference for the safe, rational, and effective use of immune detection site inhibitors. Methods: We searched for articles published in English between January 1, 2010, and May 18, 2022. All clinical trials of ICI-based therapies for digestive system cancers were investigated, including only randomized controlled trials that reported data on the overall incidence of treatment-related adverse events (trAEs) or immune-related adverse reactions (irAEs) or tables. Results: We searched 2048 records, of which 21 studies (7108 patients) were eligible for inclusion. The incidence of ICI trAEs of any grade was 82.7% (95% CI 73.9-90.0), and the incidence of grade 3 or higher trAEs was 27.5% (95% CI 21.3-34.1). The pooled rate of ICI irAEs of any grade was 26.3% (95% CI 11.8-44.0), and the incidence of grade 3 or higher irAEs was 9.4% (95% CI 1.1-24.6). In multivariate analysis, the incidence, characteristics, and distribution of AEs varied by cancer type, combination therapy modality (single/two-drug), and different agent types. Conclusion: Our meta-analysis summarizes AEs associated with ICI in digestive system cancers. The incidence, characteristics, and distribution of AEs vary by cancer type, combination therapy modality, and different agent types. These findings can be considered for the early identification of AEs and provide effective interventions to reduce the severity of these patients. It can provide a clinical reference and may contribute to clinical practice.

Open channel block of Kv1.5 channels by HMQ1611.

Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (I Kur). Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as an effective treatment of re-entrant based atrial fibrillation, because Kv1.5 is highly expressed in human cardiac atria but scarcely in ventricles. The Kv1.5 blockade is also expected to be used in cancer therapeutics since Kv1.5 is overexpressed in some types of human tumors. Here, we investigated the blockade of hKv1.5 channels by HMQ1611, a symmetrical biphenyl derivative. hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. The effects of HMQ1611 on wild-type and 13 hKv1.5 mutant channels were examined using the whole-cell patch-clamp method, and molecular docking simulation was conducted to predict the docking position of HMQ1611 within Kv1.5 channels. We showed that HMQ1611 reversibly inhibited the hKv1.5 current in a concentration-dependent manner (IC50 = 2.07 µM). HMQ1611 blockade of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. HMQ1611 inhibition was significantly reduced in the T479A, T480A, V505A, I508A, L510A, V512A, and V516A hKv1.5 mutant channels. Molecular docking analysis predicted that V505, V512, and T480 were involved in the blocking action of HMQ1611 on hKv1.5 channels. These results suggest that HMQ1611 inhibits hKv1.5 currents as an open channel blocker. Amino acid residues located at the base of the selectivity filter (T479 and T480) and in the S6 segment (V505, I508, L510, V512, and V516) of hKv1.5 appear to constitute potential binding sites for HMQ1611.

Profile analysis of circRNAs in human THP-1 derived macrophages infected with intracellular Staphylococcus aureus.

BACKGROUND: Intracellular Staphylococcus aureus (S. aureus) infection is generally persistent, recurrent and difficult to treat due to the poor availability of antibiotics within macrophages cells and the lack of ideal diagnostic markers. Circular RNAs (circRNAs), with covalently closed circular structures, exists in the serum stably and is not easily degraded by nucleases. Besides, circRNAs play a pivotal in the eukaryotic regulation of genes expression and served as biomarkers in variety disease including microbial infections. However, the function of host circRNAs in intracellular S. aureus infection remains largely unclear. METHODS: In this study, the circRNAs expression profile was investigated by RNA sequencing technology in both S. aureus-infected THP-1 derived macrophages and mock control cells. The differentially expressed circRNAs (DE circRNAs) with a fold-change >1.5 (p < 0.05) are analyzed using functional pathway clustering prediction. Then, RT-qPCR was performed to verify the top 2 up-regulated circRNAs in the THP-1 cell and human serum samples so as to evaluate the value of circRNAs for S. aureus diagnosis. RESULTS: An intracellular survival THP-1 derived macrophages model of S. aureus infection was established. A total of 5,299 circRNAs were identified in human THP-1 derived macrophages infected with intracellular S. aureus. There were 61 DE circRNAs with a fold-change >1.5 (p < 0.05) after S. aureus infection. Among them, 22 circRNAs were up-regulated while 39 circRNAs down-regulated. GO and KEGG pathway analysis demonstrated that DE circRNAs were enriched in the processes such as Neurotrophin, Pyruvate metabolism and Notch signaling pathway. Moreover, hsa_circ_0000311 and chr13:43500472-43544806-(novel) were verified to be significantly upregulated in THP-1 derived macrophages and human serum samples between two groups. Finally, the networks of circRNA-miRNA-mRNA based on these two circRNAs were constructed respectively. CONCLUSION: Our study provides the first profile analysis of host circRNAs involved in intracellular S. aureus infection, which may serve as biomarkers for S. aureus diagnosis and contribute to the understanding of S. aureus evasion mechanisms.

Identification of key serum biomarkers for the diagnosis and metastatic prediction of osteosarcoma by analysis of immune cell infiltration.

BACKGROUND: The role of circular RNAs (circRNAs) and microRNAs (miRNAs) in osteosarcoma (OS) development has not been fully elucidated. Further, the contribution of the immune response to OS progression is not well defined. However, it is known that circRNAs and miRNAs can serve as biomarkers for the diagnosis, prognosis, and therapy of many cancers. Thus, the aim of this study was to identify novel key serum biomarkers for the diagnosis and metastatic prediction of OS by analysis of immune cell infiltration and associated RNA molecules. METHODS: Human OS differentially expressed circRNAs (DEcircRNAs), differentially expressed miRNAs (DEmiRNAs), and differentially expressed mRNAs (DEmRNAs) were identified by analysis of microarray data downloaded from Gene Expression Omnibus (GEO) datasets. Further, characteristic patterns of OS-infiltrating immune cells were analyzed. On this basis, we identified statistically significant transcription factors. Moreover we performed pathway enrichment analysis, constructed protein-protein interaction networks, and devised competitive endogenous RNA (ceRNA) networks. Biological targets of the ceRNA networks were evaluated and potential OS biomarkers confirmed by RT-qPCR analysis of the patients' serum. RESULTS: Seven differentially expressed circRNAs, 166 differentially expressed miRNAs, and 175 differentially expressed mRNAs were identified. An evaluation of cellular OS infiltration identified the highest level of infiltration by M0 macrophages, M2 macrophages, and CD8+ T cells, with M0 macrophages and CD8+ T cells as the most prominent. Significant patterns of tumor-infiltrating immune cells were identified by principal component analysis. Moreover, 185 statistically significant transcription factors were associated with OS. Further, in association with immune cell infiltration, hsa-circ-0010220, hsa-miR-326, hsa-miR-338-3p, and FAM98A were identified as potential novel biomarkers for OS diagnosis. Of these, FAM98A had the most promise as a diagnostic marker for OS and OS metastasis. Most importantly, a novel diagnostic model consisting of these four biomarkers (hsa-circ-0010220, hsa-miR-326, hsa-miR-338-3p, and FAM98A) was established with a 0.928 AUC value. CONCLUSIONS: In summary, potential serum biomarkers for OS diagnosis and metastatic prediction were identified based on an analysis of immune cell infiltration. A novel diagnostic model consisting of these four promising serum biomarkers was established. Taken together, the results of this study provide a new perspective by which to understand immunotherapy of OS.

Fluid dynamics analysis and experimental study for solar radiation error correction of sounding humidity sensor.

In order to correct the solar radiation error of relative humidity, the mainstream capacitive sounding humidity sensor HC103M2 is selected and investigated by simulation analysis and experimental verification. First, the basic theories for solar radiation error and sensor error itself are elaborated, and simulational and experimental platforms are introduced. The computational fluid dynamics (CFD) method is utilized to theoretically investigate the dry error of the humidity sensor caused by solar radiation heating, which is related to radiation intensity, altitude, and solar elevation angle as well as reflectivity, thickness, and shape of the shield. Then, in order to verify the accuracy of the simulation, an experimental platform including a humidity sensor and two temperature sensors to measure the solar radiation heating is built to analyze the relative error of humidity obtained by the CFD simulation and experiment. It is found that their maximum deviation is 3.30% and the average error is 1.94%, which indicates that the calculation using the CFD method is accurate and feasible. In order to easily and operationally predict the solar radiation heating of the humidity sensor, a back propagation (BP) neural network fusion algorithm based on three inputs of radiation intensity, air pressure, and solar elevation angle is proposed. Compared with the solar radiation heating obtained by CFD simulation, the maximum absolute error is about 0.2 K, and the relative error of humidity is about ±1.30%. Finally, a case of vertical humidity profile correction considering the temperature-sensitive error of HC103M2 is analyzed. The response time of sensor measurement and the airflow into the shield are discussed as well. The corrected results after taking solar radiation heating into account are more similar to those measured by RS92 and cryogenic frost point hygrometer (CFH). This result shows that the prediction model is accurate, which may be applied to correct the dry error and further improve the accuracy of humidity measurement.

Identification of the miRNA signature and key genes in colorectal cancer lymph node metastasis.

BACKGROUND: Because its metastasis to the lymph nodes are closely related to poor prognosis, miRNAs and mRNAs can serve as biomarkers for the diagnosis, prognosis, and therapy of colorectal cancer (CRC). This study aimed to identify novel gene signatures in the lymph node metastasis of CRC. METHODS: GSE56350, GSE70574, and GSE95109 datasets were downloaded from the Gene Expression Omnibus (GEO) database, while data from 569 colorectal cancer cases were also downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed miRNAs (DE-miRNAs) were calculated using R programming language (Version 3.6.3), while gene ontology and enrichment analysis of target mRNAs were performed using FunRich ( http://www.funrich.org ). Furthermore, the mRNA-miRNA network was constructed using Cytoscape software (Version 3.8.0). Gene expression levels were verified using the GEO datasets. Similarly, quantitative real-time PCR (qPCR) was used to examine expression profiles from 20 paired non-metastatic and metastatic lymph node tissue samples obtained from patients with CRC. RESULTS: In total, five DE-miRNAs were selected, and 34 mRNAs were identified after filtering the results. Moreover, two key miRNAs (hsa-miR-99a, hsa-miR-100) and one gene (heparan sulfate-glucosamine 3-sulfotransferase 2 [HS3ST2]) were identified. The GEO datasets analysis and qPCR results showed that the expression of key miRNA and genes were consistent with that obtained from the bioinformatic analysis. A novel miRNA-mRNA network capable of predicting the prognosis and confirmed experimentally, hsa-miR-99a-HS3ST2-hsa-miR-100, was found after expression analysis in metastasized lymph node tissue from CRC samples. CONCLUSION: In summary, miRNAs and genes with potential as biomarkers were found and a novel miRNA-mRNA network was established for CRC lymph node metastasis by systematic bioinformatic analysis and experimental validation. This network may be used as a potential biomarker in the development of lymph node metastatic CRC.

Knowledge and intake of folic acid to prevent neural tube defects among pregnant women in urban China: a cross-sectional study.

BACKGROUND: The prevalence of neural tube defects (NTDs) in China declined during 2000-2017 with periconceptional folic acid (FA) supplementation, which is effective in reducing the risk of birth defects. We aimed to assess the knowledge and actual use of FA among Chinese pregnant women and to explore factors associated with FA use before pregnancy. METHODS: All data were collected in face-to-face interviews during health visits among pregnant women. We collected information about knowledge and use of FA supplements and demographic, socioeconomic, and health status. One maternity and childcare hospital was chosen in each of four cities: Beijing, Huaibei, Kunming, and Haikou. In total, 435 pregnant women were randomly recruited for interviews conducted from June to December 2016. RESULTS: A total of 428 pregnant women were included in this survey. Of these, 82.0% (351/428) knew that FA can prevent NTDs, and 75.9% (325/428) knew the correct time to take FA. Overall, 65.9% (282/428) of women knew both that FA can prevent NTDs and the recommended time to take FA before pregnancy. Approximately 95.1% (407/428) of women reported having ever taken FA, only 46.3% (198/428) had begun to take FA supplementation before conception, and 64.5% (109/169) of women from rural areas failed to take FA before pregnancy. Women living in northern China (odds ratio [OR] = 1.81, 95% confidence interval [CI], 1.18-2.77), those with unplanned pregnancy (OR = 1.99, 95% CI 1.30-3.04), and highly educated women (OR = 2.37, 95% CI 1.45-3.88) were more likely to know about FA. Women who were homemakers (OR = 1.94, 95% CI 1.21-3.11) and had unplanned pregnancy (OR = 6.18, 95% CI 4.01-9.53) were less likely to begin taking FA before pregnancy. CONCLUSIONS: Our survey showed that most pregnant women knew about FA. Although preconception intake of FA can help to reduce NTDs, improving the rate of FA intake before pregnancy is needed in urban areas of China, especially among homemakers and women from rural areas or with unplanned pregnancy. Campaigns are needed to increase awareness about FA and FA use before pregnancy among rural women, homemakers, and those with unplanned pregnancy and lower education levels.

F-box protein FBXO30 mediates retinoic acid receptor γ ubiquitination and regulates BMP signaling in neural tube defects.

Retinoic acid (RA), an active derivative of vitamin A, is critical for the neural system development. During the neural development, the RA/RA receptor (RAR) pathway suppresses BMP signaling-mediated proliferation and differentiation of neural progenitor cells. However, how the stability of RAR is regulated during neural system development and how BMP pathway genes expression in neural tissue from human fetuses affected with neural tube defects (NTDs) remain elusive. Here, we report that FBXO30 acts as an E3 ubiquitin ligase and targets RARγ for ubiquitination and proteasomal degradation. In this way, FBXO30 positively regulates BMP signaling in mammalian cells. Moreover, RA treatment leads to suppression of BMP signaling by reducing the level of FBXO30 in mammalian cells and in mouse embryos with NTDs. In samples from human NTDs with high levels of retinol, downregulation of BMP target genes was observed, along with aberrant FBXO30 levels. Collectively, our results demonstrate that RARγ levels are controlled by FBXO30-mediated ubiquitination and that FBXO30 is a key regulator of BMP signaling. Furthermore, we suggest a novel mechanism by which high-retinol levels affect the level of FBXO30, which antagonizes BMP signaling during early stage development.

Identification of a CqCaspase gene with antiviral activity from red claw crayfish Cherax quadricarinatus.

Caspase, an aspartate specific proteinase mediating apoptosis, plays a key role in immune response. In our previous study, the expression of a caspase gene was up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To further reveal the effect of caspase on WSSV infection, we cloned this caspase gene (denominated as CqCaspase) with an open reading frame of 1062 bp, which encoded 353 amino acids with a caspase domain (CASc) containing a p20 subunit and a p10 subunit. Tissue distribution analysis indicated that the mRNA transcript of CqCaspase was widely expressed in all tested tissues with the highest expression in Hpt, while the lowest expression in muscle. To further explore the effect of CqCaspase on WSSV replication, recombinant protein of CqCaspase (rCqCaspase) was delivered into Hpt cells followed by WSSV infection, which resulted in a significantly decreased expression of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV, suggesting that CqCaspase, possibly by the enhanced apoptotic activity, had a strong negative effect on the WSSV replication. These data together indicated that CqCaspase was likely to play a vital role in immune defense against WSSV infection in a crustacean C. quadricarinatus, which shed a new light on the mechanism study of WSSV infection in crustaceans.

An Ns1abp-like gene promotes white spot syndrome virus infection by interacting with the viral envelope protein VP28 in red claw crayfish Cherax quadricarinatus.

Influenza A virus non-structural-1A binding protein (named as Ns1abp) was originally identified as a host protein from human that bound to the viral NS-1 protein. In our previous study, the expression of an Ns1abp-like gene (denoted as CqNs1abp-like gene) was found to be up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To elucidate the role of CqNs1abp-like gene involved in WSSV infection, we cloned the CqNs1abp-like gene in which the open reading frame was 2232 bp, encoding 743 amino acids with two typical domains of one BTB (Broad-Complex, Tramtrack and Bric a brac) domain at N-terminal and six Kelch domains at C-terminal. The gene expression profile showed that the mRNA transcript of CqNs1abp-like gene was widely expressed in all the tested tissues with highest expression in nerve, relatively high expression in Hpt and lowest expression in eyestalk. Importantly, both the WSSV entry and the viral replication were significantly reduced in Hpt cells after gene silencing of CqNs1abp-like gene. By using protein pull-down assay, we found that the recombinant BTB domain, six Kelch domains and CqNs1abp-like intact protein were all bound to the WSSV envelope protein VP28, respectively, in which the BTB domain showed slightly less binding affinity than that of the six Kelch domains or the recombinant intact protein. Besides, the WSSV entry into Hpt cells was clearly decreased when the virus was pre-incubated with the recombinant BTB domain, six Kelch domains, or the recombinant CqNs1abp-like intact protein, respectively, suggesting that the CqNs1abp-like gene was likely to function as a putative recognition molecular towards WSSV infection in a crustacean C. quadricarinatus. Taken together, these data shed new light on the mechanism of WSSV infection and a putatively novel target on anti-WSSV infection in crustacean farming.

A thymosin repeated protein1 reduces white spot syndrome virus replication in red claw crayfish Cherax quadricarinatus.

The ß-thymosins are a group of structurally related, highly conserved intracellular small peptides in vertebrates with various biological functions, including cytoskeletal remodeling, neuronal development, cell migration, cell survival, tissue repair and inhibition of inflammation. In contrast to vertebrates, the function of ß-thymosin is not fully understood in crustaceans. Previously, we found that a thymosin-repeated protein1 (CqTRP1) gene was up-regulated after white spot syndrome virus (WSSV) challenge in hematopoietic tissue (Hpt) cells from the red claw crayfish Cherax quadricarinatus. To further identify the effect of CqTRP1 on WSSV infection, a full length cDNA sequence of ß-thymosin homologue was cloned and analyzed from red claw crayfish followed by functional study. The CqTRP1 cDNA contains an open reading frame of 387 nucleotides encoding a protein of 129 amino acids with a putative molecular mass of 14.3 kDa. The amino acid sequence showed high identity with other ß-thymosins and contained three characteristic thymosin ß actin-binding motifs, suggesting that CqTRP1 was a member of the ß-thymosin family. Tissue distribution analysis revealed a ubiquitous presence of CqTRP1 in all the examined tissues with the highest expression in hemocytes, Hpt and gonad at the transcriptional level. Interestingly, the gene silencing of endogenous CqTRP1 by RNAi enhanced the WSSV replication in Hpt cells. Meanwhile, the WSSV replication was significantly reduced in the Hpt cell cultures if overloaded with a recombinant CqTRP1. Taken together, these data clearly indicated that CqTRP1 was likely to be associated with the anti-WSSV response in a crustacean C. quadricarinatus, which provides new strategy against white spot disease in crustacean aquaculture.

A CqFerritin protein inhibits white spot syndrome virus infection via regulating iron ions in red claw crayfish Cherax quadricarinatus.

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus.