Clinical characteristics of SARS-CoV-2 Omicron variant infection in children with acute leukemia.

Hematologic diseases and various therapeutic stages can impact the presentation of SARS-CoV-2 Omicron variant infection. This study retrospectively analyzed data on Omicron infection in children with acute leukemia treated at our hospital between January 16, 2023, and February 25, 2023, using questionnaires. The prevalence of Omicron infection in children undergoing consolidation chemotherapy, maintenance chemotherapy, drug withdrawal, and healthy children was 81.8%, 75.2%, 55.2%, and 61.9%, respectively. The observed differences were statistically significant (P < 0.05). During the course of infection, children with leukemia undergoing chemotherapy, including both the consolidation and maintenance chemotherapy groups, exhibited a prolonged time to achieve SARS-CoV-2 negativity compared to the drug withdrawal and healthy groups. However, there was no significant increase in the incidence of symptoms across all body systems, and no children experienced serious sequelae or death. Furthermore, our observations indicated that all manifestations of Omicron infection in children with leukemia after drug withdrawal were not significantly different from those in healthy children. This suggested, to a certain extent, that the immune function of children with leukemia recovers effectively after the cessation of drug treatment. These findings are crucial for guiding clinical management and alleviating concerns about infection for both children with leukemia and their parents.

Relationship between subtype-specific minimal residual disease level and long-term prognosis in children with acute lymphoblastic leukemia.

Minimal residual disease (MRD) based risk stratification criteria for specific genetic subtypes remained unclear in childhood acute lymphoblastic leukemia (ALL). Among 723 children with newly diagnosed ALL treated with the Chinese Children Leukemia Group CCLG-2008 protocol, MRD was assessed at time point 1 (TP1, at the end of induction) and TP2 (before consolidation treatment) and the MRD levels significantly differed in patients with different fusion genes or immunophenotypes (P all < 0.001). Moreover, the prognostic impact of MRD varied by distinct molecular subtypes. We stratified patients in each molecular subtype into two MRD groups based on the results. For patients carrying BCR::ABL1 or KMT2A rearrangements, we classified patients with MRD < 10-2 at both TP1 and TP2 as the low MRD group and the others as the high MRD group. ETV6::RUNX1+ patients with TP1 MRD < 10-3 and TP2 MRD-negative were classified as the low MRD group and the others as the high MRD group. For T-ALL, We defined children with TP1 MRD ≥ 10-3 as the high MRD group and the others as the low MRD group. The 10-year relapse-free survival of low MRD group was significantly better than that of high MRD group. We verified the prognostic impact of the subtype-specific MRD-based stratification in patients treated with the BCH-ALL2003 protocol. In conclusion, the subtype-specific MRD risk stratification may contribute to the precise treatment of childhood ALL.

Optical Genome Mapping Reveals Novel Structural Variants in Lymphoblastic Lymphoma.

BACKGROUND: Accurate histologic and molecular genetic diagnosis is critical for the pathogenesis study of pediatric patients with lymphoblastic lymphoma (LBL). Optical genome mapping (OGM) as all-in-one process allows the detection of most major genomic risk markers, which addresses some of the limitations associated with conventional cytogenomic testing, such as low resolution and throughput, difficulty in ascertaining genomic localization, and orientation of segments in duplication, inversions, and insertions. Here, for the first time, we examined the cytogenetics of 5 children with LBL using OGM. METHODS: OGM was used to analyze 5 samples of pediatric LBL patients treated according to the modified NHL-BFM95 backbone regimen. Whole-exon Sequencing (WES) was used to confirm the existence of structural variants (SVs) identified by OGM with potentially clinical significance on MGI Tech (DNBSEQ-T7) platform. According to the fusion exon sequences revealed by WES, the HBS1L :: AHI1 fusion mRNA in case 4 was amplified by cDNA-based PCR. RESULTS: In total, OGM identified 251 rare variants (67 insertions, 129 deletions, 3 inversion, 25 duplications, 15 intrachromosomal translocations, and 12 interchromosomal translocations) and 229 copy number variants calls (203 gains and 26 losses). Besides all of the reproducible and pathologically significant genomic SVs detected by conventional cytogenetic techniques, OGM identified more SVs with definite or potential pathologic significance that were not detected by traditional methods, including 2 new fusion genes, HBS1L :: AHI1 and GRIK1::NSDHL , which were confirmed by WES and/or Reverse Transcription-Polymerase Chain Reaction. CONCLUSIONS: Our results demonstrate the feasibility of OGM to detect genomic aberrations, which may play an important role in the occurrence and development of lymphomagenesis as an important driving factor.

Longitudinal changes in body mass index, height, and weight in children with acute myeloid leukemia.

BACKGROUND: This study reported height prediction and longitudinal growth changes in Chinese pediatric patients with acute myeloid leukemia (AML) during and after treatment and their associations with outcomes. METHODS: Changes in 88 children with AML in percentages according to the growth percentile curve for Chinese boys/girls aged 2-18/0-2 years for body mass index (BMI), height, and weight from the time of diagnosis to 2 years off therapy were evaluated. The outcomes of AML were compared among patients with different BMI levels. RESULTS: The proportion of underweight children (weight < 5th percentile) increased significantly from the initial diagnosis to the end of consolidation treatment. The proportion of patients with low BMI (BMI < 5th percentile) was highest (23.08%) during the consolidation phase, and no children were underweight, but 20% were overweight (BMI > 75th percentile) after 2 years of drug withdrawal. Unhealthy BMI at the initial diagnosis and during intensive chemotherapy leads to poorer outcomes. For height, all patients were in the range of genetic height predicted based on their parents' height at final follow-up. CONCLUSIONS: Physicians should pay more attention to the changes in height and weight of children with AML at these crucial treatment stages and intervene in time.

DNMT3A low-expression is correlated to poor prognosis in childhood B-ALL and confers resistance to daunorubicin on leukemic cells.

BACKGROUND: Little is known about DNMT3A expression and its prognostic significance in childhood B cell acute lymphoblastic leukemia (B-ALL). METHODS: We determined DNMT3A mRNA expression in 102 children with B-ALL. Correlations with relapse-free survival (RFS) and common clinical characteristics were analyzed. DNMT3A was stably knocked out by CRISPR/Cas9 gene editing technology in Reh and 697 B-ALL cell lines. Cell proliferation activity after treated with daunorubicin (DNR) was determined by CCK8 assay in DNMT3A KO Reh and 697 cell lines. RESULTS: DNMT3A expression in B-ALL patients who were in continuous complete remission (CCR) was higher than in those who got relapse (P = 0.0111). Receiver operating characteristic curve showed prognostic significance of DNMT3A expression (P = 0.003). Low expression of DNMT3A (≤ 0.197) was significantly correlated with poor RFS (P < 0.001) in children with B-ALL. Knock-out of DNMT3A in Reh and 697 cell lines significantly increased IC50 of DNR (P = 0.0201 and 0.0022 respectively), indicating elevated resistance to DNR. CONCLUSION: Low expression of DNMT3A associates with poor prognosis in children with B-ALL. Knock-out of DNMT3A confers resistance to DNR on leukemic cells.

Current Anesthesia Practices of Pediatric Cardiac Surgeries in Tertiary Maternity and Children's Hospitals in China: A National Survey.

OBJECTIVES: To describe the current development of Chinese pediatric cardiac anesthesia practices. DESIGN: Descriptive research study. SETTING: This study used electronic questionnaires. The authors searched the official website of the National Health Commission of the People's Republic of China for tertiary maternity and children's hospitals across the country. PARTICIPANTS: Tertiary maternity and children's hospitals. INTERVENTIONS: All representatives of the invited hospitals were asked to report the official statistics of their hospitals whenever possible. MEASUREMENTS AND MAIN RESULTS: The survey questions were related to the geographic distribution and caseloads of pediatric cardiac surgical resources, technical capacities, anesthetic regimens, monitoring practices, and qualification requirements of anesthesiologists. A total of 130 hospitals were confirmed, using the registration information of the National Health Commission of the People's Republic of China, and 108 hospitals agreed to participate in this study. All enrolled hospitals completed the questionnaires, of which 52 could perform cardiac surgeries and were located in provinces, autonomous regions, and municipalities across the country, except for the Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region, and Tibet Autonomous Region. The authors found that most hospitals' caseload of pediatric cardiac surgeries was relatively small (<500 cases per year). Hospitals capable of performing high-risk pediatric cardiac surgeries are mainly located in Eastern China. Most hospitals prefer total intravenous anesthesia in cardiac surgeries, and commonly used anesthetics include propofol, sufentanil, rocuronium, and cisatracurium. Except for the basic intraoperative monitoring items (including electrocardiography, invasive blood pressure, central venous pressure, pulse oxygen saturation, intake-output volume, and body temperature), bispectral index and near-infrared spectroscopy are relatively commonly used in some hospitals. Postoperative analgesia for children undergoing cardiac surgeries was provided in 38 hospitals, and an intravenous analgesia pump was the most widely used analgesia measure. In addition, the most frequently mentioned qualification requirements for pediatric cardiac anesthesiologists in these hospitals specializing in cardiac surgeries were a further study in domestic hospitals specializing in cardiac surgeries and the professional titles of the attending doctors. CONCLUSIONS: Pediatric cardiac medical resources are mainly concentrated in Eastern China, and most hospitals capable of performing high-risk cardiac surgeries are located in the eastern part of the country. The authors found that pediatric cardiac anesthesia practices varied widely among the hospitals, and the main problem with pediatric cardiac anesthesia in China is that there is no systemic fellowship training curriculum at present; therefore, there is an urgent need to develop a fellowship training curriculum to further improve the quality of Chinese pediatric cardiac anesthesia.

A TRIM-like protein restricts WSSV replication in the oriental river prawn, Macrobrachium nipponense.

Tripartite motif (TRIM) proteins are a multifunctional family of ubiquitin E3 ligases involved in multiple biological processes. Studies have shown that many TRIM proteins in mammals play vital roles in the host defense against viral pathogens. In the present study, we identified a novel TRIM gene (MnTrim-like) from the oriental river prawn, Macrobrachium nipponense. Predicted MnTrim-like protein contains the characteristic RING finger domain. MnTrim-like was abundantly distributed in hepatopancreas, intestine, stomach, and gills. Upon white spot syndrome virus (WSSV) challenge, transcripts of MnTrim-like in the stomach were significantly up-regulated. Knockdown of MnTrim-like increased the expression of VP28 and decreased the synthesis of several antimicrobial peptides, including two crustins and one anti-lipopolysaccharide factor. Besides, silencing of these three antimicrobial peptides (AMPs) led to an increase in the expression of VP28 and WSSV copies. Moreover, it was found that injection of recombinant MnTrim-like protein with WSSV could decrease the transcription of VP28 and the number of virus particles. These results suggest that this MnTrim-like may restrict WSSV infection by positively regulating the expression of AMPs with antiviral activities and directly interacting with viral components. This study will broaden our understanding about the function of TRIM in crustacean during viral infection.

Eight TRIM32 isoforms from oriental river prawn Macrobrachium nipponense are involved in innate immunity during white spot syndrome virus infection.

Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. In this study, full-length MnTRIM32 cDNA was obtained from oriental river prawn Macrobrachium nipponense, and eight MnTRIM32 isoforms generated by alternative splicing were identified. The open reading frames of the eight MnTRIM32 isoforms were predicted to be separately composed of 402, 346, 347, 346, 414, 358, 359, and 358 amino acid residues. Protein structural analysis revealed that all MnTRIM32 isoforms contained a RING domain and a coiled coil region. MnTRIM32 was ubiquitously expressed in all tissues tested, with the highest expression in the hepatopancreas. The mRNA levels of MnTRIM32 in the gills, stomach, and intestine of prawns were found to undergo time-dependent enhancement following white spot syndrome virus (WSSV) stimulation. Double-stranded RNA interference studies revealed that MnTRIM32 silencing significantly downregulated the expression levels of interferon (IFN) regulatory factor MnIRF, IFN-like factor MnVago4, and tumor necrosis factor MnTNF. Furthermore, knockdown of MnTRIM32 in WSSV-challenged prawns increased the expression of VP28 and the number of WSSV copies, suggesting that MnTRIM32 plays a positive role in limiting WSSV infection. These findings provided strong evidence for the important role of MnTRIM32 in the antiviral innate immunity of M. nipponense.

Histology, ultrastructure, and differential gene expression in relation to seasonal sperm storage in the oviduct of the Chinese alligator, Alligator sinensis.

Although oviductal sperm storage are essential steps in reproduction for female animals with internal fertilisation, no systematic study on the identification of genes involving sperm storage has been performed in crocodilian species. In the present research, the relationship between morphological variation related to sperm storage in the oviduct and gene expression patterns derived from RNA sequencing analyses between active period (AP), breeding period (BP), and hibernation period (HP) were investigated. The corresponding results indicated that sperm were observed not only in the ciliated cells within infundibulum and mucosal layer of uterus during BP, but also been detected in the spermatosperm storage tube (SST) in the anterior uterus at HP stage. The further transmission electron microscopy analysis indicated that the differences in the number and activity of the secretory cells likely to attributed to the seasonal variation of microenvironment related to the sperm storage. Based on the RNA-sequecing, 13147 DEGs related to the Peroxisome proliferator-activated receptors (PPARs) and FOXO signalling were identified, including these, the down-regulated ATG12 and BCL2L11 in the HP group may thus constitute an important point of convergence between autophagy and apoptosis involving the FOXO1 pathway. The genes involved in the PPARs pathway might modulate the immune response and thereby contribute to prolong the life span of stored spermatozoa in Alligator sinensis . The outcomes of this study provide fundamental insights into the mechanism of sperm storage in A. sinensis .

Cost-effectiveness analysis of dasatinib versus imatinib in pediatric philadelphia chromosome-positive acute lymphoblastic leukemia patients in China.

BACKGROUND: Dasatinib and imatinib are the recommended tyrosine kinase inhibitors (TKIs) for treating pediatric Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL), and the one which has been approved indication in China is imatinib. Recently, clinical demand for Ph + ALL treatment is becoming unmet gradually with the increasing resistance of imatinib. There are some studies reporting the better efficacy and comparative safety of dasatinib compared with imatinib, but no economic comparison has been published. This study aims to supplement economic evidence by comparing the cost-effectiveness between imatinib and dasatinib in treating pediatric patients with Ph+ ALL in China, and to help clinical rational drug use via multi-dimensional value assessment. METHODS: A decision tree model combined with a 10-year Markov model were established based on the disease progression. The parameters were collected from published literatures and our hospital's electronic medical records. From the health system perspective, the incremental cost-effectiveness ratio (ICER) between the two treatment groups was calculated through cost-effectiveness analysis and then compared with the willingness-to-pay (WTP) threshold. The set WTP threshold in this study was 1 times per capita gross domestic product (GDP) of China, as recommended by the World Health Organization. Direct medical costs and quality-adjusted life years (QALYs) were calculated and discounted at 5%. The sensitivity analyses were conducted to assess the uncertainty and robustness of the results. RESULTS: The total costs were CNY 1,020,995.35 and CNY 1,035,788.50 in imatinib group and dasatinib group during the 10-year simulation, and the total QALYs were 2.59 and 4.84. Compared with the imatinib treatment group, the ICER was around CNY 6,575.78/ QALY, which was less than the set threshold CNY 70,892/ QALY. The sensitive analyses indicated the robustness of the results. CONCLUSIONS: The cost-effectiveness analysis shows the potential cost-effective advantages of adding dasatinib comparing with adding imatinib for pediatric Ph + ALL patients in China under the set WTP threshold, which indicates that those patients could achieve more QALYs by paying acceptable fee.

BACH2-mediated FOS confers cytarabine resistance via stromal microenvironment alterations in pediatric ALL.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer that mainly affects children. Relapse and chemoresistance result in treatment failure, underlining the need for improved therapies. BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription repressor recognized as a tumor suppressor in lymphomas, but little is known about its function and regulatory network in pediatric ALL (p-ALL). Herein, we found aberrant BACH2 expression at new diagnosis not only facilitated risk stratification of p-ALL but also served as a sensitive predictor of early treatment response and clinical outcome. Silencing BACH2 in ALL cells increased cell proliferation and accelerated cell cycle progression. BACH2 blockade also promoted cell adhesion to bone marrow stromal cells and conferred cytarabine (Ara-C)-resistant properties to leukemia cells by altering stromal microenvironment. Strikingly, we identified FOS, a transcriptional activator competing with BACH2, as a novel downstream target repressed by BACH2. Blocking FOS by chemical compounds enhanced the effect of Ara-C treatment in both primary p-ALL cells and pre-B-ALL-driven leukemia xenografts and prolonged the survival of tumor-bearing mice. These data highlight an interconnected network of BACH2-FOS, disruption of which could render current chemotherapies more effective and offer a promising therapeutic strategy to overcome Ara-C resistance in p-ALL.

Effects of age on sugammadex reversal of neuromuscular blockade induced by rocuronium in Chinese children: a prospective pilot trial.

BACKGROUND: Sugammadex reverses neuromuscular blockade induced by steroidal relaxants. We compared the recovery for neuromuscular blockade reversal with sugammadex in children aged 1-12 years. METHODS: From August 2019 to August 2020, patients who received 2.0 mg·kg- 1 sugammadex for neuromuscular blockade reversal after surgery were recruited. The primary outcome was the time for the train-of-four ratio (TOFR) to recover to 0.9; secondary outcomes included the incidence of the TOFR < 0.9, extubation time, length of stay at the post-anesthesia care unit, and adverse events. Hemodynamic parameters before and 5 min after sugammadex administration and vital signs in the recovery room were also recorded. RESULTS: Eighty-six children were recruited (1 to < 3 years, n = 23; 3 to < 5 years, n = 33; 5 to ≤12 years, n = 30). Intergroup differences in the recovery of the TOFR to 0.9 were not statistically significant (F = 0.691, p = 0.504). Recurrence of the TOFR < 0.9 was not observed in any group. Five minutes after sugammadex administration, the heart rates of patients aged 3 to < 5 and 5 to ≤12 years were significantly lower than those at baseline (p < 0.05). Extubation time was similar in patients aged 1 to ≤12 years. Length of stay and end-tidal capnography at the post-anesthesia care unit as well as adverse events did not differ significantly. CONCLUSION: A moderate (TOF count two) rocuronium-induced neuromuscular blockade can be effectively and similarly reversed with sugammadex 2 mg·kg- 1 in Chinese children aged 1-12 years. TRIAL REGISTRATION: Chinese Clinical Trial Registry: ChiCTR1900023715 (June 8, 2019).

ß-catenin promotes MTX resistance of leukemia cells by down-regulating FPGS expression via NF-κB.

BACKGROUND: Aberrant activation of ß-catenin has been shown to play important roles in the chemoresistance of acute lymphoblastic leukemia (ALL), but the involvement and mechanism of ß-catenin in methotrexate (MTX) resistance is poorly understood. In the present study, we demonstrate a critical role of ß-catenin-NF-κB-FPGS pathway in MTX resistance in the human T-lineage ALL cell lines. METHODS: Lentivirus sh-ß-catenin was used to silence the expression of ß-catenin. Flow cytometry was performed to detect apoptosis after MTX treatment. Western blot, real-time PCR, Co-immunoprecipitation (Co-IP), Chromatin immunoprecipitation (ChIP), Re-ChIP, and Luciferase assay were utilized to investigate the relationship among ß-catenin, nuclear factor (NF)-κB, and folypoly-γ-glutamate synthetase (FPGS). RESULTS: Depletion of ß-catenin significantly increased the cytotoxicity of MTX. At the molecular level, knockdown of ß-catenin caused the increase of the protein level of FPGS and NF-κB p65. Furthermore, ß-catenin complexed with NF-κB p65 and directly bound to the FPGS promoter to regulate its expression. In addition, ß-catenin repression prolonged the protein turnover of FPGS. CONCLUSIONS: Taken together, our results demonstrate that ß-catenin may contribute to MTX resistance in leukemia cells via the ß-catenin-NF-κB-FPGS pathway, posing ß-catenin as a potential target for combination treatments during ALL therapy.

Taiman negatively regulates the expression of antimicrobial peptides by promoting the transcription of cactus in Macrobrachium nipponense.

In insects, Taiman (Tai) participates in the juvenile hormone, 20-hydroxyecdysone, insulin, and Hippo signaling pathways. However, the role of Tai in crustacean innate immunity is less known. In this study, four Tai isoforms (MnTai-A, MnTai-B, MnTai-C, and MnTai-D) produced by alternative splicing were identified from Macrobrachium nipponense. The obtained genome sequences indicated that MnTai DNA has more than 20 exons and 19 introns. The second to last (-exon2) and the third to last (-exon3) exons can be alternatively spliced. The loss of -exon2 or -exon3 produces MnTai-B or MnTai-C, respectively. Both exons are absent in MnTai-D. The full-length cDNA of MnTai-A (including all exons) was 6894 bp with an open reading frame of 4998 bp that encoded a protein of 1665 amino acids. MnTaiA contains the conservative structure of the Tai family and clustered with nuclear receptor coactivator from shrimp. All these four isoforms were widely distributed in a variety of tissues with the highest expression level in the hepatopancreas except MnTaiC. The transcriptional levels of total Tai genes (designated as MnTaiT) in the hepatopancreas and gills were regulated by bacterial or viral challenge. Knockdown of MnTaiT increased the expression of anti-microbial peptides (AMPs) during Vibrio parahaemolyticus infection. Further study indicated that the negative regulation of AMP gene expression by prawn Tai was mediated through its positive regulation of cactus. Our research provides valuable information that prawn Tai isoforms are involved in innate immunity.

Activating transcription factor 2 (ATF2) negatively regulates the expression of antimicrobial peptide genes through tumor necrosis factor (TNF) in Macrobrachium nipponense.

Activating transcription factor 2 (ATF2), a member of the bZIP transcription factor family, is involved in multiple physiological and developmental processes, yet its role in the innate immunity remains unclear. In this study, two isoforms (named as MnATF2a and MnATF2b) of ATF2 gene were identified in Macrobrachium nipponense and were produced by exon skipping. The full length of MnATF2a is 2328 bp with an open reading frame of 2079 bp that encode 692 amino acids. MnATF2a has 237 bp nucleotides more than MnATF2b and the extra 237 bp is a complete exon. MnATF2a and MnATF2b proteins contain the same conserved and typical bZIP domain at the C-terminus. MnATF2a has 79 amino acids more than MnATF2b. MnATF2a and MnATF2b are widely distributed in a variety of immune tissues. After Vibrio parahaemolyticus and Staphylococcus aureus infection, the expression levels of MnATF2a and MnATF2b were significant up-regulated in the gills and stomach at 12 h. RNA interference analysis showed that knockdown of the total MnATF2 gene significantly inhibits the transcription of tumor necrosis factor (TNF) and promotes the expression of crustins (including Cru3, Cru4, and Cru7). Further study showed that knockdown of MnTNF evidently increase the expression of Cru3, Cru4, and Cru7. Our research indicates that ATF2 negatively regulate the expression of AMPs by regulating the transcription of TNF in M. nipponense. This study provides valuable information about the function of ATF2 family in the innate immunity in crustacean.

Identification of two carcinin isoforms (MnCarc1 and MnCarc2) and their function in the antimicrobial immunity of Macrobrachium nipponense.

Carcinin, a member of the crustin family, plays important roles in crustacean innate immunity. In this study, we identified two carcinin isoforms (MnCarc1 and MnCarc2) produced by alternative splicing from Macrobrachium nipponense. The full length of MnCarc1 and MnCarc2 cDNA are 1554 and 1495 bp with 687 and 609 bp open reading frame-encoding proteins that contain 228 and 202 amino acids, respectively. The genome of carcinin has nine exons and eight introns. MnCarc1 transcript contains all nine exons, whereas MnCarc2 only contains eight exons and lacks exon 4. MnCarc1 and MnCarc2 proteins contain a signal peptide, cysteine-rich regions, and a whey acidic protein domain. The phylogenetic tree shows that MnCarc1 and MnCarc2 are not grouped with other crustins and carcinins. MnCarc1 and MnCarc2 form a subgroup. MnCarc1 and MnCarc2 are widely distributed in various tissues. The expression of MnCarc1 and MnCarc2 were evidently upregulated at multiple time points in hemocytes and the intestine of M. nipponense after white spot syndrome virus, Vibrio parahaemolyticus, and Staphylococcus aureus challenges. Further studies showed that knockdown of MnDorsal or MnStat transcription factor could remarkably inhibit the upregulated expression of MnCarc1 and MnCarc2 caused by viral or bacterial challenges. In addition, recombinant MnCarc1 and MnCarc2 proteins could bind to various bacteria and polysaccharides and inhibit the growth of S. aureus and V. parahaemolyticus in vitro. This study indicated that carcinins from M. nipponense were involved in prawns innate immunity.

Two relish isoforms produced by alternative splicing participate in the regulation of antimicrobial peptides expression in Procambarus clarkii intestine.

Nuclear factor κB (NF-κB) plays a key role in the innate immunity of invertebrates. Relish belongs to the NF-κB family. In insects, alternative splicing induces the sequence diversity of the Relish gene. However, information on the roles of various relish isoforms in crustacean innate immune response is limited. Here, two alternatively spliced Relish isoforms (designated as SPcRelish and LPcRelish) were identified from freshwater crayfish (Procambarus clarkii), and functional analysis was performed. The Relish gene has 25 exons and 24 introns. The long isoform LPcRelish is fully spliced, whereas the short isoform SPcRelish is alternatively spliced and contains exon 1-9 and a retention of intron 9. LPcRelish contains the Rel homology domain (RHD), the ig-like, plexins, transcription factors (IPT), and ankyrin-repeat (ANK) inhibitory domain. However, SPcRelish contains only the RHD and IPT domain, and does not have an ANK domain. The transcripts of SPcRelish and LPcRelish can be regulated by Vibrio parahaemolyticus. The intestinal immunological barrier and bacterial balance in the intestine play crucial roles in host health. In this study, we analyzed the connection between Relish isoforms and the transcripts of antimicrobial peptides (AMPs) in intestine. The transcripts of all the tested AMPs, except ALF-41125, were upregulated by V. parahaemolyticus. The knock down of the SPcRelish gene resulted in a significant decrease in the expression levels of ALF-7032, ALF-13162, and Crustin-42012 during V. parahaemolyticus invasion. The expression levels of four AMP genes (ALF-41125, ALF-42430, Crustin-41354, and Crustin-42993) were obviously increased in V. parahaemolyticus-challenged SPcRelish-silenced crayfish. ALF-7032, ALF-9228, ALF-13162, ALF-42430, Crustin-41354, Crustin-42012, and Crustin-42993 were evidently downregulated in V. parahaemolyticus-infected LPcRelish-silenced crayfish. Overall, generating the two Relish isoforms by alternative splicing may be an important mechanism of the host immune system to promote molecular diversity, which results in the functional diversity of the relish transcription factor.

Two Wnt genes regulate the expression levels of antimicrobial peptides during Vibrio infection in Macrobrachium nipponense.

The Wnt signal transduction pathway is involved in a wide variety of cellular processes, including cell proliferation, differentiation, apoptosis, and immunity against microbial infection. In the current study, we cloned and characterized two Wnt homologues (Mn-Wnt4 and Mn-Wnt16) in Macrobrachium nipponense. The full length cDNA of Mn-Wnt4 was 3144 bp with a 1074 bp open reading frame (ORF) that encoded a protein containing 358 amino acid residues. The full length cDNA of Mn-Wnt16 transcript was 2893 bp with a 1281 bp ORF that encoded a 427 amino acid protein. Mn-Wnt4 and Mn-Wnt16 proteins contained a highly conserved WNT1 domain. Tissue distribution analysis showed that Mn-Wnt4 and Mn-Wnt16 were highly expressed in the stomach. The transcriptional levels of Mn-Wnt4 and Mn-Wnt16 in the stomach were upregulated at most tested time points after bacterial (Staphylococcus aureus and Vibrio parahaemolyticus) and viral (White spot syndrome virus) infection. Moreover, the expression levels of some antimicrobial peptides (AMPs) (including anti-lipopolysaccharide factor [ALF] and crustin [CRU]) were upregulated after V. parahaemolyticus infection. We further used dsRNA-mediated RNA interference technology to explore the relationship between these two Wnt genes and the expression levels of AMPs during V. parahaemolyticus infection. Mn-Wnt4 knockdown could significantly inhibit the expression of ALF1 and CRU4 in the stomach of V. parahaemolyticus-injected prawns, whereas Mn-Wnt16 silencing could result in the inhibition of the expression level of CRU3 and CRU4 in the stomach of V. parahaemolyticus-infected prawns. These findings indicated that the Wnt gene family might participate in the body's innate immune response to Vibrio infection by regulating the synthesis of a variety of AMPs. Our study will help to understand the role of the Wnt signaling pathway in the immune response of crustaceans.

Molecular cloning and expression analysis of two type II crustin genes in the oriental river prawn, Macrobrachium nipponense.

Innate immunity is the primary defense of crustaceans against pathogens. Crustins, as antimicrobial peptides, are important to crustacean innate immunity. In this study, two kinds of Gly-rich crustin genes were cloned from Macrobrachium nipponense and were referred to as Mn-Gly-Cru1 and Mn-Gly-Cru2. These crustins belong to type II crustins with typical type II crustin structures. The full-length cDNA of Mn-Gly-Cru1 is 677 bp and contains a 576 bp open reading frame (ORF) encoding 191 amino acids. The full-length cDNA of Mn-Gly-Cru2 is 727 bp, with 573 bp ORF encoding 190 amino acids. The constructed phylogenetic tree indicated that Mn-Gly-Cru1 and Mn-Gly-Cru2 belong to the type IIa subfamily. RT-PCR analysis showed that Mn-Gly-Cru1 and Mn-Gly-Cru2 are widely distributed in various tissues. qRT-PCR results indicated that Mn-Gly-Cru1 is mainly expressed in the gills, whereas Mn-Gly-Cru2 is expressed at the highest level in hemocytes. The transcripts of Mn-Gly-Cru1 and Mn-Gly-Cru2 respond to bacterial or white spot syndrome virus (WSSV) stimuli. After injection of 48 h dsMnRelish, the expression of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all inhibited. After WSSV, Vibrio parahaemolyticus, or Staphylococcus aureus challenge, MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all upregulated. However, the expression levels of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 at 6 h bacteria or 36 h WSSV challenge were downregulated in Relish-silenced prawns when compared with the control (bacteria or WSSV challenge only, bacteria or WSSV challenge plus dsGFP injection). Results suggest that Mn-Gly-Cru1 and Mn-Gly-Cru2 play essential roles in M. nipponense innate immunity against bacteria or WSSV, and the expression levels of both genes are regulated by Relish transcriptional factor.

Positive and negative regulatory effects of transcription factor activator protein 1 (AP1) on the expression of antimicrobial peptides in Macrobrachium nipponense.

Transcription factor activator protein 1 (AP1) plays an irreplaceable role in the response to a variety of external stimulants, such as cellar stress, bacterial and viral infections, and inflammatory cytokines. In this study, we identified a novel AP1 gene from Macrobrachium nipponense and named it MnAP1, which has a full length of 1747 bp contains an 882 bp open reading frame, and encodes a protein with 293 amino acids. The MnAP1 protein contains Pfam and bZIP domains. MnAP1 is widely distributed in hemocytes, heart, hepatopancreas, gill, stomach, and intestinal tissues. The expression levels of MnAP1 in the gills and stomach were significantly upregulated after Vibrio parahaemolyticus and Staphylococcus aureus attacks. We studied the relationship between MnAP1 and the transcripts of antimicrobial peptides (AMPs) in gills through RNA interference. Interestingly, the regulatory effects of MnAP1 on the expression of different AMPs were different. We found that the expression levels of crustins, including Cru1, Cru3, and Cru4 in the gills were evidently decreased, whereas the synthesis of Cru5 and anti-lipopolysaccharide factors (ALF3 and ALF4) were obviously increased. We further explored the effect of MnAP1 on the expression of transcription factor relish from M. nipponense. The result showed that the knockdown of MnAP1 can remarkably upregulate the expression of MnRelish. Relish as a member of the nuclear factor κB family that regulates the expression of AMPs in the innate immunity of crustacean. Hence, we also detected the expression levels of Cru5, ALF3, and ALF4 in the gills of MnRelish-silenced prawns. The Data showed that the expression levels of these three AMPs were evidently reduced after MnRelish silencing. Our results indicated that MnAP1 plays a positive role in regulating the expression of AMPs, promotes the JNK/AP1 signaling pathway, and exerts a negative regulatory effect on the synthesis of AMPs by inhibiting the transcription of NF-κB factor in the innate immunity of M. nipponense.