Injection Of Recombinant Protein Research Articles

Boosting BDNF in muscle rescues impaired axonal transport in a mouse model of DI-CMTC peripheral neuropathy

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.

Antiviral functions of IFNd against ISKNV and interaction analysis of IFNd and its receptors in spotted seabass (Lateolabrax maculatus).

Type I interferons (IFNs) play a significant role in antiviral innate immunity. But, the antiviral function of IFNd is controversial in teleosts. Here, we identified three IFNd receptors belonging to cytokine receptor family B (LmCRFB1, LmCRFB2, and LmCRFB5) in spotted seabass (Lateolabrax maculatus). LmIFNd and its receptors were highly expressed in gill, spleen and head kidney tissues. Additionally, LmIFNd, its receptors, and their downstream signal genes (LmTYK2, LmJAK1, LmSTAT1, and LmSTAT2) were induced by infectious spleen and kidney necrosis virus (ISKNV) infection. Injection of recombinant protein (LmIFNd-His) in vivo and incubation with the LmIFNd-His in vitro both induced expressions of IFN-stimulated genes (LmISGs). IFNd-His had a dose-dependent protective effect on the activity of brain cells infected by ISKNV and reduced the number of ISNKV copies. LmIFNd-His also bound to extracellular domains of the three receptors in vitro in the pull-down assay. LmIFNd-His preferentially induced ISG expression through receptor complex LmCRFB1 and LmCRFB5, followed by LmCRFB2 and LmCRFB5, to induce the expressions of LmISGs. Our results show that LmIFNd can enhance the antiviral immune response of spotted seabass, and it uses receptor complex LmCRFB1 and LmCRFB5 as well as LmCRFB2 and LmCRFB5 to induce LmISG expression. It is the first study about the antiviral function of LmIFNd and its receptor complex in spotted seabass, and it provides a reference for further studies of the controversial anti-viral function of IFNd in teleosts.

Characterization and immune functional analysis of two new type I crustins in the oriental river prawn Macrobrachium nipponense

Crustins are cationic antimicrobial peptides that are categorized into five types and play a key role in the nonspecific immunity of crustaceans. In the present study, full-length cDNA sequences of two Macrobrachium nipponense crustin genes (MnCrustin1 and MnCrustin2) were cloned and identified. The full-length cDNAs of MnCrustin1 and MnCrustin2 were 715 and 881 bp, including 351 and 330 bp open reading frames (ORFs) encoding 117 and 110 amino acids, respectively. A signal peptide, a cysteine (Cys)-rich region and a whey acidic protein (WAP) domain existed in both MnCrustin1 and MnCrustin2. Phylogenetic analysis and multiple sequence alignment indicated that MnCrustin1 and MnCrustin2 belonged to the type I crustin family. The MnCrustin1 and MnCrustin2 mRNAs were expressed in various tissues of M. nipponense and were highly expressed in hemocytes and gills. Recombinant MnCrustin1 and MnCrustin2 inhibited the growth of both gram-positive and gram-negative bacteria in vitro. After Aeromonas hydrophila and white spot syndrome virus (WSSV) infection, MnCrustin1 and MnCrustin2 expression levels were elevated in the hepatopancreas, hemocytes and gills of M. nipponense. Tests of RNA interference (RNAi) and recombinant protein injection were employed to further study the roles of MnCrustin1 and MnCrustin2 in pathogen infection. The results indicated that silencing MnCrustin1 and MnCrustin2 accelerated the mortality rate of M. nipponense after challenge with A. hydrophila or WSSV, while recombinant MnCrustin1 and MnCrustin2 decreased the mortality rate under these conditions. These results suggest that MnCrustin1 and MnCrustin2 both play prominent roles in the immune response of M. nipponense to bacterial and viral infection and are important immune effectors of this prawn against pathogen infection.

CgWnt-1 regulates haemocyte proliferation during immune response of oyster Crassostrea gigas

Recent findings regarding the immunomodulatory role of Wnt signaling suggest that it is significant in regulating the differentiation and proliferation of immune cells. In the present study, a Wnt-1 homolog (designated as CgWnt-1) with a conserved WNT1 domain was identified from oyster Crassostrea gigas. The transcripts of CgWnt-1 were barely expressed in egg to gastrula stage during early embryogenesis, and up-regulated significantly in the trochophore to juvenile stage. The mRNA transcripts of CgWnt-1 were detected in different tissues of adult oyster, with an extremely high expression level in the mantle, which was 77.38-fold (p < 0.05) of that in labial palp. After Vibrio splendidus stimulation, the mRNA expression levels of CgWnt-1 and Cgβ-catenin in haemocytes up-regulated significantly at 3, 12, 24, and 48 h (p < 0.05). After injection of recombinant protein (rCgWnt-1) into oyster in vivo, the expressions of Cgβ-catenin, cell proliferation related genes CgRunx-1 and CgCDK-2 in haemocytes significantly up-regulated, which were 4.86-fold (p < 0.05), 9.33-fold (p < 0.05), 6.09-fold (p < 0.05) of those in rTrx group, respectively. The percentage of EDU+ cells in haemocytes also significantly increased (2.88-fold of that in control group, p < 0.05) at 12 h after rCgWnt-1 treatment. When the Wnt signal inhibitor C59 was injected simultaneously with rCgWnt-1, the expressions of Cgβ-catenin, CgRunx-1, and CgCDK-2 were significantly reduced, which were 0.32-fold (p < 0.05), 0.16-fold (p < 0.05), and 0.25-fold (p < 0.05) of that in rCgWnt-1 group, respectively, and the percentage of EDU+ cells in haemocytes was also significantly inhibited (0.15-fold compared with that in rCgWnt-1 group, p < 0.05). These results suggested that the conserved CgWnt-1 could modulate haemocytes proliferation via regulating cell cycle related genes and involved in the immune response of oysters.

Assisted oocyte activation does not overcome recurrent embryo developmental problems.

Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. NCT03354013.

An Interleukin-17 Isoform from Thick Shell Mussel Mytilus coruscus Serves as a Mediator of Inflammatory Response

The inflammatory cytokine interleukin-17 (IL17) plays an important role in innate immunity by binding to its receptors (IL17Rs) to activate immune defense signals. To date, information on members of the IL17 family is still very limited in molluscan species. Here, a novel member of the IL17 family was identified and characterized from thick shell mussel Mytilus coruscus, and this gene was designated as McIL17-1 by predicting structural domains and phylogenetic analysis. McIL17-1 transcripts existed in all examined tissues with high expression levels in gills, hemocytes and digestive glands. After the stimuli of different pathogen associated molecular patterns (PAMPs) for 72 h, transcriptional expression of McIL17-1 was significantly upregulated, except for poly I:C stimulation. Cytoplasm localization of McIL17-1 was shown in HEK293T cells by fluorescence microscopy. Further, in vivo and in vitro assays were performed to evaluate the potential function of McIL17-1 played in immune response. McIL17-1 was either knocked down or overexpressed in vivo through RNA inference (RNAi) and recombinant protein injection, respectively. With the infection of living Vibrio alginolyticus, a high mortality rate was exhibited in the McIL17-1 overexpressed group compared to the control group, while a lower mortality rate was observed in the McIL17-1 knocked down group than control group. In vitro, the flow cytometric analysis showed that the apoptosis rate of McIL17-1 inhibited hemocytes was significantly lower than that of the control group after lipopolysaccharide stimulation. These results collectively suggested that the newly identified IL17 isoform is involved in the inflammatory response to bacterial infection in M. coruscus.

The sORF-Encoded Peptides, ATP Synthase Subunits, Facilitate WSSV Duplication in Shrimp.

Short open reading frames (sORFs) are a newly identified family of genes, and the functions of most sORF genes and their encoded peptides (SEPs) are still unknown. In this study, two ATP synthase subunits were identified in kuruma shrimp (Marsupenaeus japonicus) as SEPs, namely MjATP5I and MjATP5L. They were widely distributed in all of the tested tissues of shrimp and upregulated in hemocytes and intestines in response to WSSV challenge. The injection of recombinant proteins (rMjATP5I and rMjATP5L) increased the expression of Ie1 and Vp28, while the knockdown of MjATP5I and MjATP5L decreased the expression of Ie1 and Vp28. All of the results suggest that MjATP5I and MjATP5L were beneficial for WSSV replication. Further exploration found that MjATP5I and MjATP5L RNAi significantly improved the shrimp survival rates, reduced ATP production, and upregulated the expression of antimicrobial peptide genes post viral challenge, and the two ATPase subunits and Relish negatively regulated each other. These results reveal that MjATP5I and MjATP5L facilitated WSSV duplication by regulating the production of ATP contents and the expression of antimicrobial peptide genes in shrimp.

A positive feedback loop between two C-type lectins originated from gene duplication and relish promotes the expression of antimicrobial peptides in Procambarus clarkii.

Gene duplication (GD) leads to the expansion of gene families that contributes organisms adapting to stress or environment and dealing with the infection of various pathogens. C-type lectins (CTLs) in crustaceans undergo gene expansion and participate in various immune responses. However, the functions of different CTL produced by GD are not fully characterized. In the present study, two CTL genes (designated as PcLec-EPS and PcLec-QPS, respectively) were identified from Procambarus clarkii. PcLec-EPS and PcLec-QPS originate from GD and the main difference between them is exon 3. PcLec-EPS and PcLec-QPS respectively contains EPS and QPS motif in their carbohydrate recognition domain. The mRNA levels of PcLec-EPS and PcLec-QPS in hemocytes, gills, intestine and lymph underwent time-dependent enhancement after D-Mannose and D-Galactose challenge. Recombinant PcLec-EPS and PcLec-QPS could bind to carbohydrates and microbes, and agglutinate bacteria. The results of experiments on recombinant protein injection and RNA interference indicate that PcLec-EPS and PcLec-QPS can respectively strong recognize and bind D-Mannose and D-Galactose, activate the Relish transcriptional factor, and further upregulate the expression of different antimicrobial peptides (AMPs). In addition, these two CTLs and Relish could positively regulate the expression of each other, suggesting that there is a positive feedback loop between two CTLs and Relish that regulates the expression of AMPs. It may contribute to the expansion of the immune response for host quickly and efficiently eliminating pathogenic microorganisms. This study provides new knowledge for clear understanding the significance and function of different CTL generated by GD in immune defenses in crustacean.

Leukocyte and IgM Responses to Immunization with the CIDR1α-PfEMP1 Recombinant Protein in the Wistar Rat.

The malaria vaccine is an important strategy for the global malaria elimination program, but the complexity of the Plasmodium antigen is a major hurdle in malaria vaccine development. The cysteine-rich interdomain region 1α (CIDR1α) of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is crucial in malaria pathogenesis, making it a vaccine candidate. This study investigated the leukocyte and IgM response generated after administering a CIDR1α-PfEMP1 recombinant protein injection in Wistar rats. The rats were divided into a control group, who received a physiological saline solution (PSS), and a treatment group, who were subcutaneously injected with 150 µg of purified CIDR1α-PfEMP1 protein three times at the 3-week interval. Blood samples were collected every week after each injection. The number of leukocytes were counted using a Neubauer chamber, and the IgM concentration was determined using an enzyme-linked immunosorbent assay (ELISA). Data were analyzed using an independent, paired-T test, a Mann–Whitney test, and a Wilcoxon test, based on the distribution of the data. The total number of leukocytes notably increased on day 29 (p < 0.05). The percentage of neutrophils decreased, especially on day 8 (p < 0.05), whereas the percentages of monocytes and lymphocytes increased, primarily on day 14 (p < 0.05). The IgM concentration increased on day 14 (p < 0.05). In conclusion, the CIDR1α-PfEMP1 recombinant protein may induce leukocyte and IgM responses, making it a potential malaria vaccine candidate.

Transcription factors foxl2 and foxl3 regulate cyp19a1a and cyp11b in orange-spotted grouper (Epinephelus coioides)

In orange-spotted grouper (E. coioides), the transcription factors foxl2 and foxl3 are thought to play an important role in ovarian and testicular development, but how they do so remains unclear. In this study, we investigated the regulatory effect of foxl2 and foxl3 on the sex steroid genes cyp19a1a and cyp11b, and then we explored the function of foxl2 and foxl3 in the sex hormone synthesis pathway in teleost fish. First, we used FOXL2 and FOXL3 recombinant proteins to investigate the regulatory mechanism of foxl2/foxl3 on aromatase in E. coioides and found that the recombinant proteins can enter gonad cells and be expressed in the nucleus. The expression levels of cyp19a1a and cyp11b were detected by real-time fluorescent quantitative PCR when FOXL2 or FOXL3 recombinant proteins were used to stimulate primary ovarian cells of orange-spotted grouper. The FOXL2 recombinant protein significantly increased the expression of cyp19a1a mRNA, which is related to estrogen production, in a dose-dependent manner. The FOXL3 recombinant protein significantly down-regulated the expression of cyp19a1a mRNA and significantly up-regulated the expression of cyp11b mRNA, which is related to androgen synthesis. Dual luciferase analysis in vitro revealed that foxl2 directly acted on the promoter region of cyp19a1a and foxl3 directly acted on the promoter regions of both cyp19a1a and cyp11b. The FOXL2 and FOXL3 recombinant protein injection experiments in vivo by surgery further showed that foxl2 promoted ovarian differentiation by directly up-regulating the expression of the aromatase gene cyp19a1a, whereas foxl3 directly inhibited the expression of the aromatase gene cyp19a1a and up-regulated the expression of cyp11b to promote testicular differentiation.

KPI5 Is Involved in the Regulation of the Expression of Antibacterial Peptide Genes and Hemolymph Melanization in the Silkworm, Bombyx mori.

Kunitz-type protease inhibitors (KPIs) are ubiquitously found in many organisms, and participate in various physiological processes. However, their function in insects remains to be elucidated. In the present study, we characterized and functionally analyzed silkworm KPI5. Sequence analysis showed that KPI5 contains 85 amino acids with six conserved cysteine residues, and the P1 site is a phenylalanine residue. Inhibitory activity and stability analyses indicated that recombinant KPI5 protein significantly inhibited the activity of chymotrypsin and was highly tolerant to temperature and pH. The spatio-temporal expression profile analysis showed that KPI5 was synthesized in the fat body and secreted into the hemolymph. In vivo induction analysis showed that the expression of KPI5 in the fat body was significantly upregulated by pathogen-associated molecular patterns (PAMPs). Binding assays suggested that KPI5 can bind to pathogens and PAMPs. In vitro pathogen growth inhibition assay and encapsulation analysis indicated that KPI5 can neither kill pathogenic bacteria directly nor promote the encapsulation of agarose beads by silkworm hemocytes. Recombinant protein injection test and CRISPR/Cas9-mediated knockdown showed that KPI5 promotes the expression of antimicrobial peptides (AMPs) in the fat body. Moreover, the survival rate of individuals in the KPI5 knockdown group was significantly lower than that of the control group after pathogen infection. Phenoloxidase (PO) activity assays showed that KPI5 significantly inhibited the hemolymph PO activity and melanization induced by PAMPs. These findings suggested that KPI5 plays a dual regulatory role in innate immunity by promoting the expression of antimicrobial peptides in the fat body and inhibiting hemolymph melanization. Our study furthers the understanding of the function of insect KPIs and provides new insights into the regulatory mechanism of insect immune homeostasis.

Roles of Crustacean Female Sex Hormone 1a in a Protandric Simultaneous Hermaphrodite Shrimp

Crustacean female sex hormone (CFSH) plays a pivotal role in the development of secondary sex characteristics in dioecious crustaceans. However, until now the knowledge concerning its functions in hermaphroditic species is scanty. Herein, we explored the function of CFSH (Lvit-CFSH1a) in the peppermint shrimp Lysmata vittata, a species characterized by a rare reproductive system of protandric simultaneous hermaphroditism (PSH). Lvit-CFSH1a cDNA was 1,220-bp in length with a 720-bp ORF encoded a polypeptide of 239-aa. RT-PCR showed that Lvit-CFSH1a was exclusively expressed in the eyestalk ganglion. For female physiology, it was found that Lvit-CFSH1a was indispensable for the development of female gonopores, but it might not involve vitellogenesis of the species. For male physiology, Lvit-CFSH1a suppressed Lvit-IAG2 expression in short-term silencing experiment and recombinant protein injection experiment, but did not affect male sexual differentiation in long-term silencing experiment. In addition, silencing the Lvit-CFSH1a gene impeded individual growth in L. vittata.

Immune Resolution Dilemma: Host Antimicrobial Factor S100A8/A9 Modulates Inflammatory Collateral Tissue Damage During Disseminated Fungal Peritonitis.

Intra-abdominal infection (peritonitis) is a leading cause of severe disease in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial and pro-inflammatory protein complex used as a biomarker for diagnosis of numerous inflammatory disorders. Here we describe the role of S100A8/A9 in inflammatory collateral tissue damage (ICTD). Using a mouse model of disseminated intra-abdominal candidiasis (IAC) in wild-type and S100A8/A9-deficient mice in the presence or absence of S100A9 inhibitor paquinimod, the role of S100A8/A9 during ICTD and fungal clearance were investigated. S100A8/A9-deficient mice developed less ICTD than wild-type mice. Restoration of S100A8/A9 in knockout mice by injection of recombinant protein resulted in increased ICTD and fungal clearance comparable to wild-type levels. Treatment with paquinimod abolished ICTD and S100A9-deficient mice showed increased survival compared to wild-type littermates. The data indicates that S100A8/A9 controls ICTD levels and antimicrobial activity during IAC and that targeting of S100A8/A9 could serve as promising adjunct therapy against this challenging disease.

TRIM9 is involved in facilitating Vibrio parahaemolyticus infection by inhibition of relish pathway in Penaeus monodon

Tripartite motif-containing 9 (TRIM9) has been demonstrated to exert important roles in regulation of innate immune signaling. In this study, a novel TRIM9 homolog was identified from Penaeus monodon (named PmTRIM9). The open reading frame (ORF) of PmTRIM9 was 2064 bp, which encoding a 687-amino-acid polypeptide. Following Vibrio parahaemolyticus challenge, the expression levels of PmTRIM9 mRNA were significantly down-regulated in tested tissues. RNA interference and recombinant protein injection experiments were performed to explore the function of PmTRIM9, and the results showed it could facilitate V. parahaemolyticus replication and lead P. monodon more vulnerable to V. parahaemolyticus challenge. The dual-luciferase reporter assay showed that PmTRIM9 possessed the ability to inhibit the promoter activity in HEK293 T cells. Silencing of PmTRIM9 could increase the expression of the major NF-κB transcription factor, PmRelish. Further studies showed that knockdown of PmRelish promoted the V. parahaemolyticus infection and decreased the expression of specific antimicrobial peptides (AMPs), including PmCRU5, PmCRU7, PmALF6, PmALF3, PmLYZ and PmPEN5. However, knockdown of PmTRIM9 increased expression levels of the same AMPs, but except for PmCRU5, indicating that PmTRIM9 may negatively regulate the PmRelish-mediated expression of AMPs. All these results suggest that PmTRIM9 was involved in facilitating V. parahaemolyticus infection by inhibition of Relish pathway in P. monodon.

A trypsin-like serine protease domain of masquerade gene in crayfish Procambarus clarkii could activate prophenoloxidase and inhibit bacterial growth

Masquerade (Mas) is a secreted trypsin-like serine protease (SPs) and involved in immune response in some arthropods. However, according to previous studies, Mas presents different functional activities. In the present study, the functional mechanisms of Mas in crayfish Procambarus clarkii immune defense were studied. A fragment cDNA sequence of PcMas was identified and characterized. From the structural analysis, it contains a trypsin-like serine protease domain. The highest expression level of PcMas was detected in hepatopancreas. The infection of A. hydrophila could induce the expression of PcMas, while the WSSV infection did not cause changes in the expression of PcMas. Through the prokaryotic expression system, the PcMas protein was expressed in E. coli. It was verified that PcMas can bind to bacteria in vitro and inhibit the growth of the bacteria. By dsRNA interference with the expression of PcMas, the decrease expression of PcMas led to a decrease in the activity of phenoloxidase in hemolymph and an increase of mortality caused by A. hydrophila infection. The injection of recombinant protein can enhance the activity of phenoloxidase and reduce mortality caused by A. hydrophila infections. Therefore, the present study confirmed that PcMas could improve the body's immune response to eliminate bacterial pathogens by binding with bacteria and activating the prophenoloxidase system. The results will enrich the molecular mechanisms of crustaceans immune defense.

Identification of co-chaperone Cdc37 in Penaeus monodon: coordination with Hsp90 can reduce cadmium stress-induced lipid peroxidation

Cell division cycle 37 (Cdc37) is an important cytoplasmic phosphoprotein, which usually functions as a complex with heat shock protein 90 (Hsp90), to effectively reduce the damage caused by heavy metals, such as cadmium (Cd), in aquatic animals. The high toxicity of Cd in aquatic systems generally has a deleterious effect on healthy farming of shrimps. In the present study, a novel Cdc37 gene from Penaeus monodon was identified and designated as PmCdc37. Following exposure to Cd stress, the expression levels of PmCdc37 were upregulated at the transcriptional level in both the hepatopancreas and hemolymph. RNA interference and recombinant protein injection experiments were carried out to determine the function of PmCdc37 in P. monodon following Cd exposure. To clarify the correlations between PmCdc37 and PmHsp90, the respective recombinant proteins were expressed in vitro, and the ATPase activity of PmHsp90, with or without PmCdc37, was assessed. Moreover, a pull-down assay was conducted to detect the correlation between PmCdc37 and PmHsp90. After analyzing the expression patterns of PmHsp90 following Cd challenge, whether PmHsp90 can promote the ability of PmCdc37 to resist Cd stress or not was investigated. The results showed that formation of a PmHsp90/PmCdc37 complex protected shrimp against Cd stress-induced damage. Moreover, we also confirmed that PmSOD is involved in Cd stress, and that the PmHsp90/PmCdc37 complex can regulate SOD enzymatic activity. PmSOD was involved in decreasing the MDA content in shrimp hemolymph caused by Cd stress. We concluded that during exposure to Cd, the PmHsp90/PmCdc37 complex increases SOD enzyme activity, and in turn decreases the MDA content, thereby protecting shrimp against the damage caused by Cd stress. The present studies contribute to understanding the molecular mechanism underlying resistance to Cd stress in shrimp.

Neuroprotection of Retinal Ganglion Cells with AAV2-BDNF Pretreatment Restoring Normal TrkB Receptor Protein Levels in Glaucoma.

Intravitreal delivery of brain-derived neurotrophic factor (BDNF) by injection of recombinant protein or by gene therapy can alleviate retinal ganglion cell (RGC) loss after optic nerve injury (ONI) or laser-induced ocular hypertension (OHT). In models of glaucoma, BDNF therapy can delay or halt RGCs loss, but this protection is time-limited. The decreased efficacy of BDNF supplementation has been in part attributed to BDNF TrkB receptor downregulation. However, whether BDNF overexpression causes TrkB downregulation, impairing long-term BDNF signaling in the retina, has not been conclusively proven. After ONI or OHT, when increased retinal BDNF was detected, a concomitant increase, no change or a decrease in TrkB was reported. We examined quantitatively the retinal concentrations of the TrkB protein in relation to BDNF, in a course of adeno-associated viral vector gene therapy (AAV2-BDNF), using a microbead trabecular occlusion model of glaucoma. We show that unilateral glaucoma, with intraocular pressure ( IOP) increased for five weeks, leads to a bilateral decrease of BDNF in the retina at six weeks, accompanied by up to four-fold TrkB upregulation, while a moderate BDNF overexpression in a glaucomatous eye triggers changes that restore normal TrkB concentrations, driving signaling towards long-term RGCs neuroprotection. We conclude that for glaucoma therapy, the careful selection of the appropriate BDNF concentration is the main factor securing the long-term responsiveness of RGCs and the maintenance of normal TrkB levels.

Mydgf promotes Cardiomyocyte proliferation and Neonatal Heart regeneration.

Myeloid-derived growth factor (Mydgf), a paracrine protein secreted by bone marrow-derived monocytes and macrophages, was found to protect against cardiac injury following myocardial infarction (MI) in adult mice. We speculated that Mydgf might improve heart function via myocardial regeneration, which is essential for discovering the target to reverse heart failure.Methods: Two genetic mouse lines were used: global Mydgf knockout (Mydgf-KO) and Mydgf-EGFP mice. Two models of cardiac injury, apical resection was performed in neonatal and MI was performed in adult mice. Quantitative reverse transcription-polymerase chain reaction, western blot and flow cytometry were performed to study the protein expression. Immunofluorescence was performed to detect the proliferation of cardiomyocytes. Heart regeneration and cardiac function were evaluated by Masson's staining and echocardiography, respectively. RNA sequencing was employed to identify the key involved in Mydgf-induced cardiomyocyte proliferation. Mydgf recombinant protein injection was performed as a therapy for cardiac repair post MI in adult mice.Results: Mydgf expression could be significantly induced in neonatal mouse hearts after cardiac injury. Unexpectedly, we found that Mydgf was predominantly expressed by endothelial cells rather than macrophages in injured neonatal hearts. Mydgf deficiency impeded neonatal heart regeneration and injury-induced cardiomyocyte proliferation. Mydgf recombinant protein promoted primary mouse cardiomyocyte proliferation. Employing RNA sequencing and functional verification, we demonstrated that c-Myc/FoxM1 pathway mediated Mydgf-induced cardiomyocyte expansion. Mydgf recombinant protein improved cardiac function in adult mice after MI injury with inducing cardiomyocyte proliferation.Conclusion: Mydgf promotes cardiomyocyte proliferation by activating c-Myc/FoxM1 pathway and improves heart regeneration both in neonatal and adult mice after cardiac injury, providing a potential target to reverse cardiac remodeling and heart failure.

Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei.

The “eyestalk-androgenic gland (AG)-testis” endocrine axis is involved in male sexual differentiation of crustaceans. The insulin-like androgenic gland hormone (IAG), secreted from the AG, plays a central role in this axis, however key factors upstream the IAG are still poorly understood. Here, two crustacean hyperglycemic hormone (CHH) genes (LvCHH1 and LvCHH2) and their putative receptor guanylate cyclase (LvGC) were identified in Litopenaeus vannamei. LvCHH1 and LvCHH2 belonged to CHH subfamily I members and LvGC was a membrane-bound guanylate cyclase. They were all differentially expressed in eyestalks and gonads of males and females. RNA interference (RNAi) of either LvCHH1 or LvCHH2 increased LvIAG expression, while injection of their recombinant protein decreased LvIAG expression, indicating that LvCHH1 and LvCHH2 are inhibitory factors of LvIAG expression. Yeast two-hybrid assay showed that both LvCHH1 and LvCHH2 interacted with LvGC and their RNAi and recombinant protein injection exerted opposite regulatory effects on the transcriptional expression of LvGC. Meanwhile, knockdown of LvGC increased LvIAG expression. These results suggest that LvGC is the receptor of LvCHH1 and LvCHH2 and they are all involved in male sexual development by regulating LvIAG expression. The present study unveils missing upstream elements in the “eyestalk-AG-testis” endocrine axis in crustacean.

Neuroprotection of retinal ganglion cells by a novel gene therapy construct that achieves sustained enhancement of brain-derived neurotrophic factor/tropomyosin-related kinase receptor-B signaling

Previous studies have demonstrated that intravitreal delivery of brain-derived neurotrophic factor (BDNF) by injection of recombinant protein or by gene therapy can alleviate retinal ganglion cell (RGC) loss after optic nerve injury. BDNF gene therapy can improve RGC survival in experimental models of glaucoma, the leading cause of irreversible blindness worldwide. However, the therapeutic efficacy of BDNF supplementation alone is time limited at least in part due to BDNF receptor downregulation. Tropomyosin-related receptor kinase-B (TrkB) downregulation has been reported in many neurological diseases including glaucoma, potentially limiting the effect of sustained or repeated BDNF delivery.Here, we characterize a novel adeno-associated virus (AAV) gene therapy (AAV2 TrkB-2A-mBDNF) that not only increases BDNF production but also improves long-term neuroprotective signaling by increasing expression of the BDNF receptor (TrkB) within the inner retina. This approach leads to significant and sustained elevation of survival signaling pathways ERK and AKT within RGCs over 6 months and avoids the receptor downregulation which we observe with treatment with AAV2 BDNF alone. We validate the neuroprotective efficacy of AAV2 TrkB-2A-mBDNF in a mouse model of optic nerve injury, where it outperforms conventional AAV2 BDNF or AAV2 TrkB therapy, before showing powerful proof of concept neuroprotection of RGCs and axons in a rat model of chronic intraocular pressure (IOP) elevation. We also show that there are no adverse effects of the vector on retinal structure or function as assessed by histology and electroretinography in young or aged animals. Further studies are underway to explore the potential of this vector as a candidate for progression into clinical studies to protect RGCs in patients with glaucoma and progressive visual loss despite conventional IOP-lowering treatment.