Cytoplasmic TP53INP2 acts as an apoptosis partner in TRAIL treatment: the synergistic effect of TRAIL with venetoclax in TP53INP2-positive acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor outcomes, especially in older AML patients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered a promising anticancer drug because it selectively induces the extrinsic apoptosis of tumor cells without affecting normal cells. However, clinical trials have shown that the responses of patients to TRAIL are significantly heterogeneous. It is necessary to explore predictable biomarkers for the preselection of AML patients with better responsiveness to TRAIL. Here, we investigated the critical role of tumor protein p53 inducible nuclear protein 2 (TP53INP2) in the AML cell response to TRAIL treatment. METHODS: First, the relationship between TP53INP2 and the sensitivity of AML cells to TRAIL was determined by bioinformatics analysis of Cancer Cell Line Encyclopedia datasets, Cell Counting Kit-8 assays, flow cytometry (FCM) and cell line-derived xenograft (CDX) mouse models. Second, the mechanisms by which TP53INP2 participates in the response to TRAIL were analyzed by Western blot, ubiquitination, coimmunoprecipitation and immunofluorescence assays. Finally, the effect of TRAIL alone or in combination with the BCL-2 inhibitor venetoclax (VEN) on cell survival was explored using colony formation and FCM assays, and the effect on leukemogenesis was further investigated in a patient-derived xenograft (PDX) mouse model. RESULTS: AML cells with high TP53INP2 expression were more sensitive to TRAIL in vitro and in vivo. Gain- and loss-of-function studies demonstrated that TP53INP2 significantly enhanced TRAIL-induced apoptosis, especially in AML cells with nucleophosmin 1 (NPM1) mutations. Mechanistically, cytoplasmic TP53INP2 maintained by mutant NPM1 functions as a scaffold bridging the ubiquitin ligase TRAF6 to caspase-8 (CASP 8), thereby promoting the ubiquitination and activation of the CASP 8 pathway. More importantly, simultaneously stimulating extrinsic and intrinsic apoptosis signaling pathways with TRAIL and VEN showed strong synergistic antileukemic activity in AML cells with high levels of TP53INP2. CONCLUSION: Our findings revealed that TP53INP2 is a predictor of responsiveness to TRAIL treatment and supported a potentially individualized therapeutic strategy for TP53INP2-positive AML patients.

The molecular mechanism and evolutionary divergence of caspase 3/7-regulated gasdermin E activation.

Caspase (CASP) is a family of proteases involved in cleavage and activation of gasdermin, the executor of pyroptosis. In humans, CASP3 and CASP7 recognize the same consensus motif DxxD, which is present in gasdermin E (GSDME). However, human GSDME is cleaved by CASP3 but not by CASP7. The underlying mechanism of this observation is unclear. In this study, we identified a pyroptotic pufferfish GSDME that was cleaved by both pufferfish CASP3/7 and human CASP3/7. Domain swapping between pufferfish and human CASP and GSDME showed that the GSDME C-terminus and the CASP7 p10 subunit determined the cleavability of GSDME by CASP7. p10 contains a key residue that governs CASP7 substrate discrimination. This key residue is highly conserved in vertebrate CASP3 and in most vertebrate (except mammalian) CASP7. In mammals, the key residue is conserved in non-primates (e.g., mouse) but not in primates. However, mouse CASP7 cleaved human GSDME but not mouse GSDME. These findings revealed the molecular mechanism of CASP7 substrate discrimination and the divergence of CASP3/7-mediated GSDME activation in vertebrate. These results also suggested that mutation-mediated functional alteration of CASP probably enabled the divergence and specialization of different CASP members in the regulation of complex cellular activities in mammals.

Cell death is essential for an organism to develop and survive as it plays key roles in processes such as embryo development and tissue regeneration. Cell death is also an important form of defence during an infection. A form of programmed cell death known as pyroptosis can be induced in infected cells, which helps to kill the infectious agent as well as alert the immune system to the infection. Pyroptosis is driven by Gasdermin E, a protein made up of two domains. At one end of the protein, the 'N-terminal' domain punctures holes in cell membranes, which can lead to cell death. At the other end, the 'C-terminal' domain inhibits the activity of the N-terminal domain. A family of proteins called caspases activate Gasdermin E by cleaving it, which releases the N-terminal domain from the inhibitory C-terminal domain. In humans, two caspases known as CASP3 and CASP7 recognize a specific sequence of amino acids ­ the building blocks of proteins ­ in Gasdermin E. However, only CASP3 is able to cleave the protein. After discovering that, unlike in humans, pufferfish Gasdermin E can be cleaved by both CASP3 and CASP7, Xu et al. wanted to investigate the underlying mechanisms behind this difference. Swapping the domains of human and pufferfish Gasdermin E and creating different versions of CASP7 revealed that the C-terminal domain of Gasdermin E and a single amino acid in CASP7 determine whether cleavage is possible. Interestingly, the key amino acid sequence required for cleavage by CASP7 is present in most vertebrate CASP3 and CASP7 proteins. However, it is absent in most mammalian CASP7. The findings of Xu et al. suggest that the different activity of human CASP7 and CASP3 is driven by a single amino acid mutation. This change likely played an important role in the process of different CASP proteins evolving to regulate different cellular activities in mammalian cells. This knowledge will be useful for future studies on the evolution and specialization of other closely related proteins.

Dynamic monitoring of serum tumor markers as prognostic factors in patients with advanced non-small-cell lung cancer treated with first-line immunotherapy: a multicenter retrospective study.

Background: To date, no specific studies have reported the use of dynamic serum tumor markers (STMs) as prognostic factors in patients with advanced non-small-cell lung cancer (NSCLC) who receive first-line immunotherapy. Therefore, it is unclear whether STMs can be used as a prognostic factor for first-line immunotherapy in advanced NSCLC. Objectives: To elucidate the role of STMs in monitoring immunotherapy response in advanced NSCLC. Patients were treated with first-line programmed cell death-1/programmed cell death ligand-1 inhibitors at four Chinese centers. Design: This was a multicenter retrospective study. Methods: Blood samples were collected at baseline and after 6-8 weeks of treatment. Computed tomography scans were used to evaluate treatment efficacy according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. Post-treatment drops in STMs [Serum carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cytokeratin fragment 19 (CYFRA21-1), carbohydrate antigen 19-9 (CA19-9), and carbohydrate antigen 125 (CA125)] were decreased ⩾20% (Group C) over baseline was used as cutoff level for defining a marker response. If STMs were increased by ⩾20% after treatment, the therapeutic effect was limited (Group A). Patients with STM changes between a 20% increase or decrease were enrolled in Group B. In univariate and multivariate stepwise Cox regression analyses, STMs and RECIST responses were analyzed for their impact on progression-free survival (PFS) and overall survival (OS). Results: The analysis included 716 patients. By multivariate analysis, CEA, NSE, CYFRA21-1, CA19-9, and CA125 (Group A versus Group B and Group A versus Group C) were associated with significant differences in PFS. Similar results were observed in the OS analysis. Similar results were observed in the adenocarcinoma subgroup analyses. In squamous cell carcinoma subgroup analyses, there was no statistical difference in PFS (p = 0.147) or OS (p = 0.068) between Group A and Group B for CA125. Conclusion: The increase and decrease in serum levels of STMs might be reliable prognostic factors for immunotherapy efficacy in NSCLC patients.

Comparison of first-line immunotherapy efficacy between advanced lung squamous cell carcinoma and pulmonary lymphoepithelioma-like carcinoma: A propensity score matching multicenter study.

Background: Compared with other lung squamous cell carcinomas (LUSC), pulmonary lymphoepithelioma-like carcinoma (pLELC) is closely associated with Epstein-Barr virus (EBV) infections with a unique molecular profile and immune microenvironment. This study was thus established to compare the treatment response and effectiveness of immunotherapy between pLELC and LUSC. Material and Methods: We enrolled 31 patients with pLELC and 116 with LUSC receiving first-line immunotherapy at three centers in China and compared the treatment response and effectiveness of immunotherapy. Propensity score matching (PSM) was used to balance the differences in baseline data between the two groups. Results: Before PSM, progression-free survival and overall survival were longer in the pLELC group than in the LUSC group (progression-free survival: hazard ratio (HR), 1.67, 95% CI: 1.05-2.63, P = 0.028; overall survival: HR, 1.90, 95% CI: 1.06-3.40, P = 0.028). This remained unchanged after PSM (progression-free survival: HR, 1.79, 95% CI: 1.02-3.15, P = 0.044; overall survival: HR, 2.20; 95% CI: 1.10-4.37, P = 0.022). Conclusion: pLELC showed a clinically meaningful survival benefit compared with traditional LUSC following immunotherapy. Subsequent studies should consider the role of the EBV in the tumor immune microenvironment of pLELC.

Baseline serum tumor markers predict the survival of patients with advanced non-small cell lung cancer receiving first-line immunotherapy: a multicenter retrospective study.

BACKGROUND: This study aimed to investigate the association between baseline serum tumor markers (STMs) (carcinoembryonic antigen [CEA], neuron-specific enolase [NSE], cytokeratin-19 fragment [CYFRA21-1], carbohydrate antigen 19-9 [CA19-9], and carbohydrate antigen 125 [CA125]) and the efficacy of first-line immunotherapy in patients with advanced non-small cell lung cancer. METHODS: This multicenter retrospective study evaluated patients who received first-line immunotherapy between July 2017 and July 2022. The endpoints were progression-free survival (PFS) and overall survival (OS), as defined by the Response Evaluation Criteria in Solid Tumors version 1.1. We divided the patients into three groups based on STM levels: Group A ≥ threefold upper limit of normal, threefold upper limit of normal > Group B > upper limit of normal, and Group C ≤ upper limit of normal. RESULTS: In total, 716 patients were included in this study. In Cox proportional hazards analyses, the STM levels in Group C were independently associated with superior PFS and OS in patients with lung adenocarcinoma (LUAD). Except for CA19-9 level, the STM levels in Group C were independently associated with superior PFS and OS in patients with lung squamous carcinoma (LUSC). Except for CEA and CA19-9 levels, the levels in Group A were independently associated with inferior PFS and OS in patients with LUAD and LUSC. CONCLUSIONS: Serum CEA, NSE, CYFRA21-1, and CA125 levels can predict PFS and OS in patients with LUAD and LUSC, and serum CA19-9 levels can predict PFS and OS in patients with LUAD. The higher the serum NSE, CYFRA21-1, and CA125 levels, the worse the PFS and OS in patients with LUAD and LUSC. In addition, the higher the serum CA19-9 level, the worse the OS in patients with LUAD.

Pyroptotic gasdermin exists in Mollusca and is vital to eliminating bacterial infection.

Gasdermin (GSDM) is a family of proteins that execute pyroptosis in vertebrate. In invertebrate, pyroptotic GSDM was documented only in coral. Recent studies identified abundant GSDM structural homologs in Mollusca, but their functions are unclear. Herein, we report a functional GSDM from Pacific abalone Haliotis discus (HdGSDME). HdGSDME is specifically activated by abalone caspase 3 (HdCASP3) cleavage at two distinct sites, generating two active isoforms with pyroptotic and cytotoxic activities. HdGSDME possesses evolutionarily conserved residues that proved to be essential to the N-terminal pore-formation and C-terminal auto-inhibition capacities. Bacterial challenge activates the HdCASP3-HdGSDME pathway and induces pyroptosis and extracellular traps in abalone. Blockage of the HdCASP3-HdGSDME axis promotes bacterial invasion and host mortality. Collectively, this study reveals the existence of functionally conserved and yet distinct-featured GSDM in Mollusca and provides insights into the function and evolution of invertebrate GSDM.

Impairment of IgG Fc functions promotes tumor progression and suppresses NK cell antitumor actions.

Natural killer (NK) cells mediate antibody dependent cytotoxic killing of cancer cells via cross-linking FcγR on NK cells with IgG-Fc. Studies have shown that the single-hinge cleaved IgGs (scIgGs) have dysfunctional Fc and failed engagement with FcγRs on immune cells. However, little is known about how scIgGs impact on antitumor immunity in the tumor microenvironment. In this study, we revealed a significant association of tumor scIgGs with tumor progression and poor outcomes of breast cancer patients (n = 547). Using multiple mouse tumor models, we demonstrated that tumor scIgGs reduced NK cell cytotoxic activities and resulted in aggressive tumor progression. We further showed that an anti-hinge specific monoclonal antibody (AHA) rescued the dysfunctional Fc in scIgGs by providing a functional Fc and restored NK cell cytotoxic activity. These findings point to a novel immunotherapeutic strategy to enhance Fc engagement with FcγRs for activation of anticancer immunity.

Akt3-mTOR regulates hippocampal neurogenesis in adult mouse.

Akt signaling has been associated with adult neurogenesis in the hippocampal dentate gyrus (DG). We reported cognitive dysfunction in Akt3 knockout (Akt3-KO) mice with the down-regulation of mTOR activation. However, little is known about the effects of Akt3 signaling on hippocampal neurogenesis. Herein, we show that progenitor cells, neuroblasts, and mature newborn neurons in hippocampal DG expressed Akt3 protein. The Akt3 phosphorylation in hippocampal DG was increased after voluntary wheel running for 7 days in wild-type mice (running WT mice), but not in Akt3-KO mice (running Akt3-KO mice). Subsequently, we observed that the proliferation of progenitor cells was suppressed in Akt3-KO mice and the mTOR inhibitor rapamycin-treated mice, whereas enhanced in running WT mice rather than running Akt3-KO mice. Neurite growth of neuroblasts was impaired in Akt3-KO mice and rapamycin-treated mice. In contrast, neither differentiation of progenitor cells nor migrating of newly generated neurons was altered in Akt3-KO mice or running WT mice. The levels of p70S6K and 4EBP1 phosphorylation were declined in Akt3-KO mice and elevated in running WT mice depending on mTOR activation. Furthermore, telomerase activity, telomere length, and expression of telomerase reverse transcriptase (TERT) were decreased in Akt3-KO mice but increased in running WT mice rather than running Akt3-KO mice, which required the mTOR activation. The study provides in vivo evidence that Akt3-mTOR signaling plays an important role in the proliferation of progenitor cells and neurite growth through positive regulated TERT expression and activation of p70S6K and 4EBP1.

Human Endogenous Retroviruses in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most aggressive and deadly brain tumor. It is primarily diagnosed in the elderly and has a 5-year survival rate of less than 6% even with the most aggressive therapies. The lack of biomarkers has made the development of immunotherapy for GBM challenging. Human endogenous retroviruses (HERVs) are a group of viruses with long terminal repeat (LTR) elements, which are believed to be relics from ancient viral infections. Recent studies have found that those repetitive elements play important roles in regulating various biological processes. The differentially expressed LTR elements from HERVs are potential biomarkers for immunotherapy to treat GBM. However, the understanding of the LTR element expression in GBM is greatly lacking. METHODS: We obtained 1077.4 GB of sequencing data from public databases. These data were generated from 111 GBM tissue studies, 30 GBM cell lines studies, and 45 normal brain tissues studies. We analyzed repetitive elements that were differentially expressed in GBM and normal brain samples. RESULTS: We found that 48 LTR elements were differentially expressed (p-value < 0.05) between GBM and normal brain tissues, of which 46 were HERV elements. Among these 46 elements, 34 significantly changed HERVs belong to the ERV1 superfamily. Furthermore, 43 out of the 46 differentially expressed HERV elements were upregulated. CONCLUSION: Our results indicate significant differential expression of many HERV LTR elements in GBM and normal brain tissues. Expression levels of these elements could be developed as biomarkers for GBM treatments.

Presence of complete murine viral genome sequences in patient-derived xenografts.

Patient-derived xenografts are crucial for drug development but their use is challenged by issues such as murine viral infection. We evaluate the scope of viral infection and its impact on patient-derived xenografts by taking an unbiased data-driven approach to analyze unmapped RNA-Seq reads from 184 experiments. We find and experimentally validate the extensive presence of murine viral sequence reads covering entire viral genomes in patient-derived xenografts. The existence of viral sequences inside tumor cells is further confirmed by single cell sequencing data. Extensive chimeric reads containing both viral and human sequences are also observed. Furthermore, we find significantly changed expression levels of many cancer-, immune-, and drug metabolism-related genes in samples with high virus load. Our analyses indicate a need to carefully evaluate the impact of viral infection on patient-derived xenografts for drug development. They also point to a need for attention to quality control of patient-derived xenograft experiments.

Virome assembly and annotation in brain tissue based on next-generation sequencing.

The glioblastoma multiforme (GBM) is one of the deadliest tumors. It has been speculated that virus plays a role in GBM but the evidences are controversy. Published researches are mainly limited to studies on the presence of human cytomegalovirus (HCMV) in GBM. No comprehensive assessment of the brain virome, the collection of viral material in the brain, based on recently sequenced data has been performed. Here, we characterized the virome from 111 GBM samples and 57 normal brain samples from eight projects in the SRA database by a tested and comprehensive assembly approach. The annotation of the assembled contigs showed that most viral sequences in the brain belong to the viral family Retroviridae. In some GBM samples, we also detected full genome sequence of a novel picornavirus recently discovered in invertebrates. Unlike previous reports, our study did not detect herpes virus such as HCMV in GBM from the data we used. However, some contigs that cannot be annotated with any known genes exhibited antibody epitopes in their sequences. These findings provide several avenues for potential cancer therapy: the newly discovered picornavirus could be a starting point to engineer novel oncolytic virus; and the exhibited antibody epitopes could be a source to explore potential drug targets for immune cancer therapy. By characterizing the virosphere in GBM and normal brain at a global level, the results from this study strengthen the link between GBM and viral infection which warrants the further investigation.

Recognition of a highly conserved glycoprotein B epitope by a bivalent antibody neutralizing HCMV at a post-attachment step.

Human cytomegalovirus (HCMV) is one of the main causative agents of congenital viral infection in neonates. HCMV infection also causes serious morbidity and mortality among organ transplant patients. Glycoprotein B (gB) is a major target for HCMV neutralizing antibodies, yet the underlying neutralization mechanisms remain largely unknown. Here we report that 3-25, a gB-specific monoclonal antibody previously isolated from a healthy HCMV-positive donor, efficiently neutralized 14 HCMV strains in both ARPE-19 cells and MRC-5 cells. The core epitope of 3-25 was mapped to a highly conserved linear epitope on antigenic domain 2 (AD-2) of gB. A 1.8 Å crystal structure of 3-25 Fab in complex with the peptide epitope revealed the molecular determinants of 3-25 binding to gB at atomic resolution. Negative-staining electron microscopy (EM) 3D reconstruction of 3-25 Fab in complex with de-glycosylated postfusion gB showed that 3-25 Fab fully occupied the gB trimer at the N-terminus with flexible binding angles. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading in ARPE-19 cells. Interestingly, bivalency was required for HCMV neutralization by AD-2 specific antibody 3-25 but not the AD-4 specific antibody LJP538. In contrast, bivalency was not required for HCMV binding by both antibodies. Taken together, our results reveal the structural basis of gB recognition by 3-25 and demonstrate that inhibition of viral membrane fusion and a requirement of bivalency may be common for gB AD-2 specific neutralizing antibody.

Optical Microfiber Reader for Enzyme-Linked Immunosorbent Assay.

In clinical diagnosis, accurate and reliable measurement technologies for the detection of disease biomarkers at ultralow concentrations can provide guidance for the initiation of treatment and potentially improve survival for patients. Here, we demonstrate an optical microfiber reader for enhanced analytical sensitivity in enzyme-linked immunosorbent assays (ELISA) that enables the detection of tiny changes of the refractive index (RI) induced by the catalyzed oxidation of substrate, owing to the strong interaction between the evanescent field and surrounding medium. By employing the microfiber reader for the C-reaction protein (CRP) and interleukin-6 (IL-6) assays after the enzymatic signal amplification in ELISA, we experimentally investigate the biosensing capacity of the device. As a result, log-linear relations of CRP and IL-6 detection in PBS and human serum between the concentration and spectral response were obtained at both nanogram and picogram levels, respectively, and anti-CRP/HRP detection as low as 9.75 pg/mL was achieved, which was undetectable by the conventional spectrophotometry. With a stable, accurate, and color-free detection capacity, this optical microfiber reader has a promising prospect in early disease diagnosis and clinical treatment.

Reduced serotonin impairs long-term depression in basolateral amygdala complex and causes anxiety-like behaviors in a mouse model of perimenopause.

Perimenopause is characterized by a gradual depletion of ovarian follicles with increased vulnerability to anxiety. However, the underlying mechanisms remain poorly understood. Herein, we show that chronic exposure to 4-vinylcycloxene diepoxide (VCD) in adult female mice (VCD-mice) caused follicles depletion and decline of serum estradiol (E2) and progesterone levels. Serotonin (5-HT) synthesis in dorsal raphe nucleus (DRN) and serotonergic afferents to basolateral amygdala complex (BLA) were reduced in VCD-mice, which were recovered by the supplement E2. VCD-mice appeared anxiety-like behaviors, which was relieved by the treatment with E2 or the co-administration of 5-HT1Ar agonist 8-OH-DPAT and 5-HT2A/Cr agonist DOI. The bath-application of 8-OH-DPAT in the slices obtained from VCD-mice (VCD-slices) corrected the increase in presynaptic glutamate release at external capsule-BLA synaptic transmission. Threshold to induce NMDA receptor (NMDAr)-dependent long-term potentiation (LTP) was declined in VCD-mice with elevation of CaMKII phosphorylation, which was sensitive to 8-OH-DPAT. Notably, the bath-application of 8-OH-DPAT in VCD-slices caused a decrease in the GABAergic feedback inhibition, which was restored by adding DOI. In addition, NMDAr-independent long-term depression (LTD) could not be induced in VCD-mice, which was rescued by the co-application of 8-OH-DPAT with DOI or the GABAA receptor agonist muscimol. Furthermore, the treatment of VCD-mice with E2 could prevent the facilitation of LTP and recover the LTD induction. Thus, the results indicate that the 5-HT deficiency in the BLA of VCD-mice causes the facilitation of LTP via enhanced glutamate release and impairs the LTD induction through diminished GABAergic inhibition, leading to anxiety-like behaviors.

Disrupting LILRB4/APOE Interaction by an Efficacious Humanized Antibody Reverses T-cell Suppression and Blocks AML Development.

Therapeutic strategies are urgently needed for patients with acute myeloid leukemia (AML). Leukocyte immunoglobulin-like receptor B4 (LILRB4), which suppresses T-cell activation and supports tissue infiltration of AML cells, represents an attractive drug target for anti-AML therapeutics. Here, we report the identification and development of an LILRB4-specific humanized mAb that blocks LILRB4 activation. This mAb, h128-3, showed potent activity in blocking the development of monocytic AML in various models including patient-derived xenograft mice and syngeneic immunocompetent AML mice. MAb h128-3 enhanced the anti-AML efficacy of chemotherapy treatment by stimulating mobilization of leukemia cells. Mechanistic studies revealed four concordant modes of action for the anti-AML activity of h128-3: (i) reversal of T-cell suppression, (ii) inhibition of monocytic AML cell tissue infiltration, (iii) antibody-dependent cellular cytotoxicity, and (iv) antibody-dependent cellular phagocytosis. Therefore, targeting LILRB4 with antibody represents an effective therapeutic strategy for treating monocytic AML.

The Y chromosome sequence of the channel catfish suggests novel sex determination mechanisms in teleost fish.

BACKGROUND: Sex determination mechanisms in teleost fish broadly differ from mammals and birds, with sex chromosomes that are far less differentiated and recombination often occurring along the length of the X and Y chromosomes, posing major challenges for the identification of specific sex determination genes. Here, we take an innovative approach of comparative genome analysis of the genomic sequences of the X chromosome and newly sequenced Y chromosome in the channel catfish. RESULTS: Using a YY channel catfish as the sequencing template, we generated, assembled, and annotated the Y genome sequence of channel catfish. The genome sequence assembly had a contig N50 size of 2.7 Mb and a scaffold N50 size of 26.7 Mb. Genetic linkage and GWAS analyses placed the sex determination locus within a genetic distance less than 0.5 cM and physical distance of 8.9 Mb. However, comparison of the channel catfish X and Y chromosome sequences showed no sex-specific genes. Instead, comparative RNA-Seq analysis between females and males revealed exclusive sex-specific expression of an isoform of the breast cancer anti-resistance 1 (BCAR1) gene in the male during early sex differentiation. Experimental knockout of BCAR1 gene converted genetic males (XY) to phenotypic females, suggesting BCAR1 as a putative sex determination gene. CONCLUSIONS: We present the first Y chromosome sequence among teleost fish, and one of the few whole Y chromosome sequences among vertebrate species. Comparative analyses suggest that sex-specific isoform expression through alternative splicing may underlie sex determination processes in the channel catfish, and we identify BCAR1 as a potential sex determination gene.

Dorsal hypothalamic dopaminergic neurons play an inhibitory role in the hypothalamic-pituitary-adrenal axis via activation of D2R in mice.

AIM: The present study investigated the effects of dorsal hypothalamic dopamine (dh-DA) neurons on activation of hypothalamic-pituitary-adrenal (HPA) axis in adult male mice. METHODS: Tyrosine hydroxylase-labelled DA neurons, DA content, c-Fos immune-positive (c-Fos+) cells and CRH expression in paraventricular nuclei (PVN), serum CORT and ACTH were examined at 4-, 8-, and 12-hour after a signal injection of MPTP (20 mg kg-1 ) respectively. RESULTS: The dh-DA neurons and DA content in PVN at 4-hour post-MPTP were reduced with recovery at 12-hour post-MPTP, while decline of nigrostriatal DA neurons and DA content in striatum started from 12-hour post-MPTP. Number of c-Fos+ cells, and CORT/ACTH levels increased at 4-hour post-MPTP, followed by recovery at 12-hour post-MPTP. The CRH mRNA was elevated at 4-hour post-MPTP, and sustained for over 12 hours. At 2-hour post-MPTP, PVN-injection of D2R agonist quinpirole corrected the increases in c-Fos+ cells, CORT/ACTH and CRH mRNA, but D1R agonist SKF38393 did not. PVN-injection of D2R antagonist L-sulpiride alone caused increases in c-Fos+ cells, CORT/ACTH and CRH mRNA. Similarly, PVN-injection of CB1R agonist WIN552,12 prevented the increases in c-Fos+ cells and CORT/ACTH rather than CRH mRNA, which were blocked by CB1R antagonist AM251. Levels of PKA and CREB phosphorylation in PVN were increased at 4-hour post-MPTP, which were blocked by quinpirole, but not WIN552,12. PKA inhibitor H89 corrected the increase of CRH mRNA at 8-hour post-MPTP. CONCLUSION: The activation of dh-DA neurons regulates negatively HPA axis through targeting D2Rs of CRH neurons to enhance endocannabinoid release and inhibit PKA-CREB pathway.

Expression of Bcl-2 genes in channel catfish after bacterial infection and hypoxia stress.

Bcl-2 proteins are of vital importance in regulation of apoptosis, and are involved in a number of biological processes such as carcinogenesis and immune responses. Bcl-2 genes have been well studied in mammals, while they are not well investigated in teleost fish including channel catfish, the major aquaculture species in the United States. In this study, we identified 34 bcl-2 genes from the channel catfish genome, and verified their identities by conducting phylogenetic and syntenic analyses. The expression profiles of the bcl-2 genes in response to bacterial infections (Edwardsiella ictaluri and Flavobacterium columnare) and hypoxia stress were determined by performing meta-analysis using the existing RNA-Seq datasets. Differential expressions of bcl-2 genes were observed after bacterial infections and hypoxia treatment, including 22 bcl-2 genes after E. ictaluri infection, 22 bcl-2 genes after F. columnare infection, and 19 bcl-2 genes after hypoxia stress. Overall, the expression of the pro-apoptotic bcl-2 genes were repressed after bacterial infection and hypoxia stress, indicating that bcl-2 genes are potentially involved in the stress response by reducing cell apoptosis. Some bcl-2 genes, such as bcl2b, mcl1a, bmf1, and bnip3, showed different expression pattern during the E. ictaluri and F. columnare infection, suggesting the difference in the pathogenicity of diseases. This work presented the first systematic identification and annotation of bcl-2 genes in catfish, providing essential genomic resources for further immune and physiological studies.