Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy.

The activation Gq protein-coupled receptors (GPCRs) is a crucial factor contributing to maladaptive cardiac hypertrophy, and dysregulation of autophagy is implicated in its prohypertrophic effects. Previous studies have shown that diacylglycerol kinase zeta (DGKζ) can suppress cardiac hypertrophy by inhibiting the diacylglycerol (DAG)-PKC pathway in response to mechanical strain or growth agonists such as endothelin-1 (ET-1). However, the involvement of DGKζ in autophagy regulation remains poorly understood. In this study, we aimed to investigate the role of DGKζ in autophagy regulation during maladaptive cardiac hypertrophy. We found that Beclin1-mediated autophagy was involved in the development of maladaptive cardiac hypertrophy and dysfunction in response to prohypertrophic challenges of transverse aortic constriction (TAC) or ET-1. Deficiency of DGKζ promoted Beclin1-mediated autophagy, aggravated adverse cardiac remodeling, and cardiac dysfunction, which could be ameliorated by genetic deletion of Beclin1 or TFEB. Mechanistically, the deficiency of DGKζ disrupted the activation of AKT/mTOR signaling, the association between mTOR and TFEB, and favored the nuclear translocation of TFEB from the cytoplasm, leading to enhanced activation of Beclin1-mediated autophagy through ULK1/Beclin1 signaling and TFEB-dependent Beclin1 transcription. Taken together, these results suggest that the mechanisms by which DGKζ alleviates pathological cardiac hypertrophy may involve the regulation of Beclin1-mediated autophagy through the mTOR/TFEB signaling pathway.

Rational Design of Guanidinium-Based Bio-MCOF as a Multifunctional Nanocatalyst in Tumor Cells for Enhanced Chemodynamic Therapy.

Chemodynamic therapy (CDT) has emerged as a promising approach to cancer treatment, which can break the intracellular redox state balance and result in severe oxidative damage to biomolecules and organelles with the advantages of being less dependent on external stimulation, having deep tissue-healing abilities, and being resistant to drug resistance. There is considerable interest in developing CDT drugs with high efficiency and low toxicity. In this study, a new guanidinium-based biological metal covalent organic framework (Bio-MCOF), GZHMU-1@Mo, is rationally designed and synthesized as a multifunctional nanocatalyst in tumor cells for enhanced CDT. The DFT calculation and experimental results showed that due to the ability of MoO42- ion to promote electron transfer and increase the redox active site, Cu3 clusters and MoO42- ions in GZHMU-1@Mo can synergistically catalyze the production of reactive oxygen species (ROS) from oxygen and H2O2 in tumor cells, as well as degrade intracellular reducing substances, GSH and NADH, so as to disrupt the redox balance in tumor cells. Moreover, GZHMU-1@Mo exhibits a potent killing effect on tumor cells under both normal oxygen and anaerobic conditions. Further in vitro and in vivo antiproliferation studies revealed that the GZHMU-1@Mo nanoagent displays a remarkable antiproliferation effect and effectively inhibits tumor growth. Taken together, our study provides an insightful reference benchmark for the rational design of Bio-MCOF-based nanoagents with efficient CDT.

[Establishment of a microtubule-fluorescent fusion protein mosaically labeled zebrafish motor neuron system].

Motor neurons are an important type of neurons that control movement. The transgenic fluorescent protein (FP)-labeled motor neurons of zebrafish line is disadvantageous for studying the morphogenesis of motor neurons. For example, the individual motor neuron is indistinguishable in this transgenic line due to the high density of the motor neurons and the interlaced synapses. In order to optimize the in vivo imaging methods for the analysis of motor neurons, the present study was aimed to establish a microtubule-fluorescent fusion protein mosaic system that can label motor neurons in zebrafish. Firstly, the promotor of mnx1, which was highly expressed in the spinal cord motor neurons, was subcloned into pDestTol2pA2 construct combined with the GFP-α-Tubulin fusion protein sequence by Gateway cloning technique. Then the recombinant constructs were co-injected with transposase mRNA into the 4-8 cell zebrafish embryos. Confocal imaging analysis was performed at 72 hours post fertilization (hpf). The results showed that the GFP fusion protein was expressed in three different types of motor neurons, and individual motor neurons were mosaically labeled. Further, the present study analyzed the correlation between the injection dose and the number and distribution of the mosaically labeled neurons. Fifteen nanograms of the recombinant constructs were suggested as an appropriate injection dose. Also, the defects of the motor neuron caused by the down-regulation of insm1a and kif15 were verified with this system. These results indicate that our novel microtubule-fluorescent fusion protein mosaic system can efficiently label motor neurons in zebrafish, which provides a more effective model for exploring the development and morphogenesis of motor neurons. It may also help to decipher the mechanisms underlying motor neuron disease and can be potentially utilized in drug screening.

Single Cell Transcriptome Sequencing of Zebrafish Testis Revealed Novel Spermatogenesis Marker Genes and Stronger Leydig-Germ Cell Paracrine Interactions.

Spermatogenesis in testis is an important process for sexual reproduction, and worldwide about 10-15 percent of couples suffer from infertility. It is of importance to study spermatogenesis at single cell level in both of human and model organisms. Currently, single-cell RNA sequencing technologies (scRNA-seq) had been extensively applied to the study of cellular components and its gene regulations in the testes of different species, including human, monkey, mouse, and fly, but not in zebrafish. Zebrafish was a widely used model organism in biology and had been extensively used for the study of spermatogenesis in the previous studies. Therefore, it is also important to profile the transcriptome of zebrafish testis at single cell level. In this study, the transcriptomes of 14, 315 single cells from adult male zebrafish testes were profiled by scRNA-seq, and 10 cell populations were revealed, including Leydig cell, Sertoli cell, spermatogonia cell (SPG), spermatocyte, and spermatids. Notably, thousands of cell-type specific novel marker genes were identified, including sumo3b for SPG, krt18a.1 for Sertoli cells, larp1b and edrf1 for spermatids, which were also validated by RNA in situ hybridization experiments. Interestingly, through Ligand-Receptor (LR) analyses, zebrafish Leydig cells demonstrated stronger paracrine influence on germ cells than Sertoli cells. Overall, this study could be an important resource for the study of spermatogenesis in zebrafish and might also facilitate the study of the genes associated with human infertility through using zebrafish as a model organism.

RelB upregulates PD-L1 and exacerbates prostate cancer immune evasion.

BACKGROUND: The interaction between programmed death receptor (PD-1) and its ligand (PD-L1) is essential for suppressing activated T-lymphocytes. However, the precise mechanisms underlying PD-L1 overexpression in tumours have yet to be fully elucidated. Here, we describe that RelB participates in the immune evasion of prostate cancer (PCa) via cis/trans transcriptional upregulation of PD-L1. METHODS: Based on transcriptome results, RelB was manipulated in multiple human and murine PCa cell lines. Activated CD4+ and CD8+ T cells were cocultured with PCa cells with different levels of RelB to examine the effect of tumourous RelB on T cell immunity. Male mice were injected with murine PCa cells to validate the effect of RelB on the PD-1/PD-L1-mediated immune checkpoint using both tumour growth and metastatic experimental models. RESULTS: PD-L1 is uniquely expressed at a high level in PCa with high constitutive RelB and correlates with the patients' Gleason scores. Indeed, a high level of PD-L1 is associated with RelB nuclear translocation in AR-negative aggressive PCa cells. Conversely, the silencing of RelB in advanced PCa cells resulted in reduced PD-L1 expression and enhanced susceptibility of PCa cells to the T cell immune response in vitro and in vivo. Mechanistically, a proximal NF-κB enhancer element was identified in the core promoter region of the human CD274 gene, which is responsible for RelB-mediated PD-L1 transcriptional activation. This finding provides an informative insight into immune checkpoint blockade by administering RelB within the tumour microenvironment. CONCLUSION: This study deciphers the molecular mechanism by which tumourous RelB contributes to immune evasion by inhibiting T cell immunity via the amplification of the PD-L1/PD-1-mediated immune checkpoint.

Neuron navigator 3 (NAV3) is required for heart development in zebrafish.

As a tightly controlled biological process, cardiogenesis requires the specification and migration of a suite of cell types to form a particular three-dimensional configuration of the heart. Many genetic factors are involved in the formation and maturation of the heart, and any genetic mutations may result in severe cardiac failures. The neuron navigator (NAV) family consists of three vertebrate homologs (NAV1, NAV2, and NAV3) of the neural guidance molecule uncoordinated-53 (UNC-53) in Caenorhabditis elegans. Although they are recognized as neural regulators, their expressions are also detected in many organs, including the heart, kidney, and liver. However, the functions of NAVs, regardless of neural guidance, remain largely unexplored. In our study, we found that nav3 gene was expressed in the cardiac region of zebrafish embryos from 24 to 48 h post-fertilization (hpf) by means of in situ hybridization (ISH) assay. A CRISPR/Cas9-based genome editing method was utilized to delete the nav3 gene in zebrafish and loss of function of Nav3 resulted in a severe deficiency in its cardiac morphology and structure. The similar phenotypic defects of the knockout mutants could recur by nav3 morpholino injection and be rescued by nav3 mRNA injection. Dual-color fluorescence imaging of ventricle and atrium markers further confirmed the disruption of the heart development in nav3-deleted mutants. Although the heart rate was not affected by the deletion of nav3, the heartbeat intensity was decreased in the mutants. All these findings indicate that Nav3 was required for cardiogenesis in developing zebrafish embryos.

Claudin h Is Essential for Hair Cell Morphogenesis and Auditory Function in Zebrafish.

Hereditary hearing loss caused by defective hair cells is one of the most common congenital diseases, whose nosogenesis is still unclear because many of the causative genes remain unidentified. Claudins are one kind of transmembrane proteins that constitute the most important components of the tight junctions and paracellular barrier and play important roles in neurodevelopment. In this study, we investigated the function of claudin h in morphogenesis and auditory function of the hair cell in zebrafish. The results of in situ hybridization showed that claudin h was specifically localized in the otic vesicle and neuromasts in zebrafish embryos. The deficiency of claudin h caused significant reduction of otic vesicle size and loss of utricle otolith. Moreover, the startle response and vestibulo-ocular reflex experiments revealed that loss of claudin h led to serious hearing loss and vestibular dysfunction. Importantly, the confocal microscopy observation found that compared to the control zebrafish, the claudin h morphants and mutants displayed significantly reduced the number of cristae hair cells and shortened kinocilia. Besides, the deficiency of claudin h also caused the loss of hair cells in neuromasts which could be rescued by injecting claudin h mRNA into the mutant embryos at one cell stage. Furthermore, the immunohistochemistry experiments demonstrated remarkable apoptosis of hair cells in the neuromasts, which might contribute to the loss of hair cells number. Overall, these data indicated that claudin h is indispensable for the development of hair cells, vestibular function, and hearing ability of zebrafish.

RelB sustains endocrine resistant malignancy: an insight of noncanonical NF-κB pathway into breast Cancer progression.

BACKGROUND: The activation of the NF-κB pathway plays a crucial role in the progression of breast cancer (BCa) and also involved in endocrine therapy resistance. On the contrary to the canonical NF-κB pathway, the effect of the noncanonical NF-κB pathway in BCa progression remains elusive. METHODS: BCa tumor tissues and the corresponding cell lines were examined to determine the correlation between RelB and the aggressiveness of BCa. RelB was manipulated in BCa cells to examine whether RelB promotes cell proliferation and motility by quantitation of apoptosis, cell cycle, migration, and invasion. RNA-Seq was performed to identify the critical RelB-regulated genes involved in BCa metastasis. Particularly, RelB-regulated MMP1 transcription was verified using luciferase reporter and ChIP assay. Subsequently, the effect of RelB on BCa progression was further validated using BCa mice xenograft models. RESULTS: RelB uniquely expresses at a high level in aggressive BCa tissues, particularly in triple-negative breast cancer (TNBC). RelB promotes BCa cell proliferation through increasing G1/S transition and/or decreasing apoptosis by upregulation of Cyclin D1 and Bcl-2. Additionally, RelB enhances cell mobility by activating EMT. Importantly, RelB upregulates bone metastatic protein MMP1 expression through binding to an NF-κB enhancer element located at the 5'-flanking region. Accordingly, in vivo functional validation confirmed that RelB deficiency impairs tumor growth in nude mice and inhibits lung metastasis in SCID mice. Video abstract.

The Requirement of Sox2 for the Spinal Cord Motor Neuron Development of Zebrafish.

Sex-determining region Y box 2 (Sox2), expressed in neural tissues, plays an important role as a transcription factor not only in the pluripotency and proliferation of neuronal cells but also in the opposite function of cell differentiation. Nevertheless, how Sox2 is linked to motor neuron development remains unknown. Here, we showed that Sox2 was localized in the motor neurons of spinal cord by in situ hybridization and cell separation, which acted as a positive regulator of motor neuron development. The deficiency of Sox2 in zebrafish larvae resulted in abnormal PMN development, including truncated but excessively branched CaP axons, loss of MiP, and increase of undifferentiated neuron cells. Importantly, transcriptome analysis showed that Sox2-depleted embryos caused many neurogenesis, axonogenesis, axon guidance, and differentiation-related gene expression changes, which further support the vital function of Sox2 in motor neuron development. Taken together, these data indicate that Sox2 plays a crucial role in the motor neuron development by regulating neuron differentiation and morphology of neuron axons.

Necroptosis mediated by impaired autophagy flux contributes to adverse ventricular remodeling after myocardial infarction.

Loss of functional cardiomyocytes by cell death after myocardial infarction is most critical for the subsequent left ventricular remodeling, cardiac dysfunction and heart failure. Numerous studies have implicated that dysregulation of autophagy might contribute to cardiomyocyte death. However, the underlying mechanisms by which autophagy dysregulation-mediated cell death remains to be elusive. Herein, we showed that,in response to myocardial ischemic damage in vivo and in vitro, autophagy activity was increased quickly but followed by the process of impaired autophagic degradation as evidenced by the sustained higher level of beclin1 until 12 weeks after myocardial infarction, while, increased accumulation of LC3 and p62. The results from both tandem mRFP-GFP-LC3 adenovirus and lysosomal inhibitor chloroquine supported defective autophagy induction by ischemia injury. Importantly, we found that the impaired autophagy flux, induced not only pharmacologically by CQ but also genetically by beclin1 knockdown, upregulated the expression of RIP3 and aggravated OGD-induced necroptotic cardiomyocyte death and cardiac dysfunction. While, upregulation of autophagy by cardiac-specific beclin1 overexpression partially ameliorated cardiac dysfunction after MI. Furthermore, constitutive activation of necroptosis by forced cardiac-specific overexpression of RIP3 aggravated necrotic cardiomyocyte death, post-MI cardiac remodeling and cardiac dysfunction, but all of which could be ameliorated by inhibition of necroptosis by RIP3 knockdown. In conclusion, these results suggested that autophagy dysfunction-mediated necroptosis mechanistically contributed to loss of cardiomyocytes, adverse ventricular remodeling and progressive heart failure after myocardial Infarction. Inhibition of necroptosis might be the potential target for preventing post-infarction cardiac remodeling and heart failure.

Expression analysis of Rab11 during zebrafish embryonic development.

BACKGROUND: Rab proteins are GTPases responsible for intracellular vesicular trafficking regulation. Rab11 proteins, members of the Rab GTPase family, are known to regulate vesicular recycling during embryonic development. In zebrafish, there are 3 rab11 paralogues, known as rab11a, rab11ba and rab11bb, sharing high identity with each other. However, the expression analysis of rab11 is so far lacking. RESULTS: Here, by phylogeny analysis, we found the three rab11 genes are highly conserved especially for their GTPase domains. We examined the expression patterns of rab11a, rab11ba and rab11bb using RT-PCR and in situ hybridization. We found that all the three genes were highly enriched in the central nervous system, but in different areas of the brain. Apart from brain, rab11a was also expressed in caudal vein, pronephric duct, proctodeum, pharyngeal arches and digestive duct, rab11ba was detected to express in muscle, and rab11bb was expressed in kidney, fin and spinal cord. Different from rab11a and rab11ba, which both have maternal expressions in embryos, rab11bb only expresses during 24hpf to 96hpf. CONCLUSIONS: Our results suggest that rab11 genes play important but distinct roles in the development of the nervous system in zebrafish. The findings could provide new evidences for better understanding the functions of rab11 in the development of zebrafish embryos.

A cupin domain is involved in α-amylase inhibitory activity.

Proteinaceous α-amylase inhibitors have specialized activities that make some strong inhibition of α-amylases. New α-amylase inhibitors continue to be discovered so far. A proteinaceous α-amylase inhibitor CL-AI was isolated and identified from chickpea seeds. CL-AI, encoded by Q9SMJ4, was a storage legumin precursor containing one α-chain and one ß-chain, and each chain possessed a same conserved cupin domain. Amino acid mutation and deficiency of cupin domain would lead to loss of α-amylase inhibitory activity, indicating that it was essential for inhibitory activity. CL-AI(α + ß) in its single stranded state in vivo had inhibitory activity. After it was processed into one α-chain and one ß-chain, the two chains were connected to each other via disulfide bond, which would cover the cupin domains and lead to the loss of inhibitory activity. The CL-AI(α + ß), α-chain and ß-chain could inhibit various α-amylases and delay the seed germination of wheat, rice and maize as well as the growth and development of potato beetle larva. Two cupin proteins, Glycinin G1 in soybean and Glutelinin in rice were also found to have inhibitory activity. Our results indicated that the cupin domain is involved in α-amylase inhibitory activity and the proteins with a cupin domain may be a new kind of proteinaceous α-amylase inhibitor.

Isolation, Identification and Characterization of a New Type of Lectin with α-Amylase Inhibitory Activity in Chickpea (Cicer arietinum L.).

BACKGROUND: Plant lectins are a group of highly diverse proteins that possess at least one non-catalytic domain that binds reversibly to a specific mono- or oligosaccharide. So far, only seven members in the lectin-arcelin-αAI1 supergene family in legume lectins have been reported to have inhibitory activity of α-amylases. OBJECTIVE & METHODS: A proteinaceous α-amylase inhibitor was isolated and purified using Ammonium sulfate precipitation (ASP), Ion exchange chromatography (IEC) and Reversed phase liquid chromatography (RPLC) from the mature seeds of chickpea. RESULTS & CONCLUSION: Identification by Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS-MS) indicated that the purified proteinaceous α-amylase inhibitor was a chickpea lectin CAL in GenBank (accession No. AGL46982.1). CAL had 227 aa containing a hemopexin- like repeats domain and was a cytoplasm protein. It had very low (<17%) identity with seven members in the lectin-arcelin-aAI1 supergene family in legumes that have α-amylase inhibitory activity. The purified CAL derived from prokaryotic expression was confirmed to have inhibitory activity against various α-amylases. The inhibitory activity of CAL against various α-amylases was severely affected by temperature, pH, incubation time, substrate concentration and CAL protein concentration. Feeding CAL reduced the weight of potato beetle larvae by 27.21% (P<0.05) and survival rate by 6.67% (P>0.05). Our results indicated that CAL is a new type of lectin with inhibitory activity against α-amylases in legume lectins, which can be used as a candidate in genetic engineering for breeding for pest resistance.