HSF1 protects cells from cadmium toxicity by governing proteome integrity.

BACKGROUND: Cadmium toxicity has been associated with disruption of protein homeostasis by interfering with protein folding processes. Heat shock factor 1 (HSF1) coordinates the rapid and extensive cellular response to maintain proteomic balance facing the challenges from many environmental stressors. Thus, we suspect that HSF1 may shield cells from cadmium toxicity by conserving proteome integrity. RESULTS: Here, we demonstrate that cadmium, a highly poisonous metal, induces aggregation of cytosolic proteins in human cells, which disrupts protein homeostasis and activates HSF1. Cadmium exposure increases HSF1's phosphorylation, nuclear translocation and DNA bindings. Aside from this, HSF1 goes through liquid-liquid phase separation to form small nuclear condensates upon cadmium exposure. A specific regulatory domain of HSF1 is critical for HSF1's phase separation capability. Most importantly, human cells with impaired HSF1 are sensitized to cadmium, however, cells with overexpressed HSF1 are protected from cadmium toxicity. Overexpression of HSF1 in human cells reduces protein aggregates, amyloid fibrils and DNA damages to antagonize cadmium toxicity. CONCLUSIONS: HSF1 protects cells from cadmium toxicity by governing the integrity of both proteome and genome. Similar mechanisms may enable HSF1 to alleviate cellular toxicity caused by other heavy metals. HSF1's role in cadmium exposure may provide important insights into the toxic effects of heavy metals on human cells and body organs, allowing us to better manage heavy metal poisoning.

TIP30 overcomes gefitinib resistance by regulating cytoplasmic and nuclear EGFR signaling in non-small-cell lung cancer.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) (eg, gefitinib) exert potent therapeutic efficacy in non-small-cell lung cancer (NSCLC) harboring EGFR-activating mutations. However, the resistance to EGFR TKIs limits their clinical therapeutic efficacy. TIP30, a newly identified tumor suppressor, appears to be involved in the regulation of cytoplasmic and nuclear EGFR signaling in NSCLC. Our previous study demonstrated that TIP30 regulated EGF-dependent cyclin D1 transcription in human lung adenocarcinoma and suppressed tumorigenesis. In the present study, the involvement of TIP30 in combating gefitinib resistance in NSCLC was determined for the first time in vitro and in vivo. Gain and loss of function studies showed that overexpression of TIP30 effectively sensitized cells to gefitinib in vitro, whereas TIP30 inhibition promoted gefitinib cell resistance. Moreover, TIP30 negatively regulated the activation of the p-AKT and p-MEK signaling pathways in PC9/GR. Importantly, PC9/GR harbored high levels of nuclear EGFR, and overexpression of TIP30 restored irregular EGFR trafficking and degradation from early endosomes to the late endosomes, decreasing the nuclear accumulation of EGFR, which may partly or totally inhibit EGFR-mediated induction of c-Myc transcription. Xenographic tumors induced by overexpression of TIP30 by PC9/GR cells in nude mice were suppressed compared with their original counterparts. Overall, it was revealed that TIP30 overexpression restored gefitinib sensitivity in NSCLC cells and attenuated the cytoplasmic and nuclear EGFR signaling pathways and may be a promising biomarker in gefitinib resistance in NSCLC.

Defective sarcomere assembly in smyd1a and smyd1b zebrafish mutants.

Two smyd1 paralogues, smyd1a and smyd1b, have been identified in zebrafish. Although Smyd1b function has been reported in fast muscle, its function in slow muscle and the function of Smyd1a, in general, are uncertain. In this study, we generated 2 smyd1a mutant alleles and analyzed the muscle defects in smyd1a and smyd1b single and double mutants in zebrafish. We demonstrated that knockout of smyd1a alone had no visible effect on muscle development and fish survival. This was in contrast to the smyd1b mutant, which exhibited skeletal and cardiac muscle defects, leading to early embryonic lethality. The smyd1a and smyd1b double mutants, however, showed a stronger muscle defect compared with smyd1a or smyd1b mutation alone, namely, the complete disruption of sarcomere organization in slow and fast muscles. Immunostaining revealed that smyd1a; smyd1b double mutations had no effect on myosin gene expression but resulted in a dramatic reduction of myosin protein levels in muscle cells of zebrafish embryos. This was accompanied by the up-regulation of hsp40 and hsp90-α1 gene expression. Together, our studies indicate that both Smyd1a and Smyd1b partake in slow and fast muscle development although Smyd1b plays a dominant role compared with Smyd1a.-Cai, M., Han, L., Liu, L., He, F., Chu, W., Zhang, J., Tian, Z., Du, S. Defective sarcomere assembly in smyd1a and smyd1b zebrafish mutants.

Metabolic switch in energy metabolism mediates the sublethal effects induced by glyphosate-based herbicide on tadpoles of a farmland frog Microhyla fissipes.

Glyphosate-based herbicides (GBHs) are widely-used agricultural chemicals, bringing potential detriments to aquatic organisms. Currently, our understanding of sublethal effects and underlying toxicologic mechanisms of GBHs are still limited, especially in amphibians. Here, the sublethal effects of a commercial GBH (KISSUN®) on tadpoles of a farmland dwelling frog, Microhyla fissipes, were investigated. The 10-d LC50 of "KISSUN®" GBH was 77.5 mg/L. Tadpoles exposed to 60-120 mg/L showed increased preference to higher temperature. After 10 days exposure, obvious growth suppression was observed in survived GBH-stressed tadpoles, characterized by dosage depended decrement in body mass, body width, total length, etc. GBH-stressed tadpoles also showed decreased tail length/snout-vent length ratio and smaller tail muscle fiber diameter. Comparative transcriptomics (control, 60 mg/L and 90 mg/L groups) was conducted to analyze the underlying molecular processes. GBH-stressed tadpoles showed downregulated transcription of ribosomal proteins and cytoskeleton proteins, which could explain their suppressed whole body and tail muscle growth. Moreover, GBH-stressed tadpoles showed transcriptional downregulation of carbohydrate and lipid catabolism, but upregulation of amino acid catabolism. It suggested a metabolic switch from carbohydrate and lipid to amino acid in these tadpoles. Accordingly, there was a trade-off between protein synthesis and energy production in respect to amino acid allocation, and it provided a metabolic explanation for why protein synthesis was downregulated and growth was suppressed in GBH-stressed tadpoles. In combination with existing literatures, we speculated that GBH might directly target the enzymes in carbohydrate and lipid catabolism, and this metabolic effect of GBH might be common to fish and amphibians. In conclusion, our study provided a systematic insight into the sublethal symptoms of GBH-stressed tadpoles, and a metabolic switch from carbohydrate and lipid to amino acid likely underlay some common toxic symptoms of GBHs on both fish and tadpoles.

Identification and differential regulation of microRNAs during thyroid hormone-dependent metamorphosis in Microhyla fissipes.

BACKGROUND: Anuran metamorphosis, which is obligatorily initiated and sustained by thyroid hormone (TH), is a dramatic example of extensive morphological, biochemical and cellular changes occurring during post-embryonic development. Thus, it provides an ideal model to understand the actions of the hormone and molecular mechanisms underlying these developmental and apoptotic processes. In addition to transcriptional factors, microRNAs (miRNAs) play key roles in diverse biological processes via post-transcriptional repression of mRNAs. However, the possible role of miRNAs in anuran metamorphosis is not well understood. Screening and identification of TH-responding miRNAs are required to reveal the integrated regulatory mechanisms of TH during metamorphosis. Given the specific role of TRs during M. fissipes metamorphosis and the characteristics of M. fissipes as an ideal model, Illumina sequencing technology was employed to get a full scope of miRNA in M. fissipes metamorphosis treated by T3. RESULTS: Morphological and histological analysis revealed that 24 h T3 treatment M. fissipes tadpoles resembled that at the climax of natural metamorphosis. Thus, small RNA libraries were constructed from control and 24 h T3 treatment groups. A total of 164 conserved miRNAs and 36 predicted novel miRNAs were characterized. Furthermore, 5' first and ninth nucleotides of miRNAs were significantly enriched in U in our study. In all, 21 miRNAs were differentially expressed between the T3 and control groups (p < 0.01). A total of 10,206 unigenes were identified as target genes of these differentially expressed miRNAs. KEGG pathway analysis indicated that the most overrepresented miRNA target genes were enriched in the "PI3k-Akt signaling pathway". In addition, a network associated with the TH signaling pathway provides an opportunity to further understand the complex biological processes that occur in metamorphosis. CONCLUSIONS: We identified a large number of miRNAs during M. fissipes metamorphosis, and 21 of them were differentially expressed in the two groups that represented two different metamorphic stages. These miRNAs may play important roles during metamorphosis. The study gives us clues for further studies of the mechanisms of anuran metamorphosis and provides a model to study the mechanism of TH-affected biological processes in humans.

Transcriptomics reveals the molecular processes of light-induced rapid darkening of the non-obligate cave dweller Oreolalax rhodostigmatus (Megophryidae, Anura) and their genetic basis of pigmentation strategy.

BACKGROUND: Vertebrates use different pigmentation strategies to adapt to various environments. A large amount of research has been done on disclosing the mechanisms of pigmentation strategies in vertebrates either under light, or, living in constant darkness. However, less attention has been paid to non-obligate, darkness dwellers. Red-spotted toothed toads Oreolalax rhodostigmatus (Megophryidae; Anura) from the karst mountainous region of southwestern China are non-obligate cave dwellers. Most tadpoles of the species possess transparent skin as they inhabit the dark karst caves. But remarkably, the transparent tadpoles can darken just within 15 h once exposed to light. Obviously, it is very significant to reveal molecular mechanisms of the unexpected rapid-darkening phenomenon. RESULTS: We compared the transcriptomes of O. rhodostigmatus tadpoles with different durations of light exposure to investigate the cellular processes and potential regulation signals for their light-induced rapid darkening. Genes involved in melanogenesis (i.e. TYR, TYRP1 and DCT) and melanocyte proliferation, as well as their transcriptional factor (MITF), showed light-induced transcription, suggesting a dominating role of morphological color change (MCC) in this process. Transcription of genes related to growth factor, MAPK and PI3K-Akt pathways increased with time of light exposure, suggesting that light could induce significant growth signal, which might facilitate the rapid skin darkening. Most importantly, an in-frame deletion of four residues was identified in O. rhodostigmatus melanocortin-1 receptor (MC1R), a critical receptor in MCC. This deletion results in a more negatively charged ligand pocket with three stereo-tandem aspartate residues. Such structural changes likely decrease the constitutive activity of MC1R, but increase its ligands-dependent activity, thus coordinating pigment regression and rapid melanogenesis in the dark and light, respectively. CONCLUSION: Our study suggested that rapid MCC was responsible for the light-induced rapid darkening of O. rhodostigmatus tadpoles. Genetic mutations of MC1R in them could explain how these non-obligate cave dwellers coordinate pigment regression and robust melanogenesis in darkness and light, respectively. To our knowledge, this is the first study that reports the association between pigmentation phenotype adaptation and MC1R mutations in amphibians and/or in non-obligate cave dwellers.

Exploring the causal associations between diet-derived circulating antioxidants and the risk of endometriosis: a Mendelian randomization study.

Background: Numerous observational studies and randomized controlled trials have recently revealed the associations between circulating antioxidants and the risk of endometriosis, while the underlying causal relationship remains unclear. This study aimed to investigate the causal association between genetically determined circulating antioxidants and the risk of endometriosis using Mendelian randomization (MR). Methods: A two-sample MR analysis was conducted using publicly available summary data from genome-wide association studies (GWAS) to investigate the causal impact of genetically determined absolute circulating antioxidants (such as ascorbate, retinol, ß-carotene, and lycopene) and their metabolites (including α-and γ-tocopherol, ascorbate, and retinol) on the risk of endometriosis. The study used inverse variance weighted (IVW) or Wald ratio analyses as the primary estimation method and also conducted sensitivity analyses to assess heterogeneity and pleiotropy. Results: No significant causality was observed for genetically determined circulating antioxidants and the risk of endometriosis. The pooled odds ratios (ORs) for absolute circulating antioxidants were 0.62 (95% CI: 0.32-1.18, retinol), 0.95 (95% CI: 0.79-1.15, ß-carotene), 1.01 (95% CI: 0.95-1.08, lycopene), and 1.00 (95% CI: 0.99-1.02, ascorbate, expressed as a Wald ratio). The pooled ORs indicating the EM risk per unit increase in circulating antioxidant metabolites were 1.04 (95% CI: 0.82-1.33, γ-tocopherol), 0.91 (95% CI: 0.57-1.46, α-tocopherol), 1.03 (95% CI: 0.99-1.07, retinol), and 0.96 (95% CI: 0.87-1.06, ascorbate). Conclusion: Our study demonstrated that increased levels of diet-derived circulating antioxidants were not significantly associated with a reduced risk of endometriosis.

Simplifying Genotyping of Mutants from Genome Editing with a Parallel qPCR-Based iGenotype Index.

Targeted genome editing is a powerful tool in reverse genetic studies of gene function in many aspects of biological and pathological processes. The CRISPR/Cas system or engineered endonucleases such as ZFNs and TALENs are the most widely used genome editing tools that are introduced into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, triggering cellular DNA repair through either homologous recombination or non-homologous end joining (NHEJ). DNA repair through the NHEJ mechanism is usually error-prone, leading to point mutations or indels (insertions and deletions) within the targeted region. Some of the mutations in embryos are germline transmissible, thus providing an effective way to generate model organisms with targeted gene mutations. However, point mutations and short indels are difficult to be effectively genotyped, often requiring time-consuming and costly DNA sequencing to obtain reliable results. Here, we developed a parallel qPCR assay in combination with an iGenotype index to allow simple and reliable genotyping. The genotype-associated iGenotype indexes converged to three simple genotype-specific constant values (1, 0, -1) regardless of allele-specific primers used in the parallel qPCR assays or gene mutations at wide ranges of PCR template concentrations, thus resulting in clear genotype-specific cutoffs, established through statistical analysis, for genotype identification. While we established such a genotyping assay in the Xenopus tropicalis model, the approach should be applicable to genotyping of any organism or cells and can be potentially used for large-scale, automated genotyping.

Muscle Fiber Characteristics and Transcriptome Analysis in Slow- and Fast-Growing Megalobrama amblycephala.

Growth is an important trait in aquaculture that is influenced by various factors, among which genetic regulation plays a crucial role. Megalobrama amblycephala, one of the most important freshwater species in China, exhibits wide variations in body mass among individuals of the same age within the same pool. But the molecular mechanisms underlying wide variation in body mass remain unclear. Here, we performed muscle histological and transcriptome analysis of muscle tissues from Fast-Growing (FG) and Slow-Growing (SG) M. amblycephala at the age of 4 months old (4 mo) and 10 months old (10 mo) to elucidate its muscle development and growth mechanism. The muscle histological analysis showed smaller diameter and higher total number of muscle fibers in FG compared to SG at 4 mo, while larger diameter and total number of muscle fibers were detected in FG at 10 mo. The transcriptome analysis of muscle tissue detected 1171 differentially expressed genes (DEGs) between FG and SG at 4 mo, and 718 DEGs between FG and SG at 10 mo. Furthermore, 44 DEGs were consistently up-regulated in FG at both 4 mo and 10 mo. Up-regulated DEGs in FG at 4 mo were mainly enriched in the pathways related to cell proliferation, while down-regulated DEGs were significantly enriched in cell fusion and muscle contraction. Up-regulated DEGs in FG at 10 mo were mainly enriched in the pathways related to cell proliferation and protein synthesis. Therefore, these results provide novel insights into the molecular mechanism of M. amblycephala muscle growth at different stages, and will be of great guiding significance to promote the fast growth of M. amblycephala.

The Regulatory Role of Myomaker in the Muscle Growth of the Chinese Perch (Siniperca chuatsi).

The fusion of myoblasts is a crucial stage in the growth and development of skeletal muscle. Myomaker is an important myoblast fusion factor that plays a crucial role in regulating myoblast fusion. However, the function of Myomaker in economic fish during posthatching has been poorly studied. In this study, we found that the expression of Myomaker in the fast muscle of Chinese perch (Siniperca chuatsi) was higher than that in other tissues. To determine the function of Myomaker in fast muscle, Myomaker-siRNA was used to knockdown Myomaker in Chinese perch and the effect on muscle growth was determined. The results showed that the growth of Chinese perch was significantly decreased in the Myomaker-siRNA group. Furthermore, both the diameter of muscle fibers and the number of nuclei in single muscle fibers were significantly reduced in the Myomaker-siRNA group, whereas there was no significant difference in the number of BrdU-positive cells (proliferating cells) between the control and the Myomaker-siRNA groups. Together, these findings indicate that Myomaker may regulate growth of fast muscle in Chinese perch juveniles by promoting myoblast fusion rather than proliferation.

HDL-cholesterol confers sensitivity of immunotherapy in nasopharyngeal carcinoma via remodeling tumor-associated macrophages towards the M1 phenotype.

BACKGROUND: The sustained effectiveness of anti-programmed cell death protein-1/programmed death-ligand 1 treatment is limited to a subgroup of patients with advanced nasopharyngeal carcinoma (NPC), and the specific biomarker determining the response to immunotherapy in NPC remains uncertain. METHODS: We assessed the associations between pre-immunotherapy and post-immunotherapy serum lipoproteins and survival in a training cohort (N=160) and corroborated these findings in a validation cohort (N=100). Animal studies were performed to explore the underlying mechanisms. Additionally, the relationship between high-density lipoprotein-cholesterol (HDL-C) levels and M1/M2-like macrophages, as well as activated CD8+T cells in tumor tissues from patients with NPC who received immunotherapy, was investigated. RESULTS: The lipoproteins cholesterol, HDL-C, low-density lipoprotein-cholesterol, triglycerides, apolipoprotein A-1 (ApoA1), and apolipoprotein B, were significantly altered after immunotherapy. Patients with higher baseline HDL-C or ApoA1, or those with increased HDL-C or ApoA1 after immunotherapy had longer progression-free survival, a finding verified in the validation cohort (p<0.05). Multivariate analysis revealed that baseline HDL-C and elevated HDL-C post-immunotherapy were independent predictors of superior PFS (p<0.05). Furthermore, we discovered that L-4F, an ApoA1 mimetic, could inhibit tumor growth in NPC xenografts. This effect was associated with L-4F's ability to polarize M2-like macrophages towards an M1-like phenotype via the activation of mitogen-activated protein kinase (MAPK) p38 and nuclear factor-κB (NF-κB) p65, thereby alleviating immunosuppression in the tumor microenvironment. Importantly, in patients with NPC with high plasma HDL-C levels, the number of M2-like macrophages was significantly decreased, while M1-like macrophages and activated CD8+T cells were notably increased in those with high HDL-C levels. CONCLUSION: Higher baseline HDL-C levels or an increase in HDL-C post-immunotherapy can enhance immunotherapeutic responses in patients with NPC by reprogramming M2-like macrophages towards the M1 phenotype. This suggests a potential role for prospectively exploring ApoA1 mimetics as adjuvant agents in combination with immunotherapy.

Synthesis and biological evaluation of a folate-targeted rhaponticin conjugate.

To improve the therapeutic effect of rhaponticin (RHA), a folate receptor (FR) targeted RHA conjugate was synthesized by utilizing a hydrophilic peptide spacer linked to folic acid (FA) via a releasable disulfide linker. This water-soluble conjugate was found to retain high affinity for FR-positive cells, and it produced specific, dose-responsive activity in vitro. Treatment of FRHA with a reducing agent indicated that the amino-reactive derivative of RHA would be released spontaneously following disulfide bond reduction within the endosomes. FRHA also proved to be active predominantly specific against FR-positive syngeneic and xenograft models in vivo, and possible curative activity resulted with minimal to moderate toxicity. The FRHA conjugate greatly enhanced the therapeutic effects and reduced the toxicity of RHA. In conclusion, FRHA represents a folate-targeted chemotherapeutic that can produce potent activity against established sc tumors. Hence, this report has a great significance in pharmacology and clinical medicine as well as methodology.

Identification of thyroid hormone response genes in the remodeling of dorsal muscle during Microhyla fissipes metamorphosis.

Introduction: Extensive morphological, biochemical, and cellular changes occur during anuran metamorphosis, which is triggered by a single hormone, thyroid hormone (TH). The function of TH is mainly mediated through thyroid receptor (TR) by binding to the specific thyroid response elements (TREs) of direct response genes, in turn regulating the downstream genes in the cascade. The remodeling of dorsal skeletal muscle during anuran metamorphosis provides the perfect model to identify the immediate early and direct response genes that are important during apoptosis, proliferation, and differentiation of the muscle. Methods: In our current study, we performed Illumina sequencing combined with single-molecule real-time (SMRT) sequencing in the dorsal muscle of Microhyla fissipes after TH, cycloheximide (CHX), and TH_CHX treatment. Results and Discussion: We first identified 1,245 differentially expressed transcripts (DETs) after TH exposure, many of which were involved in DNA replication, protein processing in the endoplasmic reticulum, cell cycle, apoptosis, p53 signaling pathway, and protein digestion and absorption. In the comparison of the TH group vs. control group and TH_CHX group vs. CHX group overlapping gene, 39 upregulated and 6 downregulated genes were identified as the TH directly induced genes. Further analysis indicated that AGGTCAnnTnAGGTCA is the optimal target sequence of target genes for TR/RXR heterodimers in M. fissipes. Future investigations on the function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian dorsal muscle remodeling. These full-length and high-quality transcriptomes in this study also provide an important foundation for future studies in M. fissipes metamorphosis.

Predictive value of immunotherapy-induced inflammation indexes: dynamic changes in patients with nasopharyngeal carcinoma receiving immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have achieved substantial advancements in clinical care. However, there is no strong evidence for identified biomarkers of ICIs in NPC. METHODS: In this retrospective study, 284 patients were enrolled into a training or validation cohort. Inflammatory indexes based on peripheral blood parameters were evaluated, including the systemic immune-inflammation index (SII), the neutrophil-lymphocyte ratio (NLR), the platelet-lymphocyte ratio (PLR), the lymphocyte-to-C-reactive protein ratio (LCR), and the lymphocyte-monocyte ratio (LMR). The optimum cut-off value for patient stratification was identified using X-tile. The Kaplan-Meier method and Cox's proportional regression analyses were used to identify prognostic factors. RESULTS: Immunotherapy significantly changed the levels of SII, NLR, PLR, LCR and LMR in NPC patients. Patients with lower SII, NLR, and PLR, as well as those with higher LCR and LMR, before immunotherapy had superior PFS (all p < 0.05). Moreover, PFS in the decreased SII, reduced NLR and increased LMR group was significantly longer than in the opposite group (all p < 0.05). Both univariate and multivariate analyses validated that baseline SII and LMR, and the immunotherapy-related SII reduction and LMR elevation were independent prognostic factors for PFS in advanced NPC patients receiving ICIs. CONCLUSIONS: Immune checkpoint inhibitor treatments significantly changed the levels of SII, NLR, PLR, LCR and LMR in NPC patients treated with immunotherapy. A lower baseline SII and a higher baseline LMR, and a reduction in SII and an elevation in LMR after immunotherapy are favorable factors for predicting survival among advanced NPC patients.

There is no strong evidence for identified biomarkers of immune checkpoint inhibitors (ICIs) in nasopharyngeal carcinoma (NPC).Lower baseline SII and higher baseline LMR were related to better PFS. The dynamic changes of SII and LMR were independent prognostic factors for the survival of NPC patients receiving ICIs.Neutrophils, platelets, lymphocytes, and monocytes can be used as cheap and valuable biomarkers for predicting tumor response in NPC on immunotherapy.

Molecular characterization of myostatin (MSTN) gene and association analysis with growth traits in the bighead carp (Aristichthys nobilis).

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily and functions as a negative regulator of skeletal muscle development and growth. In this study, the bighead carp MSTN gene (AnMSTN for short) was cloned and characterized. The 3,769 bp genomic sequence of AnMSTN consisted of three exons and two introns, and the full length cDNA (2,141 bp) of the gene had an open reading frame encoding a polypeptide of 375 amino acids. The deduced amino acid sequence of AnMSTN showed 67.1-98.7 % homology with MSTNs of avian, mammalian and teleostean species. Sequence comparison and phylogenetic analysis confirmed the MSTNs were conserved throughout the vertebrates and AnMSTN belonged to MSNT-1 isoform. AnMSTN was expressed in various tissues with the highest expression in muscle. Two single nucleotide polymorphisms, g.1668T > C in intron 2 and g.2770C > A in 3' UTR, were identified in AnMSTN by sequencing PCR fragments, and genotyped by SSCP. Association analysis showed that g.2770C > A genotypes were significantly associated with total length, body length and body weight (P < 0.01). These results suggest that AnMSTN involves in the regulation of growth, and this polymorphism would be informative for further studies on selective breeding in bighead carp.

Synthesis and spectroscopic characterization of 4-butoxyethoxy-N-octadecyl-1,8-naphthalimide as a new fluorescent probe for the determination of proteins.

A novel 4-butoxyethoxy-N-octadecyl-1,8-naphthalimide (BON) was synthesized as a fluorescent probe for the determination of proteins. The interactions between BON and bovine serum albumin (BSA) were studied by fluorescence spectroscopy and UV-vis absorption spectroscopy. Fluorescence data revealed that the fluorescence quenching of BSA by BON was likely the result of the formation of the BON-BSA complex. According to the modified Stern-Volmer equation, the binding constants of BON with BSA at four different temperatures were obtained. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated to be -23.27 kJ mol(-1) and 10.40 J mol(-1)K(-1) according to van't Hoff equation, indicating that the hydrogen bonds and hydrophobic interactions played a dominant role in the binding of BON to BSA. Furthermore, displacement experiments using warfarin indicated that BON could bind to site I of BSA. The effect of BON on the conformation of BSA was also analyzed by synchronous fluorescence and three-dimensional fluorescence spectra. A new fluorescence quenching assay of the proteins BSA using BON in the HCl-Tris (pH 7.4) buffer solution was developed with maximum excitation and emission wavelengths of 373 and 489 nm, respectively. The linear range was 0.1-10.0×10(-5) mol L(-1) with detection limits were determined to be 1.76×10(-8) mol L(-1). The effect of metal cations on the fluorescence spectra of BON in ethanol was also investigated. Determination of protein in human serum by this method gave results which were very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry.

Transcriptome profiling reveals gene regulation programs underlying tail development in the Ornamented Pygmy frog Microhyla fissipes.

Introduction: Tadpole tail develops from the tailbud, an apparently homogenous mass of cells at the posterior of the embryo. While much progress has been made in understanding the origin and the induction of the tailbud, the subsequent outgrowth and differentiation have received much less attention, particularly with regard to global gene expression changes. Methods: By using RNA-seq with SMRT and further analyses, we report the transcriptome profiles at four key stages of tail development, from a small tailbud to the onset of feeding (S18, S19, S21 and S28) in Microhyla fissipes, an anuran with a number of advantages for developmental and genetic studies. Results: We obtained 48,826 transcripts and discovered 8807 differentially expressed transcripts (DETs, q < 0.05) among these four developmental stages. We functionally classified these DETs by using GO and KEGG analyses and revealed 110 significantly enriched GO categories and 6 highly enriched KEGG pathways (Protein digestion and absorption; ECM-receptor interaction; Pyruvate metabolism; Fatty acid degradation; Valine, leucine and isoleucine degradation; and Glyoxylate and dicarboxylate metabolism) that are likely critically involved in developmental changes in the tail. In addition, analyses of DETs between any two individual stages demonstrated the involvement of distinct biological pathways/GO terms at different stages of tail development. Furthermore, the most dramatic changes in gene expression profile are those between S28 and any of the other three stages. The upregulated DETs at S28 are highly enriched in "myosin complex" and "potassium channel activity", which are important for muscle contraction, a critical function of the tail that the animal needs by the end of embryogenesis. Additionally, many DETs and enriched pathways discovered here during tail development, such as HDAC1, Hes1 and Hippo signaling pathway, have also been reported to be vital for the tissue/organ regeneration, suggesting conserved functions between development and regeneration. Conclusion: The present staudy provides a golbal overview of gene expression patterns and new insights into the mechanism involved in anuran tail development and regeneration.

The Changes in the Frog Gut Microbiome and Its Putative Oxygen-Related Phenotypes Accompanying the Development of Gastrointestinal Complexity and Dietary Shift.

There are many examples of symbiotic and reciprocal relationships in ecological systems; animal gut microbiome-host interactions are one such kind of bidirectional and complex relationship. Here, we utilized several approaches (16S rRNA gene sequencing, metagenomics, and transcriptomics) to explore potential gut microbiome-host interactions accompanying the development of gastrointestinal complexity and a dietary shift from metamorphosis to maturity in ornamented pygmy frogs (Microhyla fissipes). We identified the possible coevolution between a particular gut microbial group (increased putative fat-digesting Erysipelotrichaceae and chitin-digesting Bacteroides and Ruminococcaceae) and the host dietary shift [from herbivore to insectivore (high proportion of dietary chitin and fat)] during metamorphosis. We also found that the remodeling and complexity of the gastrointestinal system during metamorphosis might have a profound effect on the gut microbial community (decreasing facultative anaerobic Proteobacteria and increasing anaerobic Firmicutes) and its putative oxygen-related phenotypes. Moreover, a high proportion of chitin-digesting bacteria and increased carbohydrate metabolism by gut microbiomes at the climax of metamorphosis would help the frog's nutrition and energy needs during metamorphosis and development. Considering the increased expression of particular host genes (e.g., chitinase) in juvenile frogs, we speculate that host plays an important role in amphibian metamorphosis, and their symbiotic gut microbiome may help in this process by providing the nutrition and energy needs. We provide this basic information for the amphibian conservation and managements.

MDM2 inhibitor RG7388 potently inhibits tumors by activating p53 pathway in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a high-risk head and neck cancer with poor clinical outcomes and insufficient treatments. The mouse double minute 2 homolog (MDM2) is the main molecular target in the clinical treatment of cancer. Indeed, MDM2 negatively regulates p53 through ubiquitin-dependent degradation. Thus, inhibition of MDM2-p53 interaction is a potential strategy for treating NPC. The latest generation MDM2 inhibitor, RG7388, shows increased potency and improved bioavailability compared to previous treatments. In this study, we investigated the efficacy and specificity of this inhibitor in NPC cell lines, and tumor-bearing mice were used to examine the therapeutic efficacy and effects of RG7388 treatment. The results showed that RG7388 potently decreased cell proliferation and activated p53-dependent pathway, resulting in cell cycle arrest and apoptosis. RG7388 significantly inhibited tumors in tumor-bearing mice. Activation of the p53 pathway-inhibited cell proliferation, as observed by detecting Ki67-positive cells. Additionally, the activity of apoptotic caspase family proteins was induced in the cleaved caspase-3-positive cells in vivo. Our results demonstrate that the MDM2 small-molecule inhibitor RG7388 is effective for NPC tumors, supporting further clinical investigation as a potential therapy for NPC.

Gene Expression Program Underlying Tail Resorption During Thyroid Hormone-Dependent Metamorphosis of the Ornamented Pygmy Frog Microhyla fissipes.

Thyroid hormone (T3) is essential for vertebrate development, especially during the so-called postembryonic development, a period around birth in mammals when plasma T3 level peaks and many organs mature into their adult form. Compared to embryogenesis, postembryonic development is poorly studied in mammals largely because of the difficulty to manipulate the uterus-enclosed embryos and neonates. Amphibian metamorphosis is independent of maternal influence and can be easily manipulated for molecular and genetic studies, making it a valuable model to study postembryonic development in vertebrates. Studies on amphibian metamorphosis have been largely focused on the two highly related species Xenopus laevis and Xenopus tropicalis. However, adult X. laevis and X. tropicalis animals remain aquatic. This makes important to study metamorphosis in a species in which postmetamorphic frogs live on land. In this regard, the anuran Microhyla fissipes represents an alternative model for developmental and genetic studies. Here we have made use of the advances in sequencing technologies to investigate the gene expression profiles underlying the tail resorption program during metamorphosis in M. fissipes. We first used single molecule real-time sequencing to obtain 67, 939 expressed transcripts in M. fissipes. We next identified 4,555 differentially expressed transcripts during tail resorption by using Illumina sequencing on RNA samples from tails at different metamorphic stages. Bioinformatics analyses revealed that 11 up-regulated KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and 88 Gene Ontology (GO) terms as well as 21 down-regulated KEGG pathways and 499 GO terms were associated with tail resorption. Our findings suggest that tail resorption in M. fissipes and X. laevis shares many programs. Future investigations on function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian tail resorption and adaptive evolution from aquatic to terrestrial life. Furthermore, analysis of the M. fissipes model, especially, on the changes in other organs associated with the transition from aquatic to terrestrial living, should help to reveal important mechanistic insights governing mammalian postembryonic developments.