Identification and functional analysis of Dmrt1 gene and the SoxE gene in the sexual development of sea cucumber, Apostichopus japonicus.

Members of the Doublesex and Mab-3-related transcription factor (Dmrt) gene family handle various vital functions in several biological processes, including sex determination/differentiation and gonad development. Dmrt1 and Sox9 (SoxE in invertebrates) exhibit a very conserved interaction function during testis formation in vertebrates. However, the dynamic expression pattern and functional roles of the Dmrt gene family and SoxE have not yet been identified in any echinoderm species. Herein, five members of the Dmrt gene family (Dmrt1, 2, 3a, 3b and 5) and the ancestor SoxE gene were identified from the genome of Apostichopus japonicus. Expression studies of Dmrt family genes and SoxE in different tissues of adult males and females revealed different expression patterns of each gene. Transcription of Dmrt2, Dmrt3a and Dmrt3b was higher expressed in the tube feet and coelomocytes instead of in gonadal tissues. The expression of Dmrt1 was found to be sustained throughout spermatogenesis. Knocking-down of Dmrt1 by means of RNA interference (RNAi) led to the downregulation of SoxE and upregulation of the ovarian regulator foxl2 in the testes. This indicates that Dmrt1 may be a positive regulator of SoxE and may play a role in the development of the testes in the sea cucumber. The expression level of SoxE was higher in the ovaries than in the testes, and knocking down of SoxE by RNAi reduced SoxE and Dmrt1 expression but conversely increased the expression of foxl2 in the testes. In summary, this study indicates that Dmrt1 and SoxE are indispensable for testicular differentiation, and SoxE might play a functional role during ovary differentiation in the sea cucumber.

Sexually dimorphic expression of foxl2 in the sea urchin (Mesocentrotus nudus).

Sea urchin (Mesocentrotus nudus) is an important economically mariculture species in several Asian countries, and gonads are the sole edible parts for people. In addition to commercial value, it is an excellent model for studying gonadal development, sex determination and sex differentiation. Identify sex-related genes is an effective way to reveal the molecular mechanism of gonadal development. In the present study, the foxl2 homologous gene was identified in M. nudus. Foxl2 is not a maternal factor, and is detected for the first time in two-arm stages. Additionally, the expression of foxl2 in the testis is higher than in the ovaries at the same developmental stages. The foxl2 transcripts were specifically enriched in the cytoplasm of germ cellsboth in the ovary and testis, but their proteins were more concentrated in the area near the oocyte nucleus. Overall, this study contributes to our understanding of the dynamic and sexually dimorphic expression pattern of foxl2 and provide a useful germ cell marker during gametogenesis in sea urchin.

Current Evidence and Future Directions of Berberine Intervention in Depression.

A major type of serious mood disorder, depression is currently a widespread and easily overlooked psychological illness. With the low side effects of natural products in the treatment of diseases becoming the pursuit of new antidepressants, natural Chinese medicine products have been paid more and more attention for their unique efficacy in improving depression. In a view from the current study, the positive antidepressant effects of berberine are encouraging. There is a lot of work that needs to be done to accurately elucidate the efficacy and mechanism of berberine in depression. In this review, the relevant literature reports on the treatment of depression and anxiety by berberine are updated, and the potential pharmacological mechanism of berberine in relieving depression has also been discussed.

Identification and functional analysis of foxl2 and nodal in sea cucumber, Apostichopus japonicus.

Sea cucumber (Apostichopus japonicus) is an important mariculture species in China. To date, the mechanisms of sex determination and differentiation in sea cucumber remain unclear. Identifying sex-specific molecular markers is an effective method for revealing the genetic basis of sex determination and sex differentiation. In this study, foxl2 and nodal homologous genes were identified in A. japonicus. Foxl2 exhibited dynamic and sexually dimorphic expression patterns in the gonads, with prominent expression in the ovaries and minimal expression in the testis according to real-time quantitative PCR (RT-qPCR) study. As nodal was specifically expressed in the ovary, it could serve as an ovary-specific marker in sea cucumber. Additionally, knockdown of foxl2 or nodal using RNA interference (RNAi) led to the down-regulation of piwi, germ cell-less, and dmrt1, suggesting that foxl2 and nodal may play important roles in gonad maintenance of sea cucumber. Overall, this study adds to our understanding of the roles of foxl2 and nodal in the gonadal development of A. japonicus, which provides further insight into the mechanisms of sea cucumber sex determination and differentiation.

Application of SNP in Genetic Sex Identification and Effect of Estradiol on Gene Expression of Sex-Related Genes in Strongylocentrotus intermedius.

Sea urchin (Strongylocentrotus intermedius) is an economically important mariculture species in Asia, and its gonads are the only edible part. The efficiency of genetic breeding in sea urchins is hampered due to the inability to distinguish gender by appearance. In this study, we first identified a sex-associated single nucleotide polymorphism (SNP) by combining type IIB endonuclease restriction site-associated DNA sequencing (2b-RAD-seq) and genome survey. Importantly, this SNP is located within spata4, a gene specifically expressed in male. Knocking down of spata4 by RNA interference (RNAi) in male individuals led to the downregulation of other conserved testis differentiation-related genes and germ cell marker genes. We also revealed that sex ratio in this validated culture population of S. intermedius is not 1:1. Moreover, after a 58-day feeding experiment with estradiol, the expression levels of several conserved genes that are related to testis differentiation, ovary differentiation, and estrogen metabolism were dynamically changed. Taken together, our results will contribute toward improving breeding efficiency, developing sex-controlled breeding, and providing a solid base for understanding sex determination mechanisms in sea urchins.

The influence of soy protein hydrolysate (SPH) addition to infant formula powder on Streptococcus thermophilus proliferation and metabolism.

Nowadays, more and more infants are getting allergic to cow's milk protein, so it is urgent to search for infant formula powder with milk protein alternatives. In the present work, soy protein hydrolysate (SPH) was added to protein-free infant formula powder and the effects of SPH addition on proliferation and metabolism of Streptococcus thermophilus were studied. Compared with commercially available infant formula powder (CK) and protein-free milk powder (BK), the infant formula powder with 20% SPH significantly enhanced the proliferation of S. thermophilus in MRS medium, resulting in a higher cell density and greater viable counts. Moreover, the influence of SPH on the metabolism of S. thermophilus was investigated by analyzing the content of seven organic acids and H2O2 in the medium. The higher content of organic acids and H2O2 is consistent with the stronger antibacterial activity to Escherichia coli. As a consequence, the addition of SPH to infant formula powder can effectively promote the growth of probiotics and SPH may be a promising protein alternative in the infant formula powder.

Identification and functional characterization of piwi1 gene in sea cucumber, Apostichopus japonicas.

The sea cucumber (Apostichopus japonicus) is an economically important mariculture species in Asia. However, the genetic breeding of sea cucumbers is difficult because the sexes cannot be identified by appearance. Therefore, studies on sex-related genes are helpful in revealing the mechanisms of sex determination and differentiation in sea cucumbers. P-element induced wimpy testis (piwi) is a germ cell marker involved in gametogenesis in vertebrates; however, the expression pattern and function during gametogenesis remain unclear in sea cucumbers. In this study, we identified a piwi homolog gene in A. japonicus (Ajpiwi1) and investigated its expression pattern, and function. Ajpiwi1 is a maternal factor and is ubiquitously expressed in adult tissues, including the ovary and testis. Ajpiwi1 expression is strong in early oocytes, spermatocytes, and spermatogonia; weak in mature oocytes; and undetected in spermatids and intra-gonadal somatic cells. The knockdown of Ajpiwi1 by RNA interference (RNAi) led to the downregulation of other conserved sex-related genes such as dmrt1, foxl2, and germ cell-less. Therefore, Ajpiwi1 might play a critical role during gametogenesis in A. japonicus. This study creates new possibilities for studying sex-related gene functions in the sea cucumber and builds a gene function research platform based on RNAi for the first time.

Design, synthesis and evaluation of 2-amino-imidazol-4-one derivatives as potent ß-site amyloid precursor protein cleaving enzyme 1 (BACE-1) inhibitors.

Inhibition of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) to prevent brain ß-amyloid (Aß) peptide's formation is a potential effective approach to treat Alzheimer's disease. In this report we described a structure-based optimization of a series of BACE1 inhibitors derived from an iminopyrimidinone scaffold W-41 (IC50 = 7.1 µM) by Wyeth, which had good selectivity and brain permeability but low activity. The results showed that occupying the S3 cavity of BACE1 enzyme could be an effective strategy to increase the biological activity, and five compounds exhibited stronger inhibitory activity and higher liposolubility than W-41, with L-5 was the most potent inhibitor against BACE1 (IC50 = 0.12 µM, logP = 2.49).

Modulation of Multiple Signaling Pathways of the Plant-Derived Natural Products in Cancer.

Natural compounds are highly effective anticancer chemotherapeutic agents, and the targets of plant-derived anticancer agents have been widely reported. In this review, we focus on the main signaling pathways of apoptosis, proliferation, invasion, and metastasis that are regulated by polyphenols, alkaloids, saponins, and polysaccharides. Alkaloids primarily affect apoptosis-related pathways, while polysaccharides primarily target pathways related to proliferation, invasion, and metastasis. Other compounds, such as flavonoids and saponins, affect all of these aspects. The association between compound structures and signaling pathways may play a critical role in drug discovery.

Molecular Cloning and Sexually Dimorphic Expression Analysis of nanos2 in the Sea Urchin, Mesocentrotus nudus.

Sea urchin (Mesocentrotus nudus) is an economically important mariculture species in China and the gonads are the solely edible parts to human. The molecular mechanisms of gonad development have attracted increasing attention in recent years. Although the nanos2 gene has been identified as a germ cell marker in several invertebrates, little is known about nanos2 in adult sea urchins. Hereinto, we report the characterization of Mnnano2, an M. nudus nanos2 homology gene. Mnnanos2 is a maternal factor and can be detected continuously during embryogenesis and early ontogeny. Real-time quantitative PCR (RT-qPCR) and section in situ hybridization (ISH) analysis revealed a dynamic and sexually dimorphic expression pattern of Mnnano2 in the gonads. Its expression reached the maximal level at Stage 2 along with the gonad development in both ovary and testis. In the ovary, Mnnanos2 is specifically expressed in germ cells. In contrast, Mnnanos2 is expressed in both nutritive phagocytes (NP) cells and male germ cells in testis. Moreover, knocking down of Mnnanos2 by means of RNA interference (RNAi) reduced nanos2 and boule expression but conversely increased the expression of foxl2. Therefore, our data suggest that Mnnanos2 may serve as a female germ cell marker during gametogenesis and provide chances to uncover its function in adult sea urchin.

Gonadal transcriptomic analysis and identification of candidate sex-related genes in Mesocentrotus nudus.

The edible sea urchin, Mesocentrotus nudus, is both an economically important mariculture species and an excellent model for studying reproductive development. The gonads are the only edible parts of sea urchins and increasing market demand for high-quality gonads has prompted increasing amounts of research into the molecular mechanisms of reproduction. Using a high-throughput sequencing technology, we performed transcriptome sequencing on the gonads of females and males, sampled before the spawning season, to identify genes involved in sex determination, sex differentiation and gametogenesis. Through a de novo transcriptome assembly approach, we obtained 104,039 unigenes, of which 40,471 (38.90%) showed homologies with known proteins in public databases. By comparing the expression levels of these unigenes in females and males, 15,368 differentially-expressed unigenes (DEGs) were identified. Compared with males, 9473 were up-regulated and 5895 were down-regulated in females. Multiple candidate genes were identified that may play important roles in spermatogenesis, oogenesis, and germ cell development. Furthermore, we identified and characterized several genes involved in sex determination and sex differentiation, such as dmrt1 and foxl2. The current study provides valuable molecular resources for studying the underlying mechanisms of reproduction in sea urchins.

Recognition of the toxicity of aristolochic acid.

WHAT IS KNOWN AND OBJECTIVE: Aristolochic acid (AA) is an abundant compound in Aristolochia plants and various natural herbs. In the 1990s, a slimming formula used in Belgium that contains Aristolochia fangchi was reported to cause kidney damage and bladder cancer, and aristolochic acid nephropathy (AAN) is now well recognized worldwide. In October 2017, researchers reported an AA signature that is closely associated with hepatocellular carcinoma (HCC) worldwide. COMMENT: There are differing opinions on the toxicity of AA, and different countries have taken different measures to address the issue. There is a lack of clarity on the causal role of AA in hepatocarcinogenesis and on the potential underlying mechanisms for the reported nephrotoxicity and carcinogenicity. The toxicity of AA differs depending on gender and age, and other risk factors that could explain the variability in the toxicity of AA remain to be identified. WHAT IS NEW AND CONCLUSION: Whether preparations containing AA, such as many Chinese medicines, should be used remains controversial, and this issue warrants further investigation before definite conclusions can be drawn.

[Inhibiting effect and mechanism of Lactobacillus E6-1 on oral cancer cell line Cal-27].

PURPOSE: To study the effect of Lactobacillus E6-1 on proliferation and apoptosis of human oral cancer cell line Cal-27 in vitro. METHODS: MTT assay was carried out by different concentrations of Lactobacillus E6-1 on Cal-27 to detect the inhibitory effect on cell proliferation, and the effect on DNA was detected by agarose gel electrophoresis. The expression levels of cyt-c, caspase-9 and caspase-3 in each group was detected by Western blot. The data were analyzed with SPSS 21.0 software package. RESULTS: The results of MTT assay showed that E6-1 had obvious inhibitory effect on Cal-27 in a dose-dependent manner. DNA ladder in Cal-27 cells was induced by different concentrations of E6-1 (10, 20, 40 mg/mL). Compared with blank control group, Western blot results showed that the expression of cyt-c, caspase-9 and caspase-3 increased significantly(P<0.05). CONCLUSIONS: Lactobacillus E6-1 can inhibit the proliferation of Cal-27 and effectively induce apoptosis of tumor cells In vitro.

[Effects of bond strength evaluation on different durations of adult permanent teeth and youth permanent teeth by using universal adhesives to dentin].

OBJECTIVE: To compare the dentin bonding strength evaluation between adult permanent teeth and youth permanent teeth after treatment for different durations by universal adhesives. METHODS: Ten adult permanent teeth and ten youth permanent teeth were selected for this study. The occlusal enamel layer was removed, and each tooth was cut into three pieces along the long axis. In total, 30 pieces of adult and youth teeth were prepared. The adult and youth teeth pieces were randomly divided into three groups and treated by universal adhesives for 10, 20, and 30 s. In this study, Scotchbond Universal (SBU) was selected as the universal adhesive. Slabs were treated by dual-cure resin cements. The specimens were tested by microshear strength test through a universal testing machine. Fracture modes were observed by a stereomicroscope. Other adult teeth and youth teeth were selected, two for each type, and treated and grouped in the same manner. Fluorescein (0.1% Rhodamine B) was dissolved in SBU adhesive, and the specimens were treated by the adhesives for 10, 20, and 30 s. Micromorphology of the resin protrusions on the adhesive surface was observed by laser confocal microscopy (CLSM). RESULTS: For the adult teeth, the highest micro-shear bonding strength was observed in the 20 and 30 s groups, and the values were higher than that of the 10 s group (P<0.05). For the youth teeth, the highest micro-shear bonding strength was observed in the 10 and 20 s groups, and the values were higher than that of the 30 s group (P<0.05). The micro-shear bonding strength in the 10 s youth teeth group was higher than that of the 10 s adult teeth (P<0.05) and was same as the adult teeth treated for 20 s (recommendation time of material instructions) (P>0.05). The main break patterns in different groups comprised adhesive failure fractures and several mixed failure fractures. No resin fracture mode was observed. CLSM revealed very few short resin protrusions in 10 s adult teeth group, whereas the number and length of resin protrusions significantly increased in the 20 s adult teeth group. The resin protrusions of the 30 s group were shorter than those of the 20 s adult teeth group. In different durations, the bonding interface in different youth teeth groups presented the same trend of change as the adult teeth. However, the number and length of resin protrusions in the 10 s group of youth teeth were all higher than those of the 10 s adult teeth group. CONCLUSIONS: In clinical practice, the bonding agent treatment duration shall be shortened appropriately for youth permanent teeth, and that for adult permanent teeth shall not be shortened to less than 20 s. On the whole, the bond strength of youth permanent teeth can achieve no significant difference with the adult permanent teeth.

Divergent Expression Patterns and Function of Two cxcr4 Paralogs in Hermaphroditic Epinephelus coioides.

Chemokine receptor Cxcr4 evolved two paralogs in the teleost lineage. However, cxcr4a and cxcr4b have been characterized only in a few species. In this study, we identified two cxcr4 paralogs from the orange-spotted grouper, Epinephelus coioides. The phylogenetic relationship and gene structure and synteny suggest that the duplicated cxcr4a/b should result from the teleost-specific genome duplication (Ts3R). The teleost cxcr4 gene clusters in two paralogous chromosomes exhibit a complementary gene loss/retention pattern. Ec_cxcr4a and Ec_cxcr4b show differential and biased expression patterns in grouper adult tissue, gonads, and embryos at different stages. During embryogenesis, Ec_cxcr4a/b are abundantly transcribed from the neurula stage and mainly expressed in the neural plate and sensory organs, indicating their roles in neurogenesis. Ec_Cxcr4a and Ec_Cxcr4b possess different chemotactic migratory abilities from the human SDF-1α, Ec_Cxcl12a, and Ec_Cxcl12b. Moreover, we uncovered the N-terminus and TM5 domain as the key elements for specific ligand⁻receptor recognition of Ec_Cxcr4a-Ec_Cxcl12b and Ec_Cxcr4b-Ec_Cxcl12a. Based on the biased and divergent expression patterns of Eccxcr4a/b, and specific ligand⁻receptor recognition of Ec_Cxcl12a/b⁻Ec_Cxcr4b/a, the current study provides a paradigm of sub-functionalization of two teleost paralogs after Ts3R.

Sexual dimorphic expression of dnd in germ cells during sex reversal and its requirement for primordial germ cell survival in protogynous hermaphroditic grouper.

Dead end (dnd), vertebrate-specific germ cell marker, had been demonstrated to be essential for primordial germ cell (PGC) migration and survival, and the link between PGC number and sex change had been revealed in some teleost species, but little is known about dnd in hermaphroditic vertebrates. In the present study, a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides) dnd homologue (Ecdnd) was identified and characterized. Quantitative real-time PCR and in situ hybridization analysis revealed a dynamic and sexually dimorphic expression pattern in PGCs and germ cells of gonads. During sex changing, the Ecdnd transcript sharply increased in early transitional gonad, reached the highest level at late transitional gonad stage, and decreased after testis maturation. Visualization of zebrafish PGCs by injecting with RFP-Ecdnd-3'UTR RNA and GFP-zfnanos3-3'UTR RNA confirmed importance of Ecdnd 3'UTR for the PGC distribution. In addition, knockdown of EcDnd by using antisense morpholinos (MO) caused the ablation of PGCs in orange-spotted grouper. Therefore, the current data indicate that Ecdnd is essential for PGCs survival and may serve as a useful germ cell marker during gametogenesis in hermaphroditic grouper.

Divergent Expression Patterns and Function Implications of Four nanos Genes in a Hermaphroditic Fish, Epinephelus coioides.

Multiple nanos genes have been characterized in several fishes, but the functional implications of their various expression patterns remain unclear. In this study, we identified and characterized four nanos genes from a hermaphroditic fish orange-spotted grouper, Epinephelus coioides. Ecnanos1a and Ecnanos1b show divergent expression patterns, and the dynamic expression change of Ecnanos1a in pituitaries during sex change is associated with testis differentiation and spermatogenesis. Ecnanos2 and Ecnanos3 might be germline stem cells (GSCs) and primordial germ cells (PGCs)-specific markers, respectively. Significantly, Ecnanos3 3'-untranslated region (UTR) is necessary for PGC specific expression, where a non-canonical "GCACGTTT" sequence is required for miR-430-mediated repression of Ecnanos3 RNA. Furthermore, grouper Dead end (Dnd) can relieve miR-430 repression in PGCs by associating with a 23 bp U-rich region (URR) in Ecnanos3 3'-UTR. The current study revealed the functional association of multiple nanos genes with PGC formation and germ cell development in orange-spotted grouper, and opened up new possibilities for developing biotechnologies through utilizing the associations between Ecnanos3 and PGCs or between Ecnanos2 and GSCs in the hermaphroditic fish.

[Bond strengths of absorbable polylactic acid root canal post with three different adhesives].

OBJECTIVE: To find absorbable adhesives with suitable bonding properties for the absorbable polylactic acid root canal post. To test and compare the bond strengths of absorbable polylactic acid root canal post with three different adhesives. METHODS: The absorbable polylactic acid root canal posts were used to restore the extracted teeth, using 3 different adhesives: cyanoacrylates, fibrin sealant and glass ionomer cement. The teeth were prepared into slices for micro-push-out test. The bond strength was statistically analyzed using ANOVA. The specimens were examined using microscope and the failure mode was divided into four categories: cohesive failure between absorbable polylactic acid root canal posts and adhesives, cohesive failure between dentin and adhesives, failure within the adhesives and failure within the absorbable polylactic acid root canal posts. RESULTS: The bond strength of cyanoacrylates [(16.83 ± 6.97) MPa] and glass ionomer cement [(12.10 ± 5.09) MPa] were significantly higher than fibrin sealant [(1.17 ± 0.50) MPa], P<0.001. There was no significant difference between cyanoacrylates and glass ionomer cement (P=0.156). In the group of cyanoacrylates, the cohesive failure between the absorbable polylactic acid root canal posts and the adhesives was 25.0%, the cohesive failure between the dentin and the adhesives was 16.7%, the failure within the adhesives was 33.3%, and the failure within the absorbable polylactic acid root canal posts was 25.0%. In the group of fibrin sealant, the cohesive failure between the absorbable polylactic acid root canal posts and the adhesives was 66.7%, the cohesive failure between the dentin and the adhesives was 22.2%, the failure within the adhesives was 11.1%. In the group of glass ionomer cement, the cohesive failure between the absorbable polylactic acid root canal posts and the adhesives was 87.5%, the failure within the adhesives was 12.5%. The major failure mode in fibrin sealant and glass ionomer cement was the cohesive failure between the absorbable polylactic acid root canal posts and the adhesives. No major failure modes were found in the group of cyanoacrylates. CONCLUSION: The bond strength of fibrin sealant is low, which cannot meet the requirement of clinical use. The bond strengths of cyanoacrylates and glass ionomer cement are suitable for clinical use. The cyanoacrylates are a kind of absorbable adhesive which has suitable bonding properties for the absorbable polylactic acid root canal post.

Grouper tshß promoter-driven transgenic zebrafish marks proximal kidney tubule development.

Kidney tubule plays a critical role in recovering or secreting solutes, but the detailed morphogenesis remains unclear. Our previous studies have found that grouper tshß (gtshß) is also expressed in kidney, however, the distribution significance is still unknown. To understand the gtshß role and kidney tubule morphogenesis, here, we have generated a transgenic zebrafish line Tg(gtshß:GFP) with green fluorescent protein driven by the gtshß promoter. Similar to the endogenous tshß in zebrafish or in grouper, the gtshß promoter-driven GFP is expressed in pituitary and kidney, and the developing details of proximal kidney tubule are marked in the transgenic zebrafish line. The gfp initially transcribes at 16 hours post fertilization (hpf) above the dorsal mesentery, and partially co-localizes with pronephric tubular markers slc20a1a and cdh17. Significantly, the GFP specifically localizes in proximal pronephric segments during embryogenesis and resides at kidney duct epithelium in adult fish. To test whether the gtshß promoter-driven GFP may serve as a readout signal of the tubular development, we have treated the embryos with retinoic acid signaing (RA) reagents, in which exogenous RA addition results in a distal extension of the proximal segments, while RA inhibition induces a weakness and shortness of the proximal segments. Therefore, this transgenic line provides a useful tool for genetic or chemical analysis of kidney tubule.