Germline sexual fate is determined by the antagonistic action of dmrt1 and foxl3/foxl2 in tilapia.

Germline sexual fate has long been believed to be determined by the somatic environment, but this idea is challenged by recent studies of foxl3 mutants in medaka. Here, we demonstrate that the sexual fate of tilapia germline is determined by the antagonistic interaction of dmrt1 and foxl3, which are transcriptionally repressed in male and female germ cells, respectively. Loss of dmrt1 rescued the germ cell sex reversal in foxl3Δ7/Δ7 XX fish, and loss of foxl3 partially rescued germ cell sex reversal but not somatic cell fate in dmrt1Δ5/Δ5 XY fish. Interestingly, germ cells lost sexual plasticity in dmrt1Δ5/Δ5 XY and foxl3Δ7/Δ7 XX single mutants, as aromatase inhibitor (AI) and estrogen treatment failed to rescue the respective phenotypes. However, recovery of germ cell sexual plasticity was observed in dmrt1/foxl3 double mutants. Importantly, mutation of somatic cell-specific foxl2 resulted in testicular development in foxl3Δ7/Δ7 or dmrt1Δ5/Δ5 mutants. Our findings demonstrate that sexual plasticity of germ cells relies on the presence of both dmrt1 and foxl3. The existence of dmrt1 and foxl3 allows environmental factors to influence the sex fate decision in vertebrates.

Multiple spinal gas in an 86-year-old diabetic man.

Although spinal gas is common and can be found in various sites and lesions, it should prompt a search for the underlying cause, given that the clinical significance of ectopic gas varies from benign to scary. Spinal gas can occur in the traumatic, iatrogenic, degenerative, osteoporotic, infectious, or neoplastic lesions. The imaging similarity may cause the misdiagnosis or delayed diagnosis which sometimes requires immediate attention. The pattern of gas distribution, detailed appearance, clinical history, and findings on examination can provide clues to diagnosis. Computed tomography is the best method for sensitive detection of gas.

Smoothened mediates medaka spermatogonia proliferation via Gli1-Rgcc-Cdk1 axis†.

The proliferation of spermatogonia directly affects spermatogenesis and male fertility, but its underlying molecular mechanisms are poorly understood. In this study, Smoothened (Smo), the central transducer of Hedgehog signaling pathway, was characterized in medaka (Oryzias latipes), and its role and underlying mechanisms in the proliferation of spermatogonia were investigated. Smo was highly expressed in spermatogonia. In ex vivo testicular organ culture and a spermatogonial cell line (SG3) derived from medaka mature testis, Smo activation promoted spermatogonia proliferation, while its inhibition induced apoptosis. The expression of glioma-associated oncogene homolog 1 (gli1) and regulator of cell cycle (rgcc) was significantly upregulated in SG3 after Smo activation. Furthermore, Gli1 transcriptionally upregulated the expression of rgcc, and Rgcc overexpression rescued cell apoptosis caused by Smo or Gli1 inhibition. Co-immunoprecipitation assay indicated that Rgcc could interact with cyclin-dependent kinase 1 (Cdk1) to regulate the cell cycle of spermatogonia. Collectively, our study firstly reveals that Smo mediates the proliferation of spermatogonia through Gli1-Rgcc-Cdk1 axis. In addition, Smo and Gli1 are necessary of the survival of spermatogonia. This study deepens our understanding of spermatogonia proliferation and survival at the molecular level, and provides insights into male fertility control and reproductive disease treatment.

Gata2a Mutation Causes Progressive Microphthalmia and Blindness in Nile Tilapia.

The normal development of lens fiber cells plays a critical role in lens morphogenesis and maintaining transparency. Factors involved in the development of lens fiber cells are largely unknown in vertebrates. In this study, we reported that GATA2 is essential for lens morphogenesis in Nile tilapia (Oreochromis niloticus). In this study, Gata2a was detected in the primary and secondary lens fiber cells, with the highest expression in primary fiber cells. gata2a homozygous mutants of tilapia were obtained using CRISPR/Cas9. Different from fetal lethality caused by Gata2/gata2a mutation in mice and zebrafish, some gata2a homozygous mutants of tilapia are viable, which provides a good model for studying the role of gata2 in non-hematopoietic organs. Our data showed that gata2a mutation caused extensive degeneration and apoptosis of primary lens fiber cells. The mutants exhibited progressive microphthalmia and blindness in adulthood. Transcriptome analysis of the eyes showed that the expression levels of almost all genes encoding crystallin were significantly down-regulated, while the expression levels of genes involved in visual perception and metal ion binding were significantly up-regulated after gata2a mutation. Altogether, our findings indicate that gata2a is required for the survival of lens fiber cells and provide insights into transcriptional regulation underlying lens morphogenesis in teleost fish.

Desert hedgehog mediates the proliferation of medaka spermatogonia through Smoothened signaling.

Desert hedgehog (DHH) signaling has been reported to be involved in spermatogenesis and the self-renewal of spermatogonial stem cells (SSCs). However, the role of DHH in proliferation of spermatogonia including SSCs remains to be elucidated. Here, we report that Dhh from medaka (Oryizas latipes) (named as OlDhh) could directly mediate the proliferation of spermatogonia via Smoothened (Smo) signaling. Oldhh is 1362 bp in length and encodes 453 amino acid (aa) residues with more than 50% identity with the homologs in other species. It has expression predominantly restricted to testis. The soluble and tag-free 176-aa mature OlDhh (named as mOlDhh) were successfully obtained by fusing with the N-terminal tag of cleavable 6-histidine and small ubiquitin-related modifier and then removing the tag. Notably, mOlDhh significantly promoted the proliferation of SG3 (a spermatogonial stem cell line from medaka testis) in a dose-dependent manner and spermatogonia in testicular organ culture. Furthermore, the proliferation of SG3 in the presence of mOlDhh could be inhibited by Smo antagonist (cyclopamine) resulting in apoptosis. Additionally, mOlDhh significantly upregulated the expression of smo as well as the pluripotent-related genes bcl6b and sall4. These data suggest that Smo is an indispensable downstream component in the Dhh signaling pathway. In conclusion, our findings unambiguously demonstrate that Dhh could directly mediate the proliferation of spermatogonia through Smo signaling. This study provides new knowledge about the proliferation regulation of spermatogonia.

Circ_SPG11 plays contributing effects on IL-1ß-induced chondrocyte apoptosis and ECM degradation via miR-665 inhibition-mediated GREM1 upregulation.

The dysregulation of circular RNA (circRNA) has been monitored in osteoarthritis (OA) cartilage, hinting that circRNA deregulation modulates OA progression. We thus aimed to unveil the role of circRNA spastic paraplegia 11 (circ_SPG11) in OA conditions. The upregulation of circ_SPG11 was observed in OA cartilage and IL-1ß-treated chondrocytes. Knockdown of circ_SPG11 restored IL-1ß-depleted cell proliferation and alleviated IL-1ß-induced cell apoptosis and ECM degradation. Circ_SPG11 bound to miR-665 and negatively regulated miR-665 expression. Inhibition of miR-665 reversed the inhibitory effect on IL-1ß-induced chondrocyte injury caused by circ_SPG11 knockdown. GREM1 was a target of miR-665, and circ_SPG11 knockdown depleted GREM1 expression by enriching miR-665. Overexpression of GREM1 also reversed the inhibitory effect on IL-1ß-induced chondrocyte injury caused by miR-665 enrichment. Circ_SPG11 might promote IL-1ß-induced chondrocyte apoptosis and ECM degradation via increasing GREM1 expression by decoying miR-665.

Nile Tilapia: A Model for Studying Teleost Color Patterns.

The diverse color patterns of cichlid fishes play an important role in mate choice and speciation. Here we develop the Nile tilapia (Oreochromis niloticus) as a model system for studying the developmental genetics of cichlid color patterns. We identified 4 types of pigment cells: melanophores, xanthophores, iridophores and erythrophores, and characterized their first appearance in wild-type fish. We mutated 25 genes involved in melanogenesis, pteridine metabolism, and the carotenoid absorption and cleavage pathways. Among the 25 mutated genes, 13 genes had a phenotype in both the F0 and F2 generations. None of F1 heterozygotes had phenotype. By comparing the color pattern of our mutants with that of red tilapia (Oreochromis spp), a natural mutant produced during hybridization of tilapia species, we found that the pigmentation of the body and eye is controlled by different genes. Previously studied genes like mitf, kita/kitlga, pmel, tyrb, hps4, gch2, csf1ra, pax7b, and bco2b were proved to be of great significance for color patterning in tilapia. Our results suggested that tilapia, a fish with 4 types of pigment cells and a vertically barred wild-type color pattern, together with various natural and artificially induced color gene mutants, can serve as an excellent model system for study color patterning in vertebrates.

Regulation of spermatogenesis and reproductive capacity by Igf3 in tilapia.

A novel insulin-like growth factor (igf3), which is exclusively expressed in the gonads, has been widely identified in fish species. Recent studies have indicated that Igf3 regulates spermatogonia proliferation and differentiation in zebrafish; however, detailed information on the role of this Igf needs further in vivo investigation. Here, using Nile tilapia (Oreochromis niloticus) as an animal model, we report that igf3 is required for spermatogenesis and reproduction. Knockout of igf3 by CRISPR/Cas9 severely inhibited spermatogonial proliferation and differentiation at 90 days after hatching, the time critical for meiosis initiation, and resulted in less spermatocytes in the mutants. Although spermatogenesis continued to occur later, more spermatocytes and less spermatids were observed in the igf3-/- testes when compared with wild type of testes at adults, indicating that Igf3 regulates spermatocyte to spermatid transition. Importantly, a significantly increased occurrence of apoptosis in spermatids was observed after loss of Igf3. Therefore, igf3-/- males were subfertile with drastically reduced semen volume and sperm count. Conversely, the overexpression of Igf3 in XY tilapia enhanced spermatogenesis leading to more spermatids and sperm count. Transcriptomic analysis revealed that the absence of Igf3 resulted in dysregulation of many genes involved in cell cycle, meiosis and pluripotency regulators that are critical for spermatogenesis. In addition, in vitro gonadal culture with 17α-methyltetosterone (MT) and 11-ketotestosterone (11-KT) administration and in vivo knockout of cyp11c1 demonstrated that igf3 expression is regulated by androgens, suggesting that Igf3 acts downstream of androgens in fish spermatogenesis. Notably, the igf3 knockout did not affect body growth, indicating that this Igf specifically functions in reproduction. Taken together, our data provide genetic evidence for fish igf3 in the regulation of reproductive capacity by controlling spermatogenesis.

Posttranslational Modification of Waxy to Genetically Improve Starch Quality in Rice Grain.

The waxy (Wx) gene, encoding the granule-bound starch synthase (GBSS), is responsible for amylose biosynthesis and plays a crucial role in defining eating and cooking quality. The waxy locus controls both the non-waxy and waxy rice phenotypes. Rice starch can be altered into various forms by either reducing or increasing the amylose content, depending on consumer preference and region. Low-amylose rice is preferred by consumers because of its softness and sticky appearance. A better way of improving crops other than downregulation and overexpression of a gene or genes may be achieved through the posttranslational modification of sites or regulatory enzymes that regulate them because of their significance. The impact of posttranslational GBSSI modifications on extra-long unit chains (ELCs) remains largely unknown. Numerous studies have been reported on different crops, such as wheat, maize, and barley, but the rice starch granule proteome remains largely unknown. There is a need to improve the yield of low-amylose rice by employing posttranslational modification of Wx, since the market demand is increasing every day in order to meet the market demand for low-amylose rice in the regional area that prefers low-amylose rice, particularly in China. In this review, we have conducted an in-depth review of waxy rice, starch properties, starch biosynthesis, and posttranslational modification of waxy protein to genetically improve starch quality in rice grains.

Regulation of Female Folliculogenesis by Tsp1a in Nile Tilapia (Oreochromis niloticus).

TSP1 was reported to be involved in multiple biological processes including the activation of TGF-ß signaling pathways and the regulation of angiogenesis during wound repair and tumor growth, while its role in ovarian folliculogenesis remains to be elucidated. In the present study, Tsp1a was found to be expressed in the oogonia and granulosa cells of phase I to phase IV follicles in the ovaries of Nile tilapia by immunofluorescence. tsp1a homozygous mutants were generated by CRISPR/Cas9. Mutation of tsp1a resulted in increased oogonia, reduced secondary growth follicles and delayed ovary development. Expression of the cell proliferation marker PCNA was significantly up-regulated in the oogonia of the mutant ovaries. Furthermore, transcriptomic analysis revealed that expressions of DNA replication related genes were significantly up-regulated, while cAMP and MAPK signaling pathway genes which inhibit cell proliferation and promote cell differentiation were significantly down-regulated. In addition, aromatase (Cyp19a1a) expression and serum 17ß-estradiol (E2) concentration were significantly decreased in the mutants. These results indicated that lacking tsp1a resulted in increased proliferation and inhibited differentiation of oogonia, which in turn, resulted in increased oogonia, reduced secondary growth follicles and decreased E2. Taken together, our results indicated that tsp1a was essential for ovarian folliculogenesis in Nile tilapia.

Mutation of cyp19a1b results in sterile males due to efferent duct obstruction in Nile tilapia.

The involvement of estrogen in male fertility has been well established in mammals. However, less is known about the role of estrogen in fish male reproduction. Our recent study revealed that Cyp19a1a deficiency had no effect on fertility in male fish. In this study, expression of Cyp19a1b, but not Cyp19a1a, was detected by immunohistochemistry in Leydig cells of tilapia testes. cyp19a1b mutation resulted in a significant decrease in the concentration of 17ß-estradiol in serum and sterility in XY fish, as no offspring were obtained when crossed with control XX fish at 240 days after hatching (dah). No sperm was obtained from the mature mutants by in vitro extrusion. Further examination of the mutant gonads revealed excessive semen accumulation and testicular hypertrophy. Semen collected from the mutant testes during autopsy contained sperm with a normal morphology that showed no significant differences in motility, VCL, BCF, STR, or fertility compared with control sperm. Efferent ducts from the mutant testes, which had low-convolution levels, fewer branches, and no blood vessels observed inside the walls, were significantly smaller in size. qRT-PCR analyses showed downregulated expression of ion exchange genes. There was increased apoptosis in the epithelial cells of the efferent ducts and other somatic cells of the testes as revealed by TUNEL staining, as well as upregulation of apoptosis gene expression in the mutants. At 360 dah, mutant fish showed testicular atrophy and efferent duct fibrosis. These results demonstrated that estrogen deficiency caused by Cyp19a1b mutation resulted in male sterility due to efferent duct obstruction.

Design, synthesis and biological evaluation of novel copper-chelating acetylcholinesterase inhibitors with pyridine and N-benzylpiperidine fragments.

Cholinergic depletion is the direct cause of disability and dementia among AD patients. AChE is a classical and key target of cholinergic disorders. Some new inhibitors of AChE combining pyridine, acylhydrazone and N-benzylpiperidine fragments were developed in this work. The hit structure was optimized to yield the compound 21 with an IC50 value of 6.62 nM against AChE, while almost no inhibitory effect against BChE. ADMET predictions and PAMPA permeability evaluation showed good drug-like property. The higher activity with an intermediate alkyl chain substitution indicates a new binding mode of inhibitor with AChE. This finding provides new insights into the binding mechanism and is helpful for discovery of novel high-activity AChE inhibitors.

Pharmacophore modeling, molecular docking and molecular dynamics studies on natural products database to discover novel skeleton as non-purine xanthine oxidase inhibitors.

Gout is a common inflammatory arthritis caused by the deposition of urate crystals within joints. It is increasingly in prevalence during the past few decades as shown by the epidemiological survey results. Xanthine oxidase (XO) is a key enzyme to transfer hypoxanthine and xanthine to uric acid, whose overproduction leads to gout. Therefore, inhibiting the activity of xanthine oxidase is an important way to reduce the production of urate. In the study, in order to identify the potential natural products targeting XO, pharmacophore modeling was employed to filter databases. Here, two methods, pharmacophore based on ligand and pharmacophore based on receptor-ligand, were constructed by Discovery Studio. Then GOLD was used to refine the potential compounds with higher fitness scores. Finally, molecular docking and dynamics simulations were employed to analyze the interactions between compounds and protein. The best hypothesis was set as a 3D query to screen database, returning 785 and 297 compounds respectively. A merged set of the above 1082 molecules was subjected to molecular docking, which returned 144 hits with high-fitness scores. These molecules were clustered in four main kinds depending on different backbones. What is more, molecular docking showed that the representative compounds established key interactions with the amino acid residues in the protein, and the RMSD and RMSF of molecular dynamics results showed that these compounds can stabilize the protein. The information represented in the study confirmed previous reports. And it may assist to discover and design new backbones as potential XO inhibitors based on natural products.

Discovery of FIXa inhibitors by combination of pharmacophore modeling, molecular docking, and 3D-QSAR modeling.

Human Coagulation Factor IXa (FIXa), specifically inhibited at the initiation stage of the blood coagulation cascade, is an excellent target for developing selective and safe anticoagulants. To explore this inhibitory mechanism, 86 FIXa inhibitors were selected to generate pharmacophore models and subsequently SAR models. Both best pharmacophore model and ROC curve were built through the Receptor-Ligand Pharmacophore Generation module. CoMFA model based on molecular docking and PLS factor analysis methods were developed. Model propagations values are q2 = 0.709, r2 = 0.949, and r2pred = 0.905. The satisfactory q2 value of 0.609, r2 value of 0.962, and r2pred value of 0.819 for CoMSIA indicated that the CoMFA and CoMSIA models are both available to predict the inhibitory activity on FIXa. On the basis of pharmacophore modeling, molecular docking, and 3D-QSAR modeling screening, six molecules are screened as potential FIXa inhibitors.

Discovery of novel wee1 inhibitors via structure-based virtual screening and biological evaluation.

Wee1 plays a critical role in the arrest of G2/M cell cycle for DNA repair before entering mitosis. Many cancer cells have been identified as overexpression of Wee1. In this research, pharmacophore modeling, molecular docking and molecular dynamics simulation approaches were constructed to identify novel potential Wee1 inhibitors. A compound 8 was found to have a novel skeleton against Wee1 with an IC50 value of 22.32 µM and a Ki value of 13.11 µM. Kinetic assays were employed to evaluate the compound 8 as a competitive inhibitor. Compound 8 was tested against A-549 tumor cell lines with IC50 value of 17.8 µM. To investigate the intermolecular interaction of Wee1 and compound 8, further molecular dynamics simulations were performed. It indicates that the binding mode of compound 8 and reference ligand is similar. The active core scaffold of compound 8 could represent a promising lead compound for studying Wee1 and be used for further structural optimization to design more potent Wee1 inhibitors.

Does intraoperative navigation improve the accuracy of pedicle screw placement in the apical region of dystrophic scoliosis secondary to neurofibromatosis type I: comparison between O-arm navigation and free-hand technique.

PURPOSE: To assess the accuracy of O-arm-navigation-based pedicle screw insertion in dystrophic scoliosis secondary to NF-1 and compare it with free-hand pedicle screw insertion technique. METHODS: 32 patients with dystrophic NF-1-associated scoliosis were divided into two groups. A total of 92 pedicle screws were implanted in apical region (two vertebrae above and below the apex each) in 13 patients using O-arm-based navigation (O-arm group), and 121 screws were implanted in 19 patients using free-hand technique (free-hand group). The postoperative CT images were reviewed and analyzed for pedicle violation. The screw penetration was divided into four grades: grade 0 (ideal placement), grade 1 (penetration <2 mm), grade 2 (penetration between 2 and 4 mm), and grade 3 (penetration >4 mm). RESULTS: The accuracy rate of pedicle screw placement (grade 0, 1) was significantly higher in the O-arm group (79 %, 73/92) compared to 67 % (81/121) of the free-hand group (P = 0.045). Meanwhile, a significantly lower prevalence of grade 2-3 perforation was observed in the O-arm group (21 vs. 33 %, P < 0.05), and the incidence of medial perforation was significantly minimized by using O-arm navigation compared to free-hand technique (2 vs. 15 %, P < 0.01). Moreover, the implant density in apical region was significantly elevated by using O-arm navigation (58 vs. 42 %, P < 0.001). CONCLUSION: We reported 79 % accuracy of O-arm-based pedicle screw placement in dystrophic NF-1-associated scoliosis. O-arm navigation system does facilitate pedicle screw insertion in dystrophic NF-1-associated scoliosis, demonstrating superiorities in the safety and accuracy of pedicle screw placement in comparison with free-hand technique.

A novel fluorescent probe with a phosphofluorene molecular structure for selective detection of hydrogen sulfide in living cells.

Hydrogen sulfide (H2S) gas plays a significant role in biological regulation. With advancements in technology, H2S has been discovered across diverse fields, necessitating a comprehensive understanding of its physiological functions through monitoring changes in H2S within complex environments and physiological processes. In this study, we designed a phosphofluorene-based conjugate probe PPF-CDNB with an asymmetric π-conjugated phosphine structure and utilized dinitrophenyl ether as the recognition site for H2S. PPF-CDNB exhibited exceptional resistance to interference and demonstrated stability over a broad pH range (3.0-10.0), making it suitable for various environmental conditions. Intracellular experiments revealed that PPF-CDNB effectively monitored both endogenous and exogenous levels of H2S.

Assessment of macro, trace and toxic element intake from rice: differences between cultivars, pigmented and non-pigmented rice.

Pigmented and non-pigmented rice varieties (grown in different areas) were collected in China, Yunnan, to investigate the content of macro-, trace elements and potentially toxic elements (PTEs), and to assess the health risk associated with dietary intake. The order of elemental concentrations in rice was Mn > Zn > Fe > Cu > Se for trace elements, P > K > Mg > Ca > Na for macro elements, and Cr > As > Cd for PTEs. Rice with a high concentration of essential elements also associated with a high content of PTEs. In addition, higher content of Cr, Mn and Na were found in pigmented rice. The health risk assessment showed that the daily intake of all elements was below the tolerable limit (UL). Moreover the intake of Fe, Zn and Se was far from sufficient for the nutrient requirement. The PTEs in rice dominated the health risk. Of concern is that this rice consumption is likely to contribute to carcinogenic risks in the long term and that adults are at higher health risk from pigmented rice compared to non-pigmented rice. This study confirms that the lack of essential micronutrients in rice and the health risk associated with rice diets should remain a concern.

Physicochemical properties of Tunisian pomegranate fruits Punica granatum L. grown at different climatic zones of Yunnan, China.

The artificial cultivation of Mengzi pomegranate has been conducted on a large area for more than 30 years in Yunnan, China. As the quality of pomegranate cultivars have degraded seriously, new cultivars have gradually been introduced to meet market demand. Comparative quality evaluation of different pomegranate varieties is beneficial to guide cultivar improvement and cultural program modification. The present study investigated the differences of physicochemical characteristics of the introduced Tunisian soft-seed pomegranate in different climate zones in Yunnan, China. Meanwhile, the differences between native cultivars (Tian guan yan (TGY) and Tian lv zi (TLZ)) and introduced cultivar were also compared. It was found that the Tunisian soft-seed pomegranate grown in Huize (Tunisian pomegranate in Huize, TH) in the temperate plateau monsoon climate belt had the highest fruit weight, % of seed, TAC, TPC, TSS, amino acids, and Mn, and had the lowest organic acids and Na. While grown in the subtropical monsoon climate area Shiping (Tunisian pomegranate in Shiping, TS), the Tunisian pomegranate fruits had the highest amounts of Cu. Commonly, there were significant correlations between cultivation climate regions and fruit properties. The contents of the TSS, TAC, TPC, flavor amino acids and organic acids varied with planted climate zones. The introduced Tunisian soft-seed pomegranate has comparable levels of physicochemical characteristics with the local main cultivars in the same planted climate region. Tunisian soft-seed pomegranate of higher quality can be obtained in Huize area. Collectively, the climate difference and cultivar shift have a significant effect on pomegranate production in Yunnan, and pomegranate with good quality can be obtained by using proper cultivars in optimized climate zone.