Spermatogenesis is essential for establishment and maintenance of reproduction in male vertebrates. Spermatogenesis, which is mainly regulated by the combined action of hormones, growth factors, and ...epigenetic factors, is highly conserved. Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-β superfamily. In this study, global gdnfa knockout and Tg (gdnfa: mcherry) transgenic zebrafish lines were generated. Loss of gdnfa resulted in disorganized testes, decreased gonadosomatic index, and low percentage of mature spermatozoa. In the Tg (gdnfa: mcherry) zebrafish line, we found that gdnfa was expressed in Leydig cells. The mutation in gdnfa significantly decreased Leydig cell marker gene expression and androgen secretion in Leydig cells. In addition, courtship behavior was disrupted in the male mutants. We present in vivo data showing that global knockout of gdnfa disrupts spermiogenesis and male courtship behavior in zebrafish. The first viable vertebrate model with a global gdnfa knockout may be valuable for studying the role of GDNF in animal reproduction.
•Generation of global gdnfa knockout and Tg (gdnfa: mcherry) transgenic zebrafish lines.•Loss of gdnfa disrupts spermiogenesis in zebrafish.•Loss of gdnfa disrupts male courtship behavior in zebrafish.•The development of Leydig cells and androgen synthesis were affected in gdnfa mutant males.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The mud loach (Misgurnus anguillicaudatus) is one of the most important aquaculture fishes in Asia. The application of the CRISPR/Cas9 system in the cultivation of loach strains with valuable ...economic traits presents great potential. Myostatin is a candidate factor that can be used for the cultivation of fast-growing loach strains. In this study, the appropriate microinjection dose of the Cas9 protein (600 pg) for genome editing was determined considering both the mutation rate and survival rate of loach embryos. With this microinjection dose of the Cas9 protein, both the exogenous GFP and endogenous myostatin genes were efficiently edited using the CRISPR/Cas9 system. Compared with that in wild-type siblings, the average body weight of the fish in 1-, 2- and 3-month-old myostatin mutant chimeric loaches was significantly increased by 34.9% (P < 0.001), 15.5% (P = 0.024) and 27.8% (P = 0.012), respectively. Mutation of mstn significantly increased the number of muscle fibers and total area of muscle fibers. Also, lipid accumulation was significantly higher in chimeric mutant loach. Additionally, the mRNA levels of myogenesis (myod and myog) and lipogenesis-related genes (scd, fabp1, fabp2 and dgat) were significantly upregulated in chimeric mutants. In particular, the mRNA level of the stearoyl-CoA desaturase gene, whose product catalyses the formation of monounsaturated fatty acids from saturated fatty acids, was increased by 23-fold (P = 0.034) and 736-fold (P < 0.05) in 3 dpf and 1-month-old chimeric loaches, respectively. The gene expression results indicated that the targeted disruption of myostatin activated myogenesis and adipogenesis in loach. Our study is highly valuable for cultivating fast-growing loach strains and understanding the specific role of myostatin in lipogenesis.
•The appropriate microinjection dose of the Cas9 protein for genome editing was determined in loach Misgurnus anguillicaudatus.•Fast-growing loaches were obtained through the targeted mutation of myostatin.•Loss of myostatin activated myogenesis in loach.•Loss of myostatin activated adipogenesis in loach.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Common carp is the fourth most cultivated fish species in the world. Sexually dimorphic growth of common carp makes its sex differentiation an important research topic. The main objective of this ...study was to investigate the role of germ cells in sex differentiation of common carp. We generated the germ cell-deficient common carp through knocking down dead end gene with a morpholino antisense oligonucleotide during the embryogenesis. In the common carp without germ cells, we found about half of the gonads (type I gonads) contain granulosa cells and theca cells, which were the same with somatic cells we observed in the wild-type ovaries. The remaining half of the gonads (type II gonads) contain Sertoli cells and Leydig cells, which were the same with somatic cells in the wild-type testes. Furthermore, the expression of sex differentiation related genes was detected in the gonads without germ cells. It was found cyp19a1a was mainly expressed in the type I gonads, and the cyp19a1a mRNA level is much higher in each type I gonad than in the type II gonad. While dmrt1 was mainly expressed in the type II gonads, and the dmrt1 mRNA level is much higher in each type II gonad than in the type I gonad. Our comprehensive analysis showed that the gonadal structure and expression of genes related with sex differentiation were sexually dimorphic in the germ cell-deficient carp. These findings are valuable for discovering the underlying mechanism of common carp sex differentiation which is crucial for cultivating monosexual carp for aquaculture.
•Germ cell-deficient common carp model was generated through knocking down dnd gene.•Common carp without germ cells displayed sexual dimorphism of gonadal structure.•Expression of cyp19a1a and dmrt1 genes was sexually dimorphic in germ cell-deficient gonads.•Gonadal differentiation is germ cell independent in common carp.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Background
Septic shock comprises a heterogeneous population, and individualized resuscitation strategy is of vital importance. The study aimed to identify subclasses of septic shock with ...non-supervised learning algorithms, so as to tailor resuscitation strategy for each class.
Methods
Patients with septic shock in 25 tertiary care teaching hospitals in China from January 2016 to December 2017 were enrolled in the study. Clinical and laboratory variables were collected on days 0, 1, 2, 3 and 7 after ICU admission. Subclasses of septic shock were identified by both finite mixture modeling and
K
-means clustering. Individualized fluid volume and norepinephrine dose were estimated using dynamic treatment regime (DTR) model to optimize the final mortality outcome. DTR models were validated in the eICU Collaborative Research Database (eICU-CRD) dataset.
Results
A total of 1437 patients with a mortality rate of 29% were included for analysis. The finite mixture modeling and
K
-means clustering robustly identified five classes of septic shock. Class 1 (baseline class) accounted for the majority of patients over all days; class 2 (critical class) had the highest severity of illness; class 3 (renal dysfunction) was characterized by renal dysfunction; class 4 (respiratory failure class) was characterized by respiratory failure; and class 5 (mild class) was characterized by the lowest mortality rate (21%). The optimal fluid infusion followed the resuscitation/de-resuscitation phases with initial large volume infusion and late restricted volume infusion. While class 1 transitioned to de-resuscitation phase on day 3, class 3 transitioned on day 1. Classes 1 and 3 might benefit from early use of norepinephrine, and class 2 can benefit from delayed use of norepinephrine while waiting for adequate fluid infusion.
Conclusions
Septic shock comprises a heterogeneous population that can be robustly classified into five phenotypes. These classes can be easily identified with routine clinical variables and can help to tailor resuscitation strategy in the context of precise medicine.
Monitoring ships on water surfaces encounters obstacles such as weather conditions, sunlight, and water ripples, posing significant challenges in accurately detecting target ships in real time. ...Synthetic Aperture Radar (SAR) offers a viable solution for real-time ship detection, unaffected by cloud coverage, precipitation, or light levels. However, SAR images are often affected by speckle noise, salt-and-pepper noise, and water surface ripple interference. This study introduces LCAS-DetNet, a Multi-Location Cross-Attention Ship Detection Network tailored for the ships in SAR images. Modeled on the YOLO architecture, LCAS-DetNet comprises a feature extractor, an intermediate layer (“Neck”), and a detection head. The feature extractor includes the computation of Multi-Location Cross-Attention (MLCA) for precise extraction of ship features at multiple scales. Incorporating both local and global branches, MLCA bolsters the network’s ability to discern spatial arrangements and identify targets via a cross-attention mechanism. Each branch utilizes Multi-Location Attention (MLA) and calculates pixel-level correlations in both channel and spatial dimensions, further combating the impact of salt-and-pepper noise on the distribution of objective ship pixels. The feature extractor integrates downsampling and MLCA stacking, enhanced with residual connections and Patch Embedding, to improve the network’s multi-scale spatial recognition capabilities. As the network deepens, we consider this structure to be cascaded and multi-scale, providing the network with a richer receptive field. Additionally, we introduce a loss function based on Wise-IoUv3 to address the influence of label quality on the gradient updates. The effectiveness of our network was validated on the HRSID and SSDD datasets, where it achieved state-of-the-art performance: a 96.59% precision on HRSID and 97.52% on SSDD.
Cetuximab has been approved for use for first-line treatment of patients with wild-type KRAS metastatic colorectal cancer (CRC). However, treatment with cetuximab has shown limited efficacy as a CRC ...monotherapy. In addition, natural killer (NK) cell function is known to be severely attenuated in cancer patients. The goal of this study was to develop a new strategy to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells, in combination with cetuximab against CRC cells.
Ex vivo expanded NK cells were stimulated with reovirus, and reovirus-activated NK cells mediated ADCC assay were performed on CRC cells in combination with cetuximab. The synergistic antitumor effects of reovirus-activated NK cells and cetuximab were tested on DLD-1 tumor-bearing mice. Finally, Toll-like receptor 3 (TLR3) knockdown in NK cells, along with chemical blockade of TLR3/dsRNA complex, and inhibition of the TLR3 downstream signaling pathway, were performed to explore the mechanisms by which reovirus enhances NK cell cytotoxicity.
We first confirmed that exposure of NK cells to reovirus enhanced their cytotoxicity in a dose-dependent manner.We then investigated whether reovirus-activated NK cells exposed to cetuximab-bound CRC cells exhibited greater anti-tumor efficacy than either monotherapy. Co-culture of CRC cell lines with reovirus-activated NK cells indicated that NK cytotoxicity was significantly higher in combination with cetuximab, regardless of KRAS mutation status or EGFR expression level. We also found that reovirus activation of NK cells, in conjunction with cetuximab, resulted in significantly stronger anti-tumor efficacy.Finally, TLR3 knockdown, inhibition of TLR3/dsRNA complex or TBK1/IKKε demonstrated that activation of NK cells by reovirus was dependent on TLR3 and its downstream signaling pathway.
This study demonstrated that combination treatment of reovirus-activated NK cells with cetuximab synergistically enhances their anti-tumor cytotoxicity, suggesting a strong candidate strategy for clinical treatment of CRC.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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