The vitellogenin present in the bloodstream undergoes internalization into developing oocytes through the vitellogenin receptor (VgR), a process mediated by receptor-mediated endocytosis. VgR plays a crucial role in facilitating the accumulation of vitellogenin and the maturation of oocytes. In this study, we characterized a Tachypleus tridentatus vitellogenin receptor (TtVgR) gene from the tri-spine horseshoe crab, revealing a length of 1956 bp and encoding 652 amino acid residues with 12 exons. TtVgR has a molecular weight of 64.26 kDa and an isoelectric point of 5.95. Predictions indicate 85 phosphorylation sites and 7 glycosylation sites within TtVgR. Transcriptional analysis demonstrated specific expression of TtVgR in the ovary and yellow connective tissue. TtVgR was identified and distributed in the plasma membrane of oocytes. The siRNA-mediated TtVgR knockdown significantly reduced the transcriptional activity of TtVgR. This depletion induced excessive ROS production, resulting in DNA damage in ovarian primary cells. TUNEL and flow cytometry analyses confirmed ovarian cell apoptosis following TtVgR knockdown, indicating DNA damage in ovarian primary cells. These findings underscore the importance of TtVgR in ovarian cell development, suggesting its potential involvement in vitellogenesis and oocyte maturation. This knowledge may inform innovative breeding strategies and contribute to the sustainable management and conservation of the tri-spine horseshoe crab.

DENV infection poses a major health concern globally and the pathophysiology relies heavily on host-cellular machinery. Although virus replication relies heavily on the host, the mechanistic details of DENV-host interaction is not fully characterized yet. Here, we are focusing on characterizing the mechanistic basis of virus-induced stress on the host cell. Specifically, we aim to characterize the role of the stress modulator ribonuclease Angiogenin during DENV infection. Our results suggested that the levels of Angiogenin are up-regulated in DENV-infected cells and the levels increase proportionately with DENV replication. Our efforts to knockdown Angiogenin using siRNA were unsuccessful in DENV-infected cells but not in mock-infected control. To further investigate the modulation between DENV replication and Angiogenin, we treated Huh7 cells with Ivermectin prior to DENV infection. Our results suggest a significant reduction in DENV replication specifically at the later stages as a consequence of Ivermectin treatment. Interestingly, Angiogenin levels were also found to be decreased proportionately. Our results suggest that Angiogenin modulation during DENV infection is important for DENV replication and pathogenesis.

Protein kinase C (PRKC) isozymes activate many signaling pathways and promote tumorigenesis, which can be confirmed by masking the kinase activity. In the present study, the kinase activity of PRKC ε and ζ isozymes was masked by siRNA in bladder cancer, and the consequent gene profile was evaluated. Here, we show that the commonly dysregulated genes affected by both the isozymes were the chemokines (CXCL8 & CXCL10), adhesion molecules (ICAM1, SPP1, MMP3, VEGFA) and mutated isoform of TP53. As these same genes were upregulated in bladder cancer patients, the activity of the kinase in downregulating them is confirmed. These genes are associated with regulating the tumor microenvironment, proliferation and differentiation of cancer cells and poor prognosis. The effect of kinase masking in downregulating these genes in bladder cancer indicates the benefits PRKC inhibitors may have in managing these patients.

Estrogen receptor positive (ER+) breast cancer (BCa) accounts for the highest proportion of breast cancer-related deaths. While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER+ BCa cells and is a novel, tractable therapeutic target for the treatment of ER+ BCa patients. Analyses of publicly available clinical datasets indicate that ER+ BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER+ BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER+ BCa cells, implicating a growth dependency of ER+ BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER+ breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER+ breast cancer signaling and growth that suggests ER+ BCa patients may benefit from SEMA3C-targeted therapy.

The Chinese tongue sole (Cynoglossus semilaevis) is a traditional, precious fish in China. Due to the large growth difference between males and females, the investigation of their sex determination and differentiation mechanisms receives a great deal of attention. Forkhead Box O (FoxO) plays versatile roles in the regulation of sex differentiation and reproduction. Our recent transcriptomic analysis has shown that foxo genes may participate in the male differentiation and spermatogenesis of Chinese tongue sole. In this study, six Csfoxo members (Csfoxo1a, Csfoxo3a, Csfoxo3b, Csfoxo4, Csfoxo6-like, and Csfoxo1a-like) were identified. Phylogenetic analysis indicated that these six members were clustered into four groups corresponding to their denomination. The expression patterns of the gonads at different developmental stages were further analyzed. All members showed high levels of expression in the early stages (before 6 months post-hatching), and this expression was male-biased. In addition, promoter analysis found that the addition of C/EBPα and c-Jun transcription factors enhanced the transcriptional activities of Csfoxo1a, Csfoxo3a, Csfoxo3b, and Csfoxo4. The siRNA-mediated knockdown of the Csfoxo1a, Csfoxo3a, and Csfoxo3b genes in the testicular cell line of Chinese tongue sole affected the expression of genes related to sex differentiation and spermatogenesis. These results have broadened the understanding of foxo\'s function and provide valuable data for studying the male differentiation of tongue sole.

As a highly conserved DNA polymerase (Pol), Pol δ plays crucial roles in chromosomal DNA synthesis and various DNA repair pathways. However, the function of POLD2, the second small subunit of DNA Pol δ (p50 subunit), has not been characterized in vivo during mammalian development. Here, we report for the first time, the essential role of subunit POLD2 during early murine embryogenesis. Although Pold2 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at gastrulation stages. Outgrowth assays reveal that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Notably, these phenotypes can be recapitulated by small interfering RNA (siRNA)-mediated knockdown, which also exhibit slowed cellular proliferation together with skewed primitive endoderm and epiblast allocation during the second cell lineage specification. In summary, our study demonstrates that POLD2 is essential for the earliest steps of mammalian development, and the retarded proliferation and embryogenesis may also alter the following cell lineage specifications in the mouse blastocyst embryos.

Astroglia play an important role, providing de novo synthesized cholesterol to neurons in the form of ApoE-lipidated particles; disruption of this process can increase the risk of Alzheimer\'s disease. We recently reported that glia-specific suppression of the lipolysis-stimulated lipoprotein receptor (LSR) gene leads to Alzheimer\'s disease-like memory deficits. Since LSR is an Apo-E lipoprotein receptor, our objective of this study was to determine the effect of LSR expression modulation on cholesterol and ApoE output in mouse astrocytes expressing human ApoE3. qPCR analysis showed that siRNA-mediated lsr knockdown significantly increased expression of the genes involved in cholesterol synthesis, secretion, and metabolism. Analysis of media and lipoprotein fractions showed increased cholesterol and lipidated ApoE output in HDL-like particles. Further, lsr expression could be upregulated when astrocytes were incubated 5 days in media containing high levels (two-fold) of lipoprotein, or after 8 h treatment with 1 µM LXR agonist T0901317 in lipoprotein-deficient media. In both conditions of increased lsr expression, the ApoE output was repressed or unchanged despite increased abca1 mRNA levels and cholesterol production. We conclude that LSR acts as a sensor of lipoprotein content in the medium and repressor of ApoE release, while ABCA1 drives cholesterol efflux, thereby potentially affecting cholesterol load, ApoE lipidation, and limiting cholesterol trafficking towards the neuron.

Traumatic neuromuscular injury to the pudendal nerve and urethra during childbirth does not regenerate well and contributes to stress urinary incontinence in women. Mesenchymal stem cells (MSCs) can improve neuroregeneration via their secretions, or secretome, which includes brain-derived neurotrophic factor (BDNF). In this study, we investigated whether BDNF is a key factor in the secretome of MSCs for the facilitation of functional recovery following a dual simulated childbirth injury. BDNF knockdown (KD) MSCs were created using an anti-BDNF shRNA lentivirus vector. A scrambled sequence was used as a transduction control (scrambled). Cells were cultured for 24 h before media was concentrated 50x to create concentrated conditioned media (CCM) containing MSC secretome. CCM of unmanipulated MSCs was screened for high BDNF expression (high BDNF CCM). Concentrated control media (CM) was created by concentrating media not conditioned by cells. Female Sprague-Dawley rats underwent bilateral pudendal nerve crush and vaginal distension (Injury) or sham injury. One hour and 1 week after injury, sham injured rats received CM, and injured rats received CM, high BDNF CCM, KD CCM, or scrambled CCM (300 μl intraperitoneally). Three weeks after injury, rats underwent leak point pressure (LPP) and pudendal nerve sensory branch potential (PNSBP) recordings. The urethra and pudendal nerve were harvested for anatomical assessment. ANOVA followed by the Student-Newman-Keuls test determined significant differences between groups (p < 0.05). BDNF KD CCM had significantly decreased BDNF concentration compared to scrambled CCM, while the concentration in high BDNF CCM was significantly increased. LPP was significantly decreased in CM and KD CCM treated animals compared to sham injury, but not with scrambled or high BDNF CCM. PNSBP firing rate showed a significant decrease with CM treatment compared to sham injury. Neuromuscular junctions in the urethral sphincter in KD CCM, scrambled CCM, and high BDNF CCM were healthier than CM treated rats. While anatomical and nerve function tests demonstrate regeneration of the pudendal nerve with any CCM treatment, LPP results suggest it takes longer to recover continence with reduced BDNF in CCM. BDNF in MSC CCM is an important factor for the acceleration of recovery from a dual nerve and muscle injury.

Novel cationic hyperbranched polymers were prepared from 2-aminoethyl methacrylate (AEMA) and di(ethylene glycol) methyl ether methacrylate (DEGMA) via the reversible addition-fragmentation chain transfer (RAFT) polymerization for siRNA delivery. Non-degradable and acid-degradable hyperbranched polymers were synthesized using N,N\'-methylenebis(acrylamide) (MBAm) and 2,2-dimethacroyloxy-1-ethoxypropane (DEP) cross-linkers, respectively. Both types of polymers were capable of forming very stable nanosized polyplexes with siRNA. Epidermal growth factor receptor (EGFR) silencing of 95% was achieved with the acid degradable cationic hyperbranched polymer, and no significant cytotoxicity was observed. Our results confirmed the high potency of using such hyperbranched polymers for the efficient protection and delivery of siRNA.

Tyro3 is a member of the TAM subfamily of receptor tyrosine kinases alongside Axl and MerTK, which are activated by homologous ligands Gas6 and protein S. The TAMs activate signalling pathways that mediate diverse functions including cell survival, proliferation, phagocytosis and immune regulation, and defects in TAM-dependent processes are associated with the development of human autoimmune diseases and numerous cancers. In this study, we have focused on the signalling and functional roles of Tyro3, about which much remains unknown. For this purpose, we used cultured human cancer cell lines with different levels of TAM expression to reveal the relative significance of Tyro3 amongst the TAMs. Knockdown of Tyro3 expression by siRNA in MGH-U3 cells, which express Tyro3 as sole TAM, caused a reduction in cell viability, which could not be rescued by TAM ligand, demonstrating the dependence of these cells solely on Tyro3. In contrast, the reduced viability of SCC-25 cells upon Tyro3 knockdown could be rescued by Gas6 as these cells express both Tyro3 and Axl and hence Axl expression was preserved. An increase in the fraction of Tyro3 knockdown cells in the early apoptotic phase was observed in four different cell lines each with a different TAM expression profile, revealing a broad anti-apoptotic function of Tyro3. Furthermore, in the Tyro3-dependent cells, Tyro3 depletion caused a significant increase in cells in the G0/G1 phase of the cell cycle concomitant with decreases in the G2/M and S phases. In addition, a cancer pathway gene discovery array revealed distinct sets of genes that were altered in expression in cancer cells upon Tyro3 knockdown. Together, these results have elucidated further a role of Tyro3 in promoting multiple tumour-supporting pathways in human cancer cells, which differs in extent depending on the presence of other TAMs in the same cells.