Search Results (5,114)

Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications. Full article

Background/Objectives: Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) function as effectors in the Hippo pathway and have attracted attention due to their association with tumor formation. Glucose transporter (GLUT) proteins also contribute to the proliferation of cancer cells. In this study, we investigated the effect of YAP/TAZ on GLUT1 expression in endometrial carcinoma, as well as the clinical relevance and prognostic value of YAP/TAZ. Methods: The effects of YAP and TAZ knockdown and YAP overexpression on GLUT1 expression in human endometrial carcinoma-derived HHUA and Ishikawa cells were evaluated using RT-qPCR. In addition, we performed immunohistochemical expression of 100 tissue samples of diagnosed endometrial carcinoma. Based on staining intensity and the percentage of positively stained tumor cells, the immunoreactivity score was calculated, which ranged from 0 to 12. Results: YAP/TAZ were identified as important factors in the regulation of GLUT1 expression in HHUA and Ishikawa cells. In addition, a significant correlation (progression-free survival p < 0.05) was observed between TAZ and GLUT1 expression in tissues from endometrial carcinoma patients, and nuclear expression of TAZ was associated with poor prognosis (p < 0.05). Conclusions: YAP/TAZ promote tumor growth via GLUT1. Therapeutic targeting of YAP/TAZ could therefore be useful in the development of future treatments. Full article

Molecular chaperones, especially heat shock proteins (HSPs) have vital functions in cells’ responses to stress. Here, we cloned and sequenced the complete complementary DNA encoding HSP90 (MjHSP90) from the shrimp Marsupenaeus japonicus. The MjHSP90 cDNA comprised 3162 bp, including a 2172 bp coding region encoding a 724 amino acid-protein (predicted molecular mass = 83.12 kDa). Homology and phylogenetic analyses showed that MjHSP90 was highly conserved and most homologous to Litopenaeus vannamei HSP90. MjHSP90 is expressed in all tested tissues, with high expression in gill tissue and the hepatopancreas. Cold stress significantly upregulated MjHSP90 expression in the gill and hepatopancreas (p < 0.05). Following RNA interference knockdown of MjHSP90, the cold stress-related death rate of the shrimp increased significantly, accompanied by significantly upregulated expression of apoptosis-related genes Mjcaspase-3 and Mjbcl-2 (p < 0.05) and an increase in the number of apoptotic cells. The results indicated that MjHSP90 might play a pivotal role in the shrimp’s immune response to cold stress. Full article

Acute myeloid leukemia (AML) is a malignant hematological tumor with a high prevalence in elderly people, and circular RNA (circRNA) plays an important role in age-related diseases. Induction of cancer cell senescence is a highly promising therapeutic strategy; however, the presence of senescence-associated circRNAs in AML remains to be elucidated. Here, we show that the expression patterns of circRNAs differed between elderly AML patients and healthy volunteers. circSATB1 was significantly overexpressed in elderly patients and AML cells. Knockdown of circSATB1 resulted in the inhibition of proliferation and arrest of the cell cycle in the G0/G1 phase; no effect on apoptosis or DNA integrity was observed, and precocious cellular senescence was promoted, characterized by no change in telomere length. Database analysis revealed that there may be two miRNA and nine RNA-binding proteins (RBPs) involved in regulating the cellular functions of circSATB1. Our observations uncover circSATB1-orchestrated cell senescence in AML, which provides clues for finding more modest therapeutic targets for AML. Full article

Grapholita molesta is a globally significant fruit pest. Females achieve maximal reproductive output through efficient sperm utilization following a single copulation. Post-mating maturation of eupyrene sperm is a critical step in reproductive success. Here, we report that a male accessory gland-derived serine protease (named GmAGSP1) is essential for this process. GmAGSP1 was only distantly related to other identified sperm-activating SPs, and its transcript was highly expressed in the AG at 48 h after emergence. RNAi-mediated knockdown of GmAGSP1 in males did not affect courtship rate, copulation duration, or mating frequency, whereas male fertility decreased significantly. Mating with GmAGSP1-knockdown males markedly impaired eupyrene sperm maturation in the spermatophores, with phenotypes including failure of eupyrene sperm bundles to dissociate normally and marked reduction in viability of the dissociated eupyrene sperm. Finally, untargeted metabolomic analysis preliminarily demonstrated marked alterations in multiple metabolic pathways within the spermatophore following mating with GmAGSP1-knockdown males. This study advances our understanding of the regulatory mechanism of “sperm activation in the spermatophore’s metabolic microenvironment mediated by male AG-derived SP” while providing critical insights for the development of novel genetic control strategies targeting G. molesta. Full article

Xylosyltransferase-I (XT-I) plays a crucial role in skeletal development and cartilage integrity. An XT-I deficiency is linked to severe bone disorders, such as Desbuquois dysplasia type 2. While animal models have provided insights into XT-I’s role during skeletal development, its specific effects on adult bone homeostasis, particularly in human mesenchymal stem cell (hMSC) differentiation, remain unclear. This study investigates how XT-I deficiency impacts the differentiation of hMSCs into chondrocytes, osteoblasts, and adipocytes—key processes in bone formation and repair. The aim of this study was to elucidate for the first time the molecular mechanisms by which XT-I deficiency leads to impaired bone homeostasis. Using CRISPR-Cas9-mediated gene editing, we generated XYLT1 knockdown (KD) hMSCs to assess their differentiation potential. Our findings revealed significant disruption in the chondrogenic differentiation in KD hMSCs, characterized by the altered expression of regulatory factors and extracellular matrix components, suggesting premature chondrocyte hypertrophy. Despite the presence of perilipin-coated lipid droplets in the adipogenic pathway, the overall leptin mRNA and protein expression was reduced in KD hMSCs, indicating a compromised lipid metabolism. Conversely, osteogenic differentiation was largely unaffected, with KD and wild-type hMSCs exhibiting comparable mineralization processes, indicating that critical aspects of osteogenesis were preserved despite the XYLT1 deficiency. In summary, these results underscore XT-I’s pivotal role in regulating differentiation pathways within the bone marrow niche, influencing cellular functions critical for skeletal health. A deeper insight into bone biology may pave the way for the development of innovative therapeutic approaches to improve bone health and treat skeletal disorders. Full article

The Notch signaling pathway is a critical regulator of embryonic brain development. Among its four mammalian receptors, Notch1 and Notch2 are particularly significant in the developing cortex, yet their roles in human neurodevelopment are not well understood. In murine cortex development, Notch1 primarily regulates early progenitor identity and neurogenesis, while Notch2 is required for maintaining radial glial cells at later stages. However, it is unclear whether these functions are conserved in the human developing brain. In this study, we used cerebral organoids as an in vitro model of early human corticogenesis and conducted lentiviral shRNA-mediated knockdowns of NOTCH1 and NOTCH2. Our findings indicate that NOTCH1 is essential for organoid growth, lumen morphogenesis, radial glial identity, and progenitor proliferation. In contrast, depleting NOTCH2 did not significantly affect these early developmental processes. These results demonstrate that NOTCH1 and NOTCH2 have potentially non-redundant and temporally distinct roles in early human corticogenesis, reflecting receptor-specific specialization within the Notch signaling pathway. Full article

Background/Objectives: Glioblastoma (GBM) is an aggressive brain tumor known for its profound heterogeneity and treatment resistance. Dysregulated complement signaling and epigenetic alterations have been implicated in GBM progression. This study identifies kynureninase (KYNU), a key enzyme in the kynurenine pathway, as a novel regulator of complement components and investigates its interaction with histone deacetylase 6 (HDAC6) in the context of therapeutic targeting. Methods: KYNU expression, and its association with complement signaling in GBM, were analyzed using publicly available datasets (TCGA, GTEx, HPA). Pathway enrichment was performed via LinkedOmics. In vitro studies in GBM cell lines (U87, U251, T98G) assessed the effects of KYNU silencing and treatment with an HDAC6 inhibitor (tubastatin) and a BET inhibitor (apabetalone) on gene expression and cell viability. Results: Bioinformatic analyses revealed significant overexpression of KYNU in GBM tissues compared to normal brain tissue. KYNU expression was positively associated with genes involved in complement and coagulation cascades. In vitro experiments demonstrated that KYNU silencing reduced the expression of C3, C3AR1, and C5AR1 and suppressed GBM cell viability. Treatment with tubastatin, while reducing viability, paradoxically upregulated complement genes, suggesting potential limitations in therapeutic efficacy. However, this effect was mitigated by KYNU knockdown. Combined treatment with apabetalone and tubastatin effectively suppressed KYNU expression and enhanced cytotoxicity, particularly in cells with high complement expression. Conclusions: Our findings establish the KYNU–HDAC6–complement axis as a critical regulatory pathway in GBM. Targeting KYNU-mediated complement activation through combined epigenetic approaches—such as HDAC6 and BET inhibition—represents a promising strategy to overcome complement-driven resistance in GBM therapy. Full article

Endometriosis is a disorder characterized by the presence of endometrial tissue outside the uterus, leading to dyspareunia, chronic pelvic pain, dysuria, and infertility. The latter has been related to implantation failure associated with alterations in decidualization, a process regulated by sex hormones such as progesterone. Membrane progesterone receptor β (mPRβ) exhibits a lower expression in endometriotic tissues than in normal endometrial ones. However, the role of mPRβ in decidualization is unknown. This work aimed to investigate whether mPRβ plays a role in the decidualization of endometrial stromal cells (ESCs) derived from women with and without endometriosis. The mPR agonist OrgOD-2 induced the gene expression of key decidualization markers (insulin-like growth factor binding protein 1, prolactin, transcription factor heart and neural crest derivatives-expressed transcript 2, and fork-head transcription factor) in healthy ESCs, eutopic (uterine cavity), and ectopic (outside of the uterine cavity) ESCs from women with endometriosis. Notably, the expression of the decidualization markers was lower in endometriotic cells than in healthy endometrial ones. An siRNA mediated knockdown of mPRβ reduced the expression of decidualization-associated genes in ESCs treated with a decidualization stimuli, regardless of whether cells were derived from healthy women or those with endometriosis. Our data suggest that progesterone, through mPRβ activation, regulates the decidualization process in endometrial stromal cells from women with and without endometriosis. Full article

Background and Objectives: Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies worldwide. Although chemotherapy is an effective treatment for colorectal cancer (CRC), its effectiveness is frequently hindered by the emergence of resistant cancer cells. Studies have demonstrated a linkage between drug resistance and the pregnane X receptor (PXR), which influences the metabolism and the transport of chemotherapeutic agents. Likewise, autophagy is also a well-established mechanism that contributes to chemotherapy resistance, and it is closely tied to tumor progression. This pre-clinical study aims to investigate the role of mtKRAS-dependent autophagy with PXR expression after treatment with Irinotecan in colorectal cancer. Methods: CRC lines were treated with specific inhibitors, such as 3-methyladeninee, hydroxychloroquine PI-103, and irinotecan hydrochloride, and subjected to various assays, including MTT for cell viability, Western blot for protein expression, siRNA-mediated PXR knock-out, and confocal microscopy for autophagic vacuole visualization. Protein quantification, gene knockdown, and subcellular localization studies were performed under standardized conditions to investigate treatment effects on autophagy and apoptosis pathways. Conclusions: Our experiments showed that PXR knockdown does not alter autophagy levels following Irinotecan treatment, but it promotes apoptotic cell death despite elevated autophagy. Moreover, late-stage autophagy inhibition reduces PXR expression, whereas induction through PI3K/AKT/mTOR inhibition leads to increased expression of PXR. Our experiments uncover a mechanism by which autophagy facilitates the nuclear translocation of the PXR, thereby promoting resistance to Irinotecan across multiple cell lines. Full article

Background: Peritoneal dissemination in colorectal cancer (CRC) is associated with poor prognosis due to limited efficacy of current therapeutic strategies. The cholinergic receptor nicotinic beta 2 subunit (CHRNB2), a component of the acetylcholine receptor, has been implicated in other malignancies, but its role in CRC remains unknown. Methods: This study evaluated the expression and function of CHRNB2 in CRC. CHRNB2 mRNA levels were quantified by qRT-PCR in cell lines and clinical specimens. Functional assays were conducted using CRC cell lines with high CHRNB2 expression, in which CHRNB2 was knocked down by shRNA. Cell proliferation, migration, and invasion were assessed in vitro. In vivo effects were evaluated using subcutaneous and peritoneal xenograft models. The impact of CHRNB2 monoclonal antibody (mAb) treatment on CRC cell proliferation was also examined. Clinical correlations were assessed between CHRNB2 expression and clinicopathological features, including recurrence patterns. Results: CHRNB2 expression varied among CRC cell lines, with the highest levels observed in LOVO cells. CHRNB2 knockdown significantly inhibited proliferation, migration, and invasion in vitro and suppressed tumor growth in vivo. CHRNB2 mAb treatment reduced cell proliferation. Clinically, high CHRNB2 expression correlated with a significantly higher cumulative rate of peritoneal recurrence, but not with recurrence in the liver, lungs, or lymph nodes. Multivariate analysis identified high CHRNB2 expression and T4 tumor depth as independent predictors of peritoneal recurrence. Conclusions: CHRNB2 promotes the malignant phenotype of CRC, particularly in peritoneal dissemination. These findings suggest that CHRNB2 may serve as a novel diagnostic biomarker and therapeutic target for CRC with peritoneal metastasis. Full article

Myeloid-derived growth factor (MYDGF), named in reference to its secretion from myeloid cells in bone marrow, is a novel protein with anti-apoptotic and tissue-repairing properties. MYDGF is found in various human tissues affected by different diseases. To date, however, MYDGF expression has yet to be reported in the nervous system. Herein, we demonstrate for the first time that MYDGF mRNA levels increased in the zebrafish retina 1 h after optic nerve injury (ONI). MYDGF-producing cells were located in the photoreceptors and infiltrating leukocytic cells. We prepared the retina for MYDGF gene knockdown by performing intraocular injections using either MYDGF-specific morpholino or the CRISPR/Cas9 system. Under these MYDGF-knockdown retinal conditions, anti-apoptotic Bcl-2 mRNA was suppressed; in comparison, apoptotic caspase-3 and inflammatory TNFα mRNA were significantly upregulated in the zebrafish retina after ONI compared to the control. Furthermore, heat shock factor 1 (HSF1) was evidently suppressed under these conditions, leading to a significant number of apoptotic neurons. These findings indicate that MYDGF is a key molecule in the stimulation of neuronal regeneration in the central nervous system. Full article

Tuberous sclerosis complex (TSC) is a systemic disease caused by mutations in either the TSC1 (encoding hamartin) or TSC2 (encoding tuberin) gene, with mutations in the TSC2 gene potentially leading to more severe clinical symptoms. Neurological symptoms are a common clinical manifestation of TSC, and neuroinflammation is thought to play an important role. Glial cells are a major source of neuroinflammation, but whether microglia are involved in the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and the expression of interleukin-1β (IL-1β) in TSC patients remains unclear. We used a transcriptome sequencing dataset for bioinformatics analysis to explore the differences in the expression of microglial inflammasome-associated hub genes. TSC2 knockdown (TSC2 KD) microglia (HMC3 cell line) were generated by lentivirus, and the expression of inflammasome-associated hub genes, microglial activation, and NLRP3 inflammasome activation were verified. In addition, experiments were performed to explore the regulatory effects of rapamycin. Bioinformatics analysis identified a total of eight inflammasome-associated hub genes. By detecting GFP fluorescence, TSC2 mRNA, TSC2 protein expression, and the phosphorylation of the mammalian target of rapamycin (p-mTOR)/mTOR, we confirmed that the TSC2 KD microglia model was successfully established. Compared with the control group, the TSC2 KD group presented higher mRNA levels and fluorescence intensities of microglia AIF1 and CD68, as well as greater reactive oxygen species (ROS) production. Eight inflammasome-associated hub gene mRNA assays revealed that the expression of the NLRP3 and IL1B genes was increased. Compared with the control group, the TSC2 KD group presented increased levels of NLRP3 and Pro-IL-1β proteins in cells and Cleaved-Caspase 1 and Cleaved-IL-1β proteins in the supernatant, suggesting NLRP3 inflammasome activation. Rapamycin intervention alleviated these changes, demonstrating that the TSC2 gene regulation of microglial activation and NLRP3 inflammasome activation are correlated with mTOR phosphorylation. In conclusion, microglia are activated in TSC patients and participate in the NLRP3 inflammasome-associated neuroinflammatory response, and rapamycin treatment can alleviate these changes. Full article

Osteoporosis is a multifactorial, polygenic disease characterized by reduced bone mineral density (BMD) and increased fracture risk. Genome-wide association studies (GWASs) have identified numerous loci associated with BMD and/or bone fractures, but functional characterization of these target genes is essential to understand the biological mechanisms underlying osteoporosis. This review focuses on current methodologies and key examples of successful functional studies aimed at evaluating gene function in osteoporosis research. Functional evaluation typically follows a multi-step approach. In silico analyses using omics datasets expression quantitative trait loci (eQTLs), protein quantitative trait loci (pQTLs), and DNA methylation quantitative trait loci (mQTLs) help prioritize candidate genes and predict relevant biological pathways. In vitro models, including immortalized bone-derived cell lines and primary mesenchymal stem cells (MSCs), are used to explore gene function in osteogenesis. Advanced three-dimensional culture systems provide additional physiological relevance for studying bone-related cellular processes. In situ analyses of patient-derived bone and muscle tissues offer validation in a disease-relevant context, while in vivo studies using mouse and zebrafish models enable comprehensive assessment of gene function in skeletal development and maintenance. Integration of these complementary methodologies helps translate GWAS findings into biological insights and supports the identification of novel therapeutic targets for osteoporosis. Full article

Appendage autotomy frequently occurs during the cultivation of Exopalaemon carinicauda, which severely impacts its survival and economic benefits. To investigate the molecular mechanism underlying appendage regeneration in E. carinicauda, this study presents a comparative transcriptome analysis on samples from different stages of appendage regeneration in individuals of the same family of E. carinicauda. A total of 6460 differentially expressed genes (DEGs) were identified between the samples collected at 0 h post-autotomy (D0) and those collected at 18 h post-autotomy (D18h). Additionally, 7740 DEGs were identified between D0 and 14 d post-autotomy (D14d), with 3382 DEGs identified between D18h and D14d. Among them, differentially expressed genes such as EcR, RXR, BMP1, and Smad4 are related to muscle growth or molting and may be involved in the regeneration process. qRT-PCR results revealed that EcBMPR2 was expressed at relatively high levels in the gonad and ventral nerve cord tissues and that the highest level of expression was detected in the regenerative basal tissue at 24 h post-autotomy. In situ hybridization results indicated strong signals of this gene in the cells at the wound site at 72 h post-autotomy. Following knockdown of EcBMPR2, the expression levels of both EcBMPR1B and EcSmad1 were significantly downregulated, and long-term interference with the EcBMPR2 gene resulted in a significantly slower appendage regeneration process compared to the control group. When the downstream transcription factor EcSmad1 was knocked down, the two receptor genes EcBMPR2 and EcBMPR1B were downregulated, whereas EcBMP7 was upregulated. After inhibiting the BMP signaling pathway, the degree of cell aggregation at the autotomy site in the experimental group was significantly lower than that in the control group, the wound healing rate was delayed, and the blastema regeneration time was prolonged from 5 d to 7 d. Collectively, these results indicate that the BMP signaling pathway plays a critical role in the early stages of appendage regeneration in E. carinicauda. This study provides important theoretical insights for understanding limb regeneration in crustaceans. Full article