In addition to its well-recognized antimicrobial properties, lysozyme can also modulate the inflammatory response. This ability may be particularly important in the gastrointestinal tract where inappropriate inflammatory reactions can damage the intestinal epithelium, leading to significant health problems. The consumption of milk from transgenic goats producing human lysozyme (hLZ) in their milk therefore has the potential to positively impact intestinal health. In order to investigate the effect of hLZ-containing milk on the inflammatory response, young pigs were fed pasteurized milk from hLZ or non-transgenic control goats and quantitative real-time PCR was performed to assess local expression of TNF-α, IL-8, and TGF-β1 in the small intestine. Histological changes were also investigated, specifically looking at villi width, length, crypt depth, and lamina propria thickness along with cell counts for intraepithelial lymphocytes and goblet cells. Significantly higher expression of anti-inflammatory cytokine TGF-β1 was seen in the ileum of pigs fed pasteurized milk containing hLZ (P = 0.0478), along with an increase in intraepithelial lymphocytes (P = 0.0255), and decrease in lamina propria thickness in the duodenum (P = 0.0001). Based on these results we conclude that consuming pasteurized milk containing hLZ does not induce an inflammatory response and improves the health of the small intestine in pigs.
Stearoyl-CoA desaturase enzyme converts specific medium- and long-chain saturated fatty acids to their monounsaturated form. Transgenic goats expressing a bovine beta-lactoglobulin promoter-rat stearoyl-CoA desaturase cDNA construct in mammary gland epithelial cells were produced by pronuclear microinjection. The fatty acid composition of milk from 4 female transgenic founders was analyzed on d 7, 14, and 30 of their first lactation. In 2 animals, the expression of the transgene changed the overall fatty acid composition of the resulting milk fat to a less saturated and more monounsaturated fatty acid profile at d 7 of lactation; however, this effect diminished by d 30. In addition, one animal had an increased proportion of the rumen-derived monounsaturated fatty acid C18:1 trans11 converted by stearoyl-CoA desaturase to the conjugated linoleic acid isomer C18:2 cis9 trans11. Milk that has higher proportions of monounsaturated fatty acids and conjugated linoleic acid may have benefits for human cardiovascular health.
Chemotherapeutic regimens for ovarian cancer often include the use of DNA interstrand crosslink-inducing agents (e.g., platinum drugs) or DNA double-strand break-inducing agents. Unfortunately, the majority of patients fail to maintain a durable response to treatment, in part, due to drug resistance, contributing to a poor survival rate. In this study, we report that cisplatin sensitivity can be restored in cisplatin-resistant ovarian cancer cells by targeting the chromatin-associated high-mobility group box 3 (HMGB3) protein. HMGB proteins have been implicated in the pathogenesis and prognosis of ovarian cancer, and HMGB3 is often upregulated in cancer cells, making it a potential selective target for therapeutic intervention. Depletion of HMGB3 in cisplatin-sensitive and cisplatin-resistant cells resulted in transcriptional downregu-lation of the kinases ATR and CHK1, which attenuated the ATR/CHK1/p-CHK1 DNA damage signaling pathway. HMGB3 was associated with the promoter regions of ATR and CHK1, suggesting a new role for HMGB3 in transcriptional regulation. Furthermore, HMGB3 depletion significantly increased apoptosis in cisplatin-resistant A2780/CP70 cells after cisplatin treatment. Taken together, our results indicate that targeted depletion of HMGB3 attenuates cisplatin resistance in human ovarian cancer cells, increasing tumor cell sensitivity to platinum drugs.Significance: This study shows that targeting HMGB3 is a potential therapeutic strategy to overcome chemoresistance in ovarian cancer.
Epigenetic re-programming is an important event in the development of primordial germ cells (PGC) into functional gametes, characterized by genome-wide erasure of DNA methylation and re-establishment of epigenetic marks, a process essential for restoration of the potential for totipotency. In this study changes in the methylation status of centromeric repeats and two IGF2-H19 differentially methylated domain (DMD) sequences were examined in porcine PGC between Days 24 and 31 of pregnancy. The methylation levels of centromeric repeats and IGF2-H19 DMD sequences decreased rapidly from Days 24 to 28 in both male and female PGC. At Days 30 and 31 of pregnancy centromeric repeats and IGF2-H19 DMD sequences acquired new methylation in male PGC, while in female PGC these sequences were completely demethylated by Day 30 and remained hypomethylated at Day 31. To characterize methylation changes that PGC undergo in culture, the methylation status of embryonic germ cells (EGCs) derived from PGC at Day 26 of pregnancy was examined. Centromeric repeats and IGF2-H19 DMD sequences were similarly methylated in both male and female EGC and hypermethylated in female EGC compared with female PGC at the same embryonic age. Our results show that, similar to murine PGC, porcine PGC undergo genome-wide DNA demethylation shortly after arrival in the genital ridges. When placed in culture porcine PGC terminate their demethylation program and may acquire new DNA methylation marks. To our knowledge, this is the first report regarding epigenetic re-programming of genital ridge PGC in the pig.
Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that protects the genome from chromosomal instability. RAD51 mediator proteins (i.e. paralogs) are critical for efficient HR in mammalian cells. However, how HR-deficient cells process DSBs is not clear. Here, we utilized a loss-of-function HR-reporter substrate to simultaneously monitor HR-mediated gene conversion and non-conservative mutation events. The assay is designed around a heteroallelic duplication of the Aprt gene at its endogenous locus in isogenic Chinese hamster ovary cell lines. We found that RAD51D-deficient cells had a reduced capacity for HR-mediated gene conversion both spontaneously and in response to I-SceI-induced DSBs. Further, RAD51D-deficiency shifted DSB repair toward highly deleterious single-strand annealing (SSA) and end-joining processes that led to the loss of large chromosomal segments surrounding site-specific DSBs at an exceptionally high frequency. Deletions in the proximity of the break were due to a non-homologous end-joining pathway, while larger deletions were processed via a SSA pathway. Overall, our data revealed that, in addition to leading to chromosomal abnormalities, RAD51D-deficiency resulted in a high frequency of deletions advancing our understanding of how a RAD51 paralog is involved in maintaining genomic stability and how its deficiency may predispose cells to tumorigenesis.
Gene targeting by homologous recombination involves the exchange of genetic information between genomic and exogenous deoxyribonucleic acid (DNA) molecules via crossover events. These exchanges are guided by homologous sequences acted on by enzymatic machinery of the cell. Homologous recombination provides a mechanism for targeting defined modifications into genes of interest, making gene‐targeting technologies valuable tools to explore gene function and to develop human, genetic disease models. Gene targeting, however, is inefficient, making the process challenging. Advances in technology now allow us to direct repair enzymes to the targeted site by inducing site‐specific DNA damage using zinc‐finger nucleases, transcription activator‐like effector nucleases, clustered regularly interspaced short palindromic repeats and triplex‐forming oligonucleotides. Further, antirecombinogenic pathways can now be transiently suppressed using ribonucleic acid (RNA) interference (RNAi) and other small molecule approaches. These and other techniques can greatly enhance gene‐targeting efficiency. The coincident development of human stem cell technology brings forth the potential of gene‐targeting strategies for therapeutic application. Key Concepts: Homology‐directed gene targeting utilises homologous recombination to introduce defined modifications into sequences of interest in mammalian genomes. Gene‐targeted knockout and knock‐in models are instrumental in elucidating gene function and studying human genetic diseases. Combining stem cell and gene‐targeting technologies opens potential avenues for gene therapy. Recent technological advances have greatly enhanced gene‐targeting efficiencies. Due to technological advances, most genes in a variety of mammalian species can now be manipulated by homology‐directed gene targeting.
<div>Abstract<p>Chemotherapeutic regimens for ovarian cancer often include the use of DNA interstrand crosslink–inducing agents (e.g., platinum drugs) or DNA double-strand break–inducing agents. Unfortunately, the majority of patients fail to maintain a durable response to treatment, in part, due to drug resistance, contributing to a poor survival rate. In this study, we report that cisplatin sensitivity can be restored in cisplatin-resistant ovarian cancer cells by targeting the chromatin-associated high-mobility group box 3 (HMGB3) protein. HMGB proteins have been implicated in the pathogenesis and prognosis of ovarian cancer, and HMGB3 is often upregulated in cancer cells, making it a potential selective target for therapeutic intervention. Depletion of HMGB3 in cisplatin-sensitive and cisplatin-resistant cells resulted in transcriptional downregulation of the kinases ATR and CHK1, which attenuated the ATR/CHK1/p-CHK1 DNA damage signaling pathway. HMGB3 was associated with the promoter regions of <i>ATR</i> and <i>CHK1</i>, suggesting a new role for HMGB3 in transcriptional regulation. Furthermore, HMGB3 depletion significantly increased apoptosis in cisplatin-resistant A2780/CP70 cells after cisplatin treatment. Taken together, our results indicate that targeted depletion of HMGB3 attenuates cisplatin resistance in human ovarian cancer cells, increasing tumor cell sensitivity to platinum drugs.</p>Significance:<p>This study shows that targeting HMGB3 is a potential therapeutic strategy to overcome chemoresistance in ovarian cancer.</p></div>
<p>The Supplementary Materials include figures, which show siRNA-mediated depletion of HMGB1, HMGB2 and HMGB3 proteins in U2OS cells (Supplementary Figure 1), expression levels of HMGB3 in human ovarian cancer cells (Supplementary Figure 2), LD50 values determined from cisplatin treatment of the A2780 and the A2780/CP70 cells via MTT assays (Supplementary Figure 3), and a luciferase assay to demonstrate transcriptional repression by HMGB3 (Supplementary Figure 4). In addition, there are 3 tables showing the siRNA sequences and the primer sequences used to measure gene expression levels and used for ChIP assays.</p>
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