ABSTRACT
Irgarol 1051 is an herbicide extensively utilized in antifouling paint due to its ability to inhibit photosynthesis. Irgarol and its photodegradation products are highly persistent in waters and sediments, although they are present in low concentrations. However, our understanding of the harmful effects of Irgarol on non-target organisms remains limited. In this study, we assessed the effects of acute (24 h) and chronic (14 days across three generations) exposure to different concentrations (including the 1/10 NOEC, NOEC, and 1/10 LC50 calculated from the 24-h acute toxicity test) of Irgarol using the water flea Moina macrocopa. Acute exposure to 1/10 LC50 significantly decreased survival, feeding rate, thoracic limb activity, heart rate, and acetylcholinesterase activity. Elevated levels of intracellular reactive oxygen species and malondialdehyde, along with a significant increase in catalase and superoxide dismutase activity, suggested the induction of oxidative stress in response to 1/10 LC50. An initial boost in glutathione level and the enzymatic activities of glutathione peroxidase and glutathione reductase, followed by a plunge, implies some compromise in the antioxidant defense system. Upon chronic exposure to the NOEC value, both generations F1 and F2 displayed a significant decrease in survival rate, body length, number of neonates per brood, and delayed sexual maturation, suggesting maternal transfer of potential damage through generations. Taken together, Irgarol induced acute toxicity through physiological and cholinergic damage, accompanied by the induction of oxidative stress, in the water flea. Even its sub-lethal concentrations can induce detrimental effects across generations when consistently exposed.
ABSTRACT
The implantation of good-quality embryos to the receptive endometrium is essential for successful live birth through in vitro fertilization (IVF). The higher the quality of embryos, the higher the live birth rate per cycle, and so efforts have been made to obtain as many high-quality embryos as possible after fertilization. In addition to an effective controlled ovarian stimulation process to obtain high-quality embryos, the composition of the embryo culture medium in direct contact with embryos in vitro is also important. During embryonic development, under the control of female sex hormones, the fallopian tubes and endometrium create a microenvironment that supplies the nutrients and substances necessary for embryos at each stage. During this process, the development of the embryo is finely regulated by signaling molecules, such as growth factors and cytokines secreted from the epithelial cells of the fallopian tube and uterine endometrium. The development of embryo culture media has continued since the first successful human birth through IVF in 1978. However, there are still limitations to mimicking a microenvironment similar to the reproductive organs of women suitable for embryo development in vitro. Efforts have been made to overcome the harsh in vitro culture environment and obtain high-quality embryos by adding various supplements, such as antioxidants and growth factors, to the embryo culture medium. Recently, there has been an increase in the number of studies on the effect of supplementation in different clinical situations such as old age, recurrent implantation failure (RIF), and unexplained infertility; in addition, anticipation of the potential benefits from individuation is rising. This article reviews the effects of representative supplements in culture media on embryo development.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor , Melatonin , Female , Humans , Pregnancy , Culture Media/chemistry , Culture Media/pharmacology , Cytokines , Insulin-Like Growth Factor I , Melatonin/pharmacologyABSTRACT
Liver transplantation is the most effective treatment option for patients with acute or chronic liver failure. However, the applicability and effectiveness of this modality are often limited by a shortage of donors, surgical complications, high medical costs, and the need for continuing immunosuppressive therapy. An alternative approach is liver cell transplantation. LIGHT (a member of the tumor necrosis factor superfamily) could be a promising candidate for promoting the differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) into hepatocyte-like cells. In this study, we investigated the effect of LIGHT on hBM-MSC differentiation into hepatocyte-like cells. Our previous results showed that LIGHT receptor lymphotoxin-ß receptor (LTßR) is constitutively expressed on the surface of hBM-MSCs. Upon treatment with recombinant human LIGHT (rhLIGHT), the phenotype of hBM-MSCs changed to round or polygonal cells. In addition, the cells exhibited high levels of hepatocyte-specific markers, including albumin, cytokeratin-18 (CK-18), CK-19, cytochrome P450 family 1 subfamily A member 1 (CYP1A1), CYP1A2, CYP3A4, SRY-box transcription factor 17 (SOX17), and forkhead box A2 (FOXA2). These results indicate that rhLIGHT enhances the differentiation of hBM-MSCs into functional hepatocyte-like cells. Furthermore, rhLIGHT-induced hepatocyte-like cells showed a higher ability to store glycogen and uptake indocyanine green compared with control cells, indicating functional progression. Additionally, treatment with rhLIGHT increased the number, viability, and proliferation of cells by inducing the S/G2/M phase and upregulating the expression of various cyclin and cyclin dependent kinase (CDK) proteins. We also found that the hepatogenic differentiation of hBM-MSCs induced by rhLIGHT was mediated by the activation of signal transducer and activator of transcription 3 (STAT3) and STAT5 pathways. Overall, our findings suggest that LIGHT plays an essential role in promoting the hepatogenic differentiation of hBM-MSCs. Hence, LIGHT may be a valuable factor for stem cell therapy.
Subject(s)
Bone Marrow , Mesenchymal Stem Cells , Humans , Bone Marrow Cells , Cell Differentiation , Hepatocytes/metabolism , Cyclin-Dependent Kinases/metabolism , Cells, Cultured , Tumor Necrosis Factor Ligand Superfamily Member 14/pharmacologyABSTRACT
Fermentation of red ginseng (RG) produces fermented red ginseng (FRG), thereby increasing the relative amount of downstream ginsenosides, including compound Y (CY), F2, Rh2, compound K (CK), compound O, protopanaxadiol (PPD), and protopanaxatriol (PPT). These downstream ginsenosides have beneficial pharmacological effects, and are easily absorbed by the human body. Based on these expectations, a randomized, single-dose, two-period, crossover clinical trial was planned to compare the pharmacokinetic characteristics of seven types (Rb1, CY, F2, CK, Rh2, PPD, and PPT) of ginsenoside components after FRG and RG administration. The safety and tolerability profiles were assessed in this clinical trial. Sixteen healthy Korean male subjects were administered 6 g of FRG or RG. All ginsenosides except Rb1 showed higher systemic exposure after FRG administration than after RG administration, based on comparisons of ginsenoside Cmax and area under the concentration-time curve (AUC) between FRG and RG. CK, the main ginsenoside component produced during the fermentation process, had 69.23/74.53-fold higher Cmax/AUClast after administration of FRG than RG, and Rh2 had 20.27/18.47-fold higher Cmax/AUClast after administration of FRG than RG. In addition, CY and F2 were detected in FRG; however, all plasma concentrations of CY and F2, except in one subject, were below the lower limit of quantification in RG. There were no clinically significant findings with respect to clinical laboratory tests, blood pressures, or adverse events. Therefore, regular administration of FRG may exert better pharmacological effects than RG.
ABSTRACT
In this study, the potential effects of 30-day exposure to environmentally relevant concentrations of chlorothalonil (0, 5, 10, and 20 µg L-l) were tested in the blood of the economically important olive flounder, Paralichthys olivaceus, using biochemical endpoints. Significant decreases in the enzymatic activities of immunity markers, alternative complements, and lysozymes were detected in the fish exposed to 10 or 20 µg L-l of chlorothalonil at day 20 or 30. The total immunoglobulin content was lowered in response to 20 µg L-l chlorothalonil at day 10 and 20, even when later exposed to 5 µg L-l at day 30. Among the essential blood components, the cortisol level was increased in response to chlorothalonil throughout the study with a decrease in white blood cells, while no changes were observed in hemoglobin, red blood cells, total protein concentration, and glucose in all exposures. The enzymatic activities of the three hepatic toxicity markers, alanine transferases, aspartate transaminase, and alkaline phosphatase, increased by 10 and/or 20 µg L-l of chlorothalonil. Significant oxidative stress was induced by chlorothalonil in the fish exposed to 10 or 20 µg L-l of chlorothalonil, as revealed by increased malondialdehyde and fluctuating glutathione levels with increase in the enzymatic activities of antioxidant defense system, including catalase and superoxide dismutase, during exposure. Taken together, these results suggest that long-term exposure to environmentally relevant concentrations of chlorothalonil can affect susceptibility to pathogens through immunosuppression, hepatic toxicity, and oxidative stress in olive flounder. These results can contribute to the monitoring of aquatic environments and ecotoxicological research through the measurement of blood components against waterborne chlorothalonil.
Subject(s)
Biofouling , Disinfectants , Flounder , Water Pollutants, Chemical , Animals , Antioxidants/metabolism , Biofouling/prevention & control , Disinfectants/toxicity , Flounder/metabolism , Nitriles , Oxidative Stress , Water Pollutants, Chemical/metabolismABSTRACT
Here, we report the complete mitogenome information of the terebellid polychaete, Thelepus plagiostoma (Schmarda, 1861). Genome sequencing by Illumina HiSeq platform permitted assembly of a circular mitochondrial genome of 15,628 bp from T. plagiostoma consisting of 67% AT nucleotides, 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a non-coding region in the typical annelid gene composition. Gene order of the T. plagiostoma mitochondrion is identical to those of the Terebelliformia mitogenomes. Phylogenetic reconstruction places T. plagiostoma within the monophyletic subclass Sedentaria, a sister to Pista cristata in the suborder Terebelliformia.
ABSTRACT
The common sunstar, Crossaster papposus, belongs to the family Solasteridae whose ordinal classification has been unstable. Here, for the first time, we assembled and annotated the complete mitochondrial genome of the common sunstar, C. papposus Linnaeus, 1767. The circular genome of C. papposus is 16,335 bp in length and contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, a control region, and large and small ribosomal subunits. The overall genomic structure and gene arrangement were identical to the reported mitochondrial genomes of sea star species, and a phylogenetic analysis of 13 PCGs recovers a closest relationship with the derived cluster of the paraphyletic order Valvatida.
ABSTRACT
The mammalian Target of Rapamycin (mTOR) pathway regulates a variety of physiological processes, including cell growth and cancer progression. The regulatory mechanisms of these signals are extremely complex and comprise many feedback loops. Here, we identified the deubiquitinating enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We demonstrated that OTUD5 stabilized ß-transducin repeat-containing protein 1 (ßTrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a dRheb-induced curled wing phenotype by RNA interference of Duba, a fly ortholog of OTUD5, in Drosophila melanogaster. Furthermore, OTUD5 knockdown inhibited the proliferation of the cancer cell lines with mutations activating mTOR pathway. Our results suggested a positive feedback loop between OTUD5 and mTOR signaling pathway.
Subject(s)
Cell Proliferation , Endopeptidases/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , Signal Transduction , Animals , Autophagy , Deubiquitinating Enzymes/metabolism , Drosophila melanogaster , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , MCF-7 Cells , Phosphorylation , RNA Interference , UbiquitinationABSTRACT
Aryl hydrocarbon receptor nuclear translocator (ARNT) plays an essential role in maintaining cellular homeostasis in response to environmental stress. Under conditions of hypoxia or xenobiotic exposure, ARNT regulates the subset of genes involved in adaptive responses, by forming heterodimers with hypoxia-inducible transcription factors (HIF1α and HIF2α) or aryl hydrocarbon receptor (AhR). Here, we have shown that ARNT interacts with DDB1 and CUL4-associated factor 15 (DCAF15), and the aryl sulfonamides, indisulam and E7820, induce its proteasomal degradation through Cullin-RING finger ligase 4 containing DCAF15 (CRL4DCAF15) E3 ligase. Moreover, the two known neo-substrates of aryl sulfonamide, RNA-binding motif protein 39 (RBM39) and RNA-binding motif protein 23 (RBM23), are not required for ARNT degradation. In line with this finding, aryl sulfonamides inhibited the transcriptional activities of HIFs and AhR associated with ARNT. Our results collectively support novel regulatory roles of aryl sulfonamides in both hypoxic and xenobiotic responses.
Subject(s)
Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Sulfonamides/therapeutic use , Ubiquitin-Protein Ligases/metabolism , Animals , Humans , Sulfonamides/pharmacology , TransfectionABSTRACT
Immunomodulatory drugs (IMiDs) exert anti-myeloma activity by binding to the protein cereblon (CRBN) and subsequently degrading IKZF1/3. Recently, their ability to recruit E3 ubiquitin ligase has been used in the proteolysis targeting chimera (PROTAC) technology. Herein, we design and synthesize a novel IMiD analog TD-106 that induces the degradation of IKZF1/3 and inhibits the proliferation of multiple myeloma cells in vitro as well as in vivo. Moreover, we demonstrate that TD-428, which comprises TD-106 linked to a BET inhibitor, JQ1 efficiently induce BET protein degradation in the prostate cancer cell line 22Rv1. Consequently, cell proliferation is inhibited due to suppressed C-MYC transcription. These results, therefore, firmly suggest that the newly synthesized IMiD analog, TD-106, is a novel CRBN modulator that can be used for targeted protein degradation.
Subject(s)
Immunologic Factors/pharmacology , Peptide Hydrolases/metabolism , Proteolysis/drug effects , Adaptor Proteins, Signal Transducing , Animals , Cell Line, Tumor , Female , Humans , Immunologic Factors/chemical synthesis , Immunologic Factors/chemistry , Mice , Piperidones/chemical synthesis , Piperidones/chemistry , Piperidones/pharmacology , Ubiquitin-Protein Ligases , Xenograft Model Antitumor AssaysABSTRACT
In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL or BCL2L1). This binding disrupted an interaction between Bcl-XL and the pro-apoptotic protein BH3-interacting domain death agonist (Bid). In particular, we found that a 16-mer p73-TAD peptide motif (p73-TAD16) mediates transcription-independent apoptosis, accompanied by cytochrome c release from the mitochondria, by interacting with Bcl-XL Interestingly, the structure of the Bcl-XL-p73-TAD16 peptide complex revealed a novel mechanism of Bcl-XL recognition by p73-TAD. We observed that the α-helical p73-TAD16 peptide binds to a noncanonical site in Bcl-XL, comprising the BH1, BH2, and BH3 domains in an orientation opposite to those of pro-apoptotic BH3 peptides. Taken together, our results indicate that the cytoplasmic apoptotic function of p73 is mediated through a noncanonical mode of Bcl-XL recognition. This finding sheds light on a critical transcription-independent, p73-mediated mechanism for apoptosis induction, which has potential implications for anticancer therapy.
Subject(s)
Apoptosis , Cytoplasm/metabolism , Tumor Protein p73/metabolism , bcl-X Protein/metabolism , Cell Line, Tumor , Cytoplasm/pathology , Humans , Mitochondria/metabolism , Mitochondria/pathology , Models, Molecular , Protein Binding , Protein Domains , Transcription, Genetic , Tumor Protein p73/chemistry , bcl-X Protein/geneticsABSTRACT
MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression. For example, miRNAs repress gene expression by recruiting the miRNA-induced silencing complex (miRISC), a ribonucleoprotein complex that contains miRNA-engaged Argonaute (Ago) and the scaffold protein GW182. Recently, ubiquitin-protein ligase E3 component N-recognin 5 (UBR5) has been identified as a component of miRISC. UBR5 directly interacts with GW182 proteins and participates in miRNA silencing by recruiting downstream effectors, such as the translation regulator DEAD-box helicase 6 (DDX6) and transducer of ERBB2,1/2,2 (Tob1/2), to the Ago-GW182 complex. However, the regulation of miRISC-associated UBR5 remains largely elusive. In the present study, we showed that UBR5 down-regulates the levels of TNF receptor-associated factor 3 (TRAF3), a key component of Toll-like receptor signaling, via the miRNA pathway. We further demonstrated that p90 ribosomal S6 kinase (p90RSK) is an upstream regulator of UBR5. p90RSK phosphorylates UBR5 at Thr637, Ser1227, and Ser2483, and this phosphorylation is required for the translational repression of TRAF3 mRNA. Phosphorylated UBR5 co-localized with GW182 and Ago2 in cytoplasmic speckles, which implies that miRISC is affected by phospho-UBR5. Collectively, these results indicated that the p90RSK-UBR5 pathway stimulates miRNA-mediated translational repression of TRAF3. Our work has added another layer to the regulation of miRISC.
Subject(s)
Autoantigens/metabolism , MicroRNAs/metabolism , RNA-Binding Proteins/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Signal Transduction , TNF Receptor-Associated Factor 3/metabolism , Toll-Like Receptors/metabolism , Ubiquitin-Protein Ligases/metabolism , 3' Untranslated Regions , Amino Acid Substitution , Animals , Autoantigens/genetics , COS Cells , Chlorocebus aethiops , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Mutation , Phosphorylation , Protein Processing, Post-Translational , RNA Interference , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/genetics , TNF Receptor-Associated Factor 3/antagonists & inhibitors , TNF Receptor-Associated Factor 3/genetics , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/geneticsABSTRACT
OBJECTIVE: The aim of the present study was to examine the relationship among male age, strict morphology, and sperm chromatin structure and condensation. METHODS: Sperm samples from a total of 100 men underwent semen analysis, and sperm chromatin structure and condensation were assessed with toluidine blue (TB) and aniline blue (AB) tests. RESULTS: Prevalence of strict morphology of less than 4%, and abnormal sperm chromatin structure and condensation did not show any statistically significant differences according to male age (p=0.605, p=0.235, and p=0.080). No significant correlation was demonstrated among age of male partners, strict morphology, and abnormal sperm chromatin structure using TB and AB tests. However, abnormal sperm chromatin condensation was positively associated with sperm chromatin structure (r=0.594, p=0.000) and showed negative correlation with strict morphology (r=-0.219, p=0.029). CONCLUSION: The tests for sperm chromatin condensation showed a significant association with strict morphology. Further study is needed to elucidate the relationship between clinical outcome and sperm chromatin tests.
ABSTRACT
Hypochondroplasia (HCH) is an autosomal dominant inherited skeletal dysplasia, usually caused by a heterozygous mutation in the fibroblast growth factor receptor 3 gene (FGFR3). A 27-year-old HCH woman with a history of two consecutive abortions of HCH-affected fetuses visited our clinic for preimplantation genetic diagnosis (PGD). We confirmed the mutation in the proband (FGFR3:c.1620C>A, p.N540K), and established a nested allele-specific PCR and sequence analysis for PGD using single lymphocyte cells. We performed this molecular genetic analysis to detect the presence of mutation among 20 blastomeres from 18 different embryos, and selected 9 embryos with the wild-type sequence (FGFR3:c.1620C). A successful pregnancy was achieved through a frozen-thawed cycle and resulted in the full-term birth of a normal neonate. To the best of our knowledge, this is the first report of a successful pregnancy and birth using single-cell allele-specific PCR and sequencing for PGD in an HCH patient.
ABSTRACT
We performed activation tagging screen to isolate abscisic acid (ABA) response mutants. One of the mutants, designated ahs10 (ABA-hypersensitive 10), exhibited ABA-hypersensitive phenotypes. TAIL-PCR analysis of the mutant revealed that T-DNA was inserted in the promoter region of the Arabidopsis gene, At2g01430, which encodes a homeodomain-leucine zipper protein ATHB17. Subsequent expression analysis indicated that ATHB17 was activated in ahs10. To recapitulate the mutant phenotypes, we prepared ATHB17 OX lines and investigated their phenotypes. The results showed that ATHB17 confers ABA-hypersensitivity and drought tolerance. On the contrary, ATHB17 knockout lines were ABA-insensitive and drought-sensitive, further demonstrating that ATHB17 is involved in ABA and water-stress responses. Interestingly, the ATHB17 effect on seedling growth in the presence of ABA was observed only during the postgermination seedling establishment stage, suggesting that it functions during a narrow developmental window of early seedling growth.
Subject(s)
Abscisic Acid/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , DNA, Bacterial/genetics , Seeds/genetics , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Droughts , Gene Expression Regulation, Plant , Gene Knockout Techniques , Genes, Plant , Mutation , Phenotype , Plants, Genetically Modified , Seeds/growth & development , Stress, Physiological , Transcription Factors/geneticsABSTRACT
BACKGROUND: Robotic surgery for rectal cancer may be a way to overcome the limitations of laparoscopic surgery. However, totally robotic surgery for rectal cancer is still technically challenging. This report describes the technical details and outcomes of totally robotic rectal surgery. METHODS: The authors developed a totally robotic surgery technique for rectal cancer and performed it for 45 patients. We designed a six-port system, including a camera port, to perform rectal cancer surgery from the splenic flexure to the pelvic diaphragm in one setup. To check the feasibility and safety of the procedure, perioperative outcomes including conversion rate, morbidity, and mortality were analyzed. RESULTS: The mean body mass index of the 45 patients was 23.6 kg/m(2) (range, 18.8-31.6 kg/m(2)). There was one case (2.2%) of conversion to laparotomy because of a common iliac artery injury. The 30-day morbidity rate was 11.1%. There was no operation-related mortality. CONCLUSIONS: Totally robotic surgery for rectal cancer using the described technique was feasible and safe. This result could facilitate the spread of robotic surgery for rectal cancer and maximize the advantages of robotic surgery.
Subject(s)
Laparoscopy/methods , Rectal Neoplasms/surgery , Robotics/instrumentation , Adult , Aged , Aged, 80 and over , Colon, Transverse/surgery , Feasibility Studies , Female , Humans , Laparoscopy/mortality , Male , Middle Aged , Pelvic Floor/surgery , Postoperative Complications , Rectal Neoplasms/mortality , Treatment OutcomeABSTRACT
OBJECTIVE: To evaluate methods of DNA extraction from single cells for their suitability to amplify and provide a correct diagnosis of target disease genes. DESIGN: Experimental study. SETTING: University hospital laboratory. PATIENT(S): Two normal adult male and female blood donors. INTERVENTION(S): Exon 51 of the dystrophin gene and the ZFX/ZFY gene were amplified from single lymphocytes using nested PCR. Five different methods of DNA extraction were tested (lysis in distilled water with freezing and thawing using liquid nitrogen, lysis in distilled water, alkaline lysis buffer, Proteinase K/sodium dodecyl sulfate (SDS) buffer, and N-lauroylsarcosine salt solution). MAIN OUTCOME MEASURE(S): Allele drop out and amplification rate. RESULT(S): The amplification efficiency from single unaffected lymphocytes was 89.0% using the liquid nitrogen method, 88.1% with the distilled water lysis method, 97.5% with the alkaline lysis buffer method, 91.5% with the Proteinase K/SDS lysis buffer method, and 84.8% using the N-lauroylsarcosine salt solution method. The mean allele drop out rate was 16.7%, 43.9%, 2.0%, 9.8%, and 18.9%, respectively, for each lysis method using single male lymphocytes as a template. CONCLUSION(S): Based on these results, DNA extraction using an alkaline lysis buffer results in more efficient rates of DNA amplification and less allele drop out than the other methods of DNA extraction tested. This method is suitable for the lysis of single cells in clinical preimplantation genetic diagnosis.
Subject(s)
Cell Fractionation/methods , Chemical Fractionation/methods , DNA/genetics , DNA/isolation & purification , Gene Frequency/genetics , Polymerase Chain Reaction/methods , Preimplantation Diagnosis/methodsABSTRACT
This study was performed to evaluate the efficiency of simplified EM grid vitrification, skipping the step of removing the cryoprotectant (5.5M EG + 1.0M sucrose) droplet on the grid after loading oocytes, compared to conventional cryopreservation protocols for mouse mature oocytes. Firstly, the recovery, survival, fertilization and hatching rates of simplified EM grid vitrification were compared with those of the slow freezing method using 1.5M DMSO. Then, conventional EM grid vitrification was compared with simplified EM grid vitrification. Simplified EM grid vitrification showed higher survival, fertilization and hatching rates than those of the slow freezing method (85.6% vs. 63.2%; 51.0% vs. 22.3%; 38.7% vs. 12.5%, p < 0.01, respectively). Moreover, simplified EM grid vitrification showed higher recovery, survival and fertilization rates than those of conventional EM grid vitrification (100% vs. 95.0%, p=0.024; 90.0% vs. 78.9%, p=0.033; 56.7% vs. 38.7%, p=0.021, respectively). Hatching rate tended to be higher for simplified EM grid vitrification compared to conventional EM grid vitrification (41.1% vs. 24.1%). In conclusion, simplified EM grid vitrification is a convenient and efficient method for cryopreservation of mouse mature oocytes, compared to conventional EM grid vitrification and slow freezing methods.