The objective of this study was to elucidate the dynamics of microtubules in post-ovulatory aging in vivo and in vitro of mouse oocytes. The fresh ovulated oocytes were obtained from oviducts of superovulated female ICR mice at 16 hours after hCG injection. The post-ovulatory aged oocytes were collected at 24 and 48 hours after hCG injection from in vivo and in vitro, respectively. Immunocytochemistry was performed on s-tubulin and acetylated a-tubulin. The microtubules were localized in the spindle assembly, which was barrel-shaped or slightly pointed at its poles and located peripherally in the fresh ovulated oocytes. The frequency of misaligned metaphase chromosomes were significantly increased in post-ovulatory aged oocytes after 48 hours of hCG injection. The spindle length and width of post-ovulatory aged oocytes were significantly different from those of fresh ovulated oocytes, respectively. The staining intensity of acetylated α-tubulin showed stronger in post-ovulatory aged oocytes than that in the fresh ovulated oocytes. In the aged oocytes, the spindles had moved towards the center of the oocytes from their original peripheral position and elongated, compared with the fresh ovulated oocytes. Microtubule organizing centers were formed and observed in the cytoplasm of the aged oocytes. On the contrary, it was not observed in the fresh ovulated oocytes. The alteration of spindle formation and chromosomes alignment substantiates the poor development and the increase of disorders from the post-ovulatory aged oocytes. It might be important to fertilize on time in ovulated oocytes for the developmental competence of embryos with normal karyotypes.
AbstractScientific methods of analysis are used to reconstruct crime scenes. However, a lack of sufficient bloodfor analysis is common at crime scenes. To apply scientific analysis methods to crime scenes, it is importantto extract components from the very small amounts of blood samples without damage. We previouslydeveloped a novel extraction reagent for analyzing bloodstains at crime scenes. The developedextraction reagent contains Tris-EDTA (TE), which preserves DNA and phosphate-buffered saline (PBS),which preserves proteins. The developed extraction reagent exhibited superior performance with respectto DNA extraction. Blood was collected from 23 healthy adult men and women in vacutainers that didnot contain coagulants or anticoagulants. These samples were used to generate DBSs under differenttemperature and humidity conditions. Proteins were extracted at five time points (day 1, 7, 14, 21, and30) using the developed extraction reagent, previously used agents TE and PBS, as well as theconventional agent double distilled water (D.D.W). The protein extraction performances of theseextraction agents were compared and analyzed using western blotting. We also quantitatively confirmedthe amount of protein extracted using commercialized GAPDH. We cross-validated the protein extractioncapability of the extraction reagent developed using two verification substances from the blood cells ofthe crime scene and confirmed that the protein extraction performance was excellent.
Objective Hyperstimulation methods are broadly used for in vitro fertilization (IVF) in patients with infertility; however, the side effects associated with these therapies, such as ovarian hyperstimulation syndrome (OHSS), have not been well studied. N-glycoproteomes are subproteomes used for the remote sensing of ovarian stimulation in follicular growth. Glycoproteomic variation in human follicular fluid (hFF) has not been evaluated. In this study, we aimed to identify and quantify the glycoproteomes and N-glycoproteins (N-GPs) in natural and stimulated hFF using label-free nano-liquid chromatography/electrospray ionization-quad time-of-flight mass spectrometry. Methods For profiling of the total proteome and glycoproteome, pooled protein samples from natural and stimulated hFF samples were selectively isolated using hydrazide chemistry to obtain the total proteomes and glycoproteomes. N-GPs were validated by the consensus sequence N-X-S/T (92.2% specificity for the N-glycomotif at p<0.05). All data were compared between natural versus hyperstimulated hFF samples. Results We detected 41 and 44 N-GPs in the natural and stimulated hFF samples, respectively. Importantly, we identified 11 N-GPs with greater than two-fold upregulation in stimulated hFF samples compared to natural hFF samples. We also validated the novel N-GPs thyroxine-binding globulin, vitamin D-binding protein, and complement proteins C3 and C9. Conclusion We identified and classified N-GPs in hFF to improve our understanding of follicular physiology in patients requiring assisted reproduction. Our results provided important insights into the prevention of hyperstimulation side effects, such as OHSS. Keywords: Fertilization in vitro; Human follicular fluid; Hyper stimulation; N-glycoprotein; Natural cycle; Proteomics
A circadian rhythm disturbance is one of the essential components of the phenotype of bipolar disorder. It has been reported that casein kinase 1 epsilon (CSNK1E), a member of the clock gene family, is associated with psychiatric phenotypes.We performed a genetic association study to determine the genetic role of CSNK1E in bipolar disorder and circadian rhythm disturbances in the Korean population.The present study included 215 patients with bipolar disorder and 773 controls. Circadian characteristics were measured by the Korean version of the Composite Scale of Morningness (CS). Single-nucleotide polymorphisms (SNPs) of CSNK1E, rs1534891 and rs2075984, were genotyped. Chi-square analyses were performed to evaluate associations involving alleles and genotypes. Haplotype analysis was also performed, and the permutation p value was calculated. We also tested further associations involving these SNPs and scores on the CS.We found a positive association between SNP rs2075984 and bipolar disorder in both the allelic (p = .003) and genotypic (p = .006) distributions. No allelic or genotypic association between SNP rs1534891 and bipolar disorder was observed. A significant association of haplotype with bipolar disorder was found (p = .033). However, no association between the CS and the genotype of either SNP was found in the total sample.CSNK1E SNP rs2075984 seemed to play a significant role in the development of bipolar disorder in this Korean sample. This association does not seem to relate to the phase preference measured by the CS. Further studies on CSNK1E with larger samples and more SNPs are necessary.
Ischemic stroke is caused by blood clot formation and consequent vessel blockage. Proteomic approaches provide a cost-effective alternative to current diagnostic methods, including computerized tomography (CT) scans and magnetic resonance imaging (MRI). To identify diagnostic biomarkers associated with ischemic stroke risk factors, we performed individual proteomic analysis of serum taken from 20 healthy controls and 20 ischemic stroke patients. We then performed SWATH analysis, a data-independent method, to assess quantitative changes in protein expression between the two experimental conditions. Our analysis identified several candidate protein biomarkers, 11 of which were validated by multiple reaction monitoring (MRM) analysis as novel diagnostic biomarkers associated with ischemic stroke risk factors. Our study identifies new biomarkers associated with the risk factors and pathogenesis of ischemic stroke which, to the best of our knowledge, were previously unknown. These markers may be effective in not only the diagnosis but also the prevention and management of ischemic stroke.
Rheumatoid arthritis (RA) is a chronic autoimmune disease that progresses into systemic inflammation and joint deformity. RA diagnosis is a complicated procedure, and early diagnostic methods are insufficient. Therefore, in this study, we attempted to identify new markers to improve the accuracy of RA prescreening. e identified differentially expressed proteins (DEPs) by using liquid chromatography tandem-mass spectrometry in health-prescreening sera with high rheumatoid factor (RF) values, and compared the findings with those from sera with normal RF values. We identified 93 DEPs; of these, 36 were upregulated, and 57 were downregulated in high-RF sera. Pathway analysis revealed that these DEPs were related to immune responses. Additionally, four DEPs were statistically analyzed by proteomic analysis; of these, SAA4 was significantly validated in individual enzyme-linked immunosorbent assays. Moreover, SAA4 was significantly upregulated in RA patients (n = 40, 66.43 ± 12.97 ng/mL) compared with normal controls (n = 40, 4.79 ± 0.95 ng/mL) and had a higher area under the curve than C-reactive protein. Thus, we identified SAA4 as a protein that was positively correlated with RF and RA. SAA4 may represent a novel prescreening marker for the diagnosis of RA.
Humans now have a life expectancy of nearly 90 years and, as a result, society is rapidly aging. These longer life spans have, however, increased the average length of hospitalization for elderly adults suffering from chronic diseases such as cancer, diabetes, and encephalopathy. In a recent survey of 10 well-being indices for elderly adults, the top demands for well-being are physical, spiritual, and psychological. Thus, we developed the WISH (Well-aging Indexing for Senior Health) Platform to enhance the normalizing exponents using survey data. Nowadays, the incidence of many chronic diseases is increasing. Thus, we designed the WISH Platform using clinical research on depression, cerebral infarction, coronary artery, and rheumatism, which are common diseases in Koreans. By applying this study to chronic diseases, which manifest differently among elderly adults across different countries, it is possible to determine influential nutrition and life patterns to create a standardized index. Such an index will help create value for the happiness of active elderly people, which in turn will aid in their efforts to maintain health; it may even benefit health promotion in other age groups.
Prostate cancer is the most common cancer in men, and before it progresses and metastasizes, the anticancer drug bicalutamide is often administered to patients. Many cases of androgen-dependent prostate cancer develop resistance during treatment with bicalutamide. Therefore, the effect of bicalutamide on androgen-dependent LNCaP prostate cancer cells is of clinical interest. The aim of this study was to demonstrate the effects of the anticancer drug bicalutamide on LNCaP prostate cancer cells by using a proteomics approach. Based on the results, 314 proteins were differentially expressed between the LNCaP and LNCaP treated with bicalutamide. The apoptosis pathway associated with differentially expressed proteins was shown in the Kyoto Encyclopedia of Gene and Genome pathway mapper. The Kyoto Encyclopedia of Gene and Genome pathway mapper results revealed that the fodrin-mediated apoptosis pathway is associated with the actions of bicalutamide and Western blotting was performed to validate these results. Impact statement We studied bicalutamide’s anticancer action by using proteomics. The effect of bicalutamide on androgen-exposed LNCaP cells was also studied. KEGG identified >1.8-fold differentially expressed proteins between test group cells. KEGG mapper showed fodrin-mediated apoptosis involvement in bicalutamide’s action. The anticancer effects of bicalutamide, which was further confirmed using Western blotting. Therefore, this drug is a potential candidate for understanding bicalutamide’s effect on LNCaP and fodrin can be used as a biomarker monitoring status in metastatic carcinoma.
Acute coronary syndrome (ACS) results from inadequate supply of blood flow from the coronary arteries to the heart or ischemia. ACS has an extremely high morbidity and mortality. The levels of biomarkers currently used for detection of ACS also increase in response to myocardial necrosis and other diseases and are not elevated immediately after symptoms appear, thus limiting their diagnostic capacity. Therefore, we aimed to discover new ACS diagnostic biomarkers with high sensitivity and specificity that are specifically related to ACS pathogenesis. Sera from 50 patients with ACS and healthy controls (discovery cohort) each were analyzed using mass spectrometry (MS) to identify differentially expressed proteins, and protein candidates were evaluated as ACS biomarkers in 120 people in each group (validation cohort). α-1-acid glycoprotein 1 (AGP1), complement C5 (C5), leucine-rich α-2-glycoprotein (LRG), and vitronectin (VN) were identified as biomarkers whose levels increase and gelsolin (GSN) as a biomarker whose levels decrease in patients with ACS. We concluded that these biomarkers are associated with the pathogenesis of ACS and can predict the onset of ACS prior to the appearance of necrotic biomarkers.
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