N-glycoproteomic analysis of human follicular fluid during natural and stimulated cycles in patients undergoing in vitro fertilization / 대한생식의학회지

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.

Proteomic Analysis of Serum from Patients with Major Depressive Disorder to Compare Their Depressive and Remission Statuses

OBJECTIVE: Currently, there are a few biological markers to aid in the diagnosis and treatment of depression. However, it is not sufficient for diagnosis. We attempted to identify differentially expressed proteins during depressive moods as putative diagnostic biomarkers by using quantitative proteomic analysis of serum. METHODS: Blood samples were collected twice from five patients with major depressive disorder (MDD) at depressive status before treatment and at remission status during treatment. Samples were individually analyzed by liquid chromatography-tandem mass spectrometry for protein profiling. Differentially expressed proteins were analyzed by label-free quantification. Enzyme-linked immunosorbent assay (ELISA) results and receiver-operating characteristic (ROC) curves were used to validate the differentially expressed proteins. For validation, 8 patients with MDD including 3 additional patients and 8 matched normal controls were analyzed. RESULTS: The quantitative proteomic studies identified 10 proteins that were consistently upregulated or downregulated in 5 MDD patients. ELISA yielded results consistent with the proteomic analysis for 3 proteins. Expression levels were significantly different between normal controls and MDD patients. The 3 proteins were ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4 and complement component 1qC, which were upregulated during the depressive status. The depressive status could be distinguished from the euthymic status from the ROC curves for these proteins, and this discrimination was enhanced when all 3 proteins were analyzed together. CONCLUSION: This is the first proteomic study in MDD patients to compare intra-individual differences dependent on mood. This technique could be a useful approach to identify MDD biomarkers, but requires additional proteomic studies for validation.

Evaluation of a ChromID C. difficile Agar for the Isolation of Clostridium difficile / 대한임상미생물학회지

BACKGROUND: Clostridium difficile is the main etiologic agent of antibiotic-associated diarrhea and the most common cause of hospital-acquired diarrhea. Recently, the incidence of C. difficile infections (CDI) has increased and new highly virulent C. difficile strains have emerged. Therefore, accurate and rapid diagnosis is needed. We compared the results of using chromID C. difficile (chromID CD, bioMerieux, France) with the conventional C. difficile Selective Agar (CDSA; BD, USA) for the isolation of C. difficile. METHODS: A total of 738 stool specimens of suspected CDI patients at the Severance Hospital from July to August 2011 were inoculated onto CDSA. Among them, 104 stool specimens revealed colonies on CDSA that were then re-inoculated onto chromID CD. The stool samples were stored at -20degrees C until the time of the re-inoculation. Cultured agars were interpreted after 24 hrs and 48 hrs, respectively. Species identification was performed on the basis of colony characteristics on agar plates as well as the ATB 32A system (API System SA, France). RESULTS: The recovery rates of CDSA and chromID CD were 30.1% and 77.5% after 24 hrs, and 77.5% and 98.6% after 48 hrs, respectively. All of the C. difficile isolates were recovered as typical gray/black colonies on chromID CD. CONCLUSION: The performance of chromID CD for the isolation of C. difficile was better than that of conventional CDSA. The chromID CD could provide easy and sensitive detection of C. difficile even after 24hrs of incubation.

VEGF Expression of Cultured Keloid Fibroblasts from Different Donor Sites Under Normoxic and Hypoxic Conditions / 대한피부과학회지

BACKGROUND: The pathophysiological events resulting in keloid formation remain unclear. Overabundant levels of VEGF have been reported to contribute to excessive wound healing. There have been many studies describing the relationship between keloids and VEGF expression. However, there have been no reports about VEGF expression related to donor sites. OBJECTIVE: We investigated VEGF expression of cultured normal and keloid fibroblasts obtained from different body areas under normoxic and hypoxic culture conditions. METHODS: Normal fibroblasts from the earlobe (n=2), shoulder (n=2) and chest (n=2) as well as keloid fibroblasts from the earlobe (n=3), shoulder (n=3) and chest (n=3) were collected and cultured. VEGF expression of fibroblasts at 6 hours, 12 hours, 24 hours and 48 hours for cells maintained under normoxic and hypoxic conditions was measured by the use of RT-PCR. Paraffin-embedded tissues (normal and keloid tissue) were assayed by immunohistochemical staining. RESULTS: For the cultured normal fibroblasts, VEGF expression for cells in the hypoxic condition was higher as compared to VEGF expression in cells in the normoxic condition, irrespective of the donor site and time. However, for the cultured keloid fibroblasts, VEGF expression for cells in the hypoxic condition was higher as compared to VEGF expression in cells in the normoxic condition for cultured shoulder fibroblasts. For each donor site, VEGF expression was highest in the shoulder, followed by the chest and earlobe for cultured normal fibroblasts, irrespective of time. For the cultured keloid fibroblasts, the highest VEGF expression occurred at 6 hours for cells in the normoxic condition and the highest VEGF expression occurred at 6 hours and 12 hours for cells in the hypoxic condition. Based on immunohistochemical staining, VEGF expression of paraffin-embedded normal tissue was lower as compared to paraffin-embedded keloid tissue. For each donor site in paraffin-embedded keloid tissue, VEGF expression was highest in the shoulder, followed by the chest and earlobe. CONCLUSION: Oxygen tension and the nature of fibroblasts from different donor sites are involved in keloid pathogenesis.