Exploring the Role of FUS Mutants from Stress Granule Incorporation to Nucleopathy in Amyotrophic Lateral Sclerosis: A Dissertation

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by preferential motor neuron death in the brain and spinal cord. The rapid disease progression results in death due to respiratory failure, typically within 3-5 years after disease onset. While ~90% of cases occur sporadically, remaining 10% of ALS cases show familial inheritance, and the number of genes linked to ALS has increased dramatically over the past decade. FUS/TLS (Fused in Sarcoma/ Translocated to liposarcoma) is a nucleic acid binding protein that may regulate several cellular functions, including RNA splicing, transcription, DNA damage repair and microRNA biogenesis. More than 50 mutations in the FUS gene are linked to 4% of familial ALS, and many of these may disrupt the nuclear localization signal, leading to variable amounts of FUS accumulation in the cytoplasm. However, the mechanism by which FUS mutants cause motor neuron death is still unknown. The studies presented in this dissertation focused on investigating the properties of FUS mutants in the absence and presence of stress conditions. We first examined how ALS-linked FUS mutants behaved in response to imposed stresses in both cell culture and zebrafish models of ALS. We found that FUS mutants were prone to accumulate in stress granules in proportion to their degree of cytoplasmic mislocalization under conditions of oxidative stress, ER stress, and heat shock. However, many FUS missense mutants are retained predominantly in the nucleus, and this suggested the possibility that these mutants might also perturb one or more nuclear functions. In a human cell line expressing FUS variants and in human fibroblasts from an ALS patient, mutant FUS expression was associated with enlarged promyelocytic leukemia nuclear bodies (PML-NBs) under basal condition. Upon oxidative insult with arsenic trioxide (ATO), PML-NBs in control cells increased acutely in size and were turned over within 12-24 h, as expected. However, PML-NBs in FUS mutant cells did not progress through the expected turnover but instead continued to enlarge over 24 h. We also observed a persistent accumulation of the transcriptional repressor Daxx and the 11S proteasome regulator in association with these enlarged PML-NBs. Furthermore, the peptidase activities of the 26S proteasome were decreased in FUS mutant cells without any changes in the expression of proteasome subunits. These results demonstrate that FUS mutant expression may alter cellular stress responses as manifested by (i) accumulation of mutant FUS into stress granules and (ii) inhibition of PML-NB dynamics. These findings suggest a novel nuclear pathology specific to mutant FUS expression that may perturb nuclear homeostasis and thereby contribute to ALS pathogenesis

In Utero Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Affects the Development of Reproductive System in Mouse

PURPOSE: Exposure of male reproductive organs to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) has been reported to cause developmental changes. In this study, we evaluated the effects of in utero TCDD exposure on male reproductive development. MATERIALS AND METHODS: Pregnant C57BL/6 mice were administered a single intraperitoneal injection of TCDD (1microgram/kg) on gestation day (GD) 15. The offspring were examined in the immature stage on postnatal day (PND) 30 and in the mature stage on PND 60. The testes were examined for histological changes, androgen receptor (AR), proliferating cell nuclear antigen (PCNA) and apoptosis following the measurement of morphological changes. RESULTS: Anogenital distance (AGD) and testis weights were reduced by TCDD exposure both on PND 30 and PND 60 while body weights and length of male offspring were not affected by TCDD. The regular sperm developmental stage was impaired with TCDD treatment on PND 30. However, no difference was found between the control group and TCDD groups on PND 60. Simultaneously, the expression of AR was also reduced on PND 30, while it was increased on PND 60 compared with the control group. The expression of PCNA was decreased whereas apoptosis was not affected by TCDD both on PND 30 and PND 60. CONCLUSION: These results suggest that in utero exposure to TCDD influences the development of testes by inhibiting the expression of AR and PCNA. Moreover, the adverse effects of TCDD on male offspring reduced over timeope

The Effects of Glyburide on Apoptosis and Endoplasmic Reticulum Stress in INS-1 Cells in a Glucolipotoxic Condition

Backgroundβ-cell death due to endoplasmic reticulum (ER) stress has been regarded as an important pathogenic component of type 2 diabetes. The possibility has been suggested that sulfonylurea, currently being used as one of the main oral hypoglycemic agents of type 2 diabetes, increases ER stress, which could lead to sulfonylurea failure. The authors of the present study examined ER stress of β-cells in a glucolipotoxic condition using glyburide (GB) in an environment mimicking type 2 diabetes.MethodsApoptosis was induced by adding various concentrations of GB (0.001 to 200 µM) to a glucolipotoxic condition using 33 mM glucose, and the effects of varied concentrations of palmitate were evaluated via annexin V staining. The markers of ER stress and pro-apoptotic markers were assessed by Western blotting and semi-quantitative reverse transcription-polymerase chain reaction. Additionally, the anti-apoptotic markers were evaluated.ResultsAddition of any concentration of GB in 150 µM palmitate and 33 mM glucose did not increase apoptosis. The expression of phosphorylated eukaryotic initiation factor (eIF-2α) was increased and cleaved caspase 3 was decreased by adding GB to a glucolipotoxic condition. However, other ER stress-associated markers such as Bip-1, X-box binding protein-1, ATF-4 and C/EBP-homologous protein transcription factor and anti-apoptotic markers phosphor-p85 phosphatidylinositol 3-kinase and phosphorylation of Akt did not change significantly.ConclusionGB did not show further deleterious effects on the degree of apoptosis or ER stress of INS-1 cells in a glucolipotoxic condition. Increased phosphorylation of eIF-2α may attenuate ER stress for adaptation to increased ER protein load

Non-mass breast lesions on ultrasound: final outcomes and predictors of malignancy

Background Breast cancer can present as non-mass lesions (NMLs) on ultrasound. However, knowledge of and understanding about NMLs are scarce. Purpose To retrospectively investigate the final outcomes of sonographic breast NMLs and determine the clinical and radiologic variables associated with malignancy Material and Methods In our radiologic database of breast ultrasound examinations between 2011 and 2014, we found 119 women with 121 NMLs with available histopathologic or sonographic follow-up (over 2 years) data. We collected the clinical variables (patient's age, symptoms, and mammographic density) and histopathologic data as well as radiologic variables (mammographic and ultrasound findings) after retrospective review by two radiologists, the authors of the current paper, in consensus. We classified the ultrasound findings according to distribution (focal, linear or segmental, and regional) and associated features (calcification, architectural distortion, and ductal changes) and analyzed the associations between variables and malignancy using the t test and χ2 test. Results Of the 121 NMLs, 88 (72.7%) were benign and 33 (27.3%) were malignant. Ductal carcinoma in situ (DCIS) (17/33, 51.5%) and invasive ductal cancer with or without DCIS (13/33, 39.4%) comprised the main malignancies, and malignancy was significantly associated with palpability ( P = 0.000). Mammographic findings and sonographic distribution and associated features were significantly different between benign and malignant lesions ( P = 0.000, P = 0.004, and P = 0.001, respectively). Malignant lesions showed more frequent calcifications combined with asymmetry ( P = 0.000) on mammography and linear-segmental distributions ( P = 0.001) and associated calcifications ( P = 0.019) or architectural distortions ( P = 0.015) on ultrasound. Conclusion Breast NMLs on ultrasound showed high risk of malignancy. Symptoms and mammographic and ultrasound findings can be possible predictors of malignancy in NMLs

Concordant or Discordant? Imaging-Pathology Correlation in a Sonography-Guided Core Needle Biopsy of a Breast Lesion

An imaging-guided core needle biopsy has been proven to be reliable and accurate for the diagnosis of both benign and malignant diseases of the breast, and has replaced surgical biopsy. However, the possibility of a false-negative biopsy still remains. Imaging-pathology correlation is of critical importance in imaging-guided breast biopsies to detect such a possible sampling error and avoid a delay in diagnosis. We will review five possible categories and corresponding management after performing an imaging-pathology correlation in a sonography-guided core needle biopsy of a breast lesion, as well as illustrate the selected images for each category in conjunction with the pathologic finding. Radiologists should be familiar with the imaging features of various breast pathologies and be able to appropriately correlate imaging findings with pathologic results after a core needle biopsy

The Results of Cervical Nucleoplasty in Patients with Cervical Disc Disorder: A Retrospective Clinical Study of 22 Patients

Nucleoplasty is a minimally invasive spinal surgery using a Coblation Ⓡ technique that creates small voids within the disc. The purpose of this study was to evaluate the efficacy of cervical nucleoplasty in patients with cervical disc disorder. Methods: Between March 2008 and December 2009, 22 patients with cervical disc disorders were treated with cervical nucleoplasty after failed conservative treatment. All procedures were performed under local anesthesia, and fluoroscopic guidance and voids were created in the disc with the Perc TM DC Spine Wand TM. Clinical outcomes were evaluated by the Modified Macnab criteria and VAS score at preprocedure, postprocedure 1 month, and 6 months. Results: Six patients had one, eight patients had two and eight patients had three discs treated; a total of 46 procedures was performed. Mean VAS reduced from 9.3 at preprocedure to 3.7 at postprocedure 1 month and to 3.4 at postprocedure 6 months. There was no significant complication related to the procedure within the first month. Outcomes were good or excellent in 17/22 (77.3%) cases. Postprocedure magnetic resonance imaging was acquired in two patients after two months showing morphologic evidence of volume reduction of protruded disc material in one patient but not in the other. Conclusions: Percutaneous decompression with a nucleoplasty using a Coblation Ⓡ technique in the treatment of cervical disc disorder is a safe, minimally-invasive and less uncomfortable procedure, with an excellent short-term clinical outcome. (Korean J Pain 2011; 24: 36-43

The invasive lobular carcinoma as a prototype luminal A breast cancer: A retrospective cohort study

<p>Abstract</p> <p>Background</p> <p>Although the invasive lobular carcinoma (ILC) is the second most frequent histologic subtype in Western countries, its incidence is much lower in Asia, and its characteristics are less well known.</p> <p>Methods</p> <p>We assessed the clinical characteristics and outcomes of 83 Korean patients (2.8%) with ILC for comparison with 2,833 (97.2%) with the invasive ductal carcinoma (IDC), including 1,088 (37.3%) with the luminal A subtype (LA-IDC).</p> <p>Results</p> <p>The mean age of all patients was 48.2 years, with no significant differences among the groups. Compared to IDC, ILC showed a larger tumor size (≥T2, 59.8% vs. 38.8%, <it>P </it>= 0.001), a lower histologic grade (HG 1/2, 90.4% vs. 64.4%, <it>P </it>< 0.001), more frequent estrogen receptor positive (90.4% vs. 64.4%, <it>P </it>< 0.001), progesterone receptor positive (71.1% vs. 50.1%, <it>P </it>< 0.001) and HER2 negative (97.5% vs. 74.6%, <it>P </it>< 0.001) status, and lower Ki-67 expression (10.3% ± 10.6% vs. 20.6% ± 19.8%, <it>P </it>< 0.001), as well as being more likely to be of the luminal A subtype (91.4% vs. 51.2%, <it>P </it>< 0.001). Six (7.2%) ILC and 359 (12.7%) IDC patients developed disease recurrence, with a median follow-up of 56.4 (range 4.9-136.6) months. The outcome of ILC was close to LA-IDC (HR 0.77 for recurrence, 95% CI 0.31-1.90, <it>P </it>= 0.57; HR 0.75 for death, 95% CI 0.18-3.09, <it>P </it>= 0.70) and significantly better than for the non-LA-IDC (HR 1.69 for recurrence, 95% CI 1.23-2.33, <it>P </it>= 0.001; HR 1.50 for death, 95% CI 0.97-2.33, <it>P </it>= 0.07).</p> <p>Conclusions</p> <p>ILC, a rare histologic type of breast cancer in Korea, has distinctive clinicopathological characteristics similar to those of LA-IDC.</p

Purification of plant-derived anti-virus mAb through optimized pH conditions for coupling between protein A and epoxy-activated beads

The main goal of this research was to determine optimum pH conditions for coupling between protein A and epoxy-activated Sepharose beads for purification of monoclonal antibodies (mAbs) expressed in plants. To confirm the effect of pH conditions on purification efficacy, epoxy-activated agarose beads were coupled to protein A under the pH conditions of 8.5, 9.5, 10.5, and 11.5 (8.5R, 9.5R, 10.5R, and 11.5R, respectively). A total of 300 g of fresh leaf tissue of transgenic Arabidopsis expressing human anti-rabies mAb (mAbP) SO57 were harvested to isolate the total soluble protein (TSP). An equal amount of TSP solution was applied to five resin groups including commercial protein A resin (GR) as a positive control. The modified 8.5R, 9.5R, 10.5R, and 11.5R showed delayed elution timing compared to the GR control resin. Nano-drop analysis showed that the total amount of purified mAbPSO57 mAbs from 60 g of fresh leaf mass were not significantly different among 8.5R (400 μg), 9.5R (360 μg), 10.5R (380 μg), and GR (350 μg). The 11.5R (25 μg) had the least mAbPSO57. SDS–PAGE analysis showed that the purity of mAbPSO57 was not significantly different among the five groups. Rapid fluorescent focus inhibition tests revealed that virus-neutralizing efficacies of purified mAbPSO57 from all the five different resins including the positive control resin were similar. Taken together, both pH 8.5 and 10.5 coupling conditions with high recovery rate should be optimized for purification of mAbPSO57 from transgenic Arabidopsis plant, which will eventually reduce down-stream cost required for mAb production using the plant system