Role of Transcription in Mammalian Copy Number Variant Formation

Genome instability, defined as an increased tendency of genome alteration, is the cause of many human diseases and conditions. It is a hallmark of human cancer and plays a role in aging and the development and function of the nervous system. Genome instability can manifest in several ways, including gaps and breaks at Common Fragile Sites (CFSs) and Copy Number Variants (CNVs). CFSs are sites on human metaphase chromosomes prone to forming gaps or breaks following replication stress. CNVs are submicroscopic genomic alternations that change the copy number of the affected region, also often following replication stress. The genome regions most prone to replication stress-induced CNVs, called “hotspots,” coincide with CFSs. In spite of their implications for human health, mechanisms leading to instability at CFSs and CNV hotspots are unclear. CFSs/CNV hotspots are AT-rich and late replicating, but those properties are not sufficient for the sites’ instability. DNA sequence at CFSs/CNV hotspots is shared among all cells, but instability is cell line-specific. We also found that while about 20% of the genome replicates late, hotspots only comprise 0.4% of the genome. Hence, instability at hotspots is determined by properties that vary between different cell lines and genomic regions. Transcription is one such property. We found that CFSs/CNV hotspots are enriched in large (>500kb), transcribed genes and that given a cell line’s transcription profile we can predict where CFSs/CNV hotspots will be in that cell line. I further show that abrogating expression of a large hotspot gene leads to a reduced number of aphidicolin-induced CNVs. These results established transcription of large genes as a determining factor for instability at hotspots. We propose that a conflict between transcription of large genes and DNA replication drives hotspot instability. I tested a model in which R-loops (RNA/DNA hybrids) create a physical interference for the replication fork and cause the fork to stall and initiate genomic alteration. R-loop manipulation by altering expression of RNase H1 had no significant effect on the frequency of APH-induced instability at hotspots, implying that R-loops do not play a central role in driving APH-induced CNVs, unlike a prior study showing that R-loop manipulation changes CFS instability. However, R-loop accumulation changes the location of breakpoints of these CNVs and change the frequency of the spontaneous CNVs, suggesting that R-loops may still play a role in both APH-induced and spontaneous CNV formation. In sum, the studies in this dissertation reveal that transcription of unusually large genes plays a pivotal role in instability at CFSs/CNV hotspots during replication stress, but not via an R-loop-associated mechanism. Nonetheless, R-loops threaten genome instability and affect CNV formation outside of hotspots. Future studies are necessary to explore other transcription-replication conflict models at CFSs/CNV hotspots and further characterize R-loop induced CNVs.PHDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147729/1/sohae_1.pd

Calpain-mediated proteolysis of polycystin-1 C-terminus induces JAK2 and ERK signal alterations

AbstractAutosomal dominant polycystic kidney disease (ADPKD), a hereditary renal disease caused by mutations in PKD1 (85%) or PKD2 (15%), is characterized by the development of gradually enlarging multiple renal cysts and progressive renal failure. Polycystin-1 (PC1), PKD1 gene product, is an integral membrane glycoprotein which regulates a number of different biological processes including cell proliferation, apoptosis, cell polarity, and tubulogenesis. PC1 is a target of various proteolytic cleavages and proteosomal degradations, but its role in intracellular signaling pathways remains poorly understood. Herein, we demonstrated that PC1 is a novel substrate for μ- and m-calpains, which are calcium-dependent cysteine proteases. Overexpression of PC1 altered both Janus-activated kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signals, which were independently regulated by calpain-mediated PC1 degradation. They suggest that the PC1 function on JAK2 and ERK signaling pathways might be regulated by calpains in response to the changes in intracellular calcium concentration

Premixed Calcium Silicate-Based Root Canal Sealer Reinforced with Bioactive Glass Nanoparticles to Improve Biological Properties

Recently, bioactive glass nanoparticles (BGns) have been acknowledged for their ability to promote interactions with the periapical tissue and enhance tissue regeneration by releasing therapeutic ions. However, there have been no studies on calcium silicate sealers with bioactive glass nanoparticle (BGn) additives. In the present study, a premixed calcium silicate root canal sealer reinforced with BGn (pre-mixed-RCS@BGn) was developed and its physicochemical features and biological effects were analyzed. Three specimens were in the trial: 0%, 0.5%, and 1% bioactive glass nanoparticles (BGns) were gradually added to the premixed type of calcium silicate-based sealer (pre-mixed-RCS). To elucidate the surface properties, scanning electron microscopy, X-ray diffraction, and energy-dispersive spectroscopy were used and flowability, setting time, solubility, and radiopacity were analyzed to evaluate the physical properties. Chemical properties were investigated by water contact angle, pH change, and ion release measurements. The antibacterial effects of the bioactive set sealers were tested with Enterococcus faecalis and the viability of human bone marrow-derived mesenchymal stem cells (hMSCs) with this biomaterial was examined. In addition, osteogenic differentiation was highly stimulated, which was confirmed by ALP (Alkaline phosphatase) activity and the ARS (Alizarin red S) staining of hMSCs. The pre-mixed-RCS@BGn satisfied the ISO standards for root canal sealers and maintained antimicrobial activity. Moreover, pre-mixed-RCS@BGn with more BGns turned out to have less cytotoxicity than pre-mixed-RCS without BGns while promoting osteogenic differentiation, mainly due to calcium and silicon ion release. Our results suggest that BGns enhance the biological properties of this calcium silicate-based sealer and that the newly introduced pre-mixed-RCS@BGn has the capability to be applied in dental procedures as a root canal sealer. Further studies focusing more on the biocompatibility of pre-mixed-RCS@BGn should be performed to investigate in vivo systems, including pulp tissue

Prevention of mitochondrial impairment by inhibition of protein phosphatase 1 activity in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of motor neurons (MNs) and subsequent muscle weakness. These pathological features are associated with numerous cellular changes, including alteration in mitochondrial morphology and function. However, the molecular mechanisms associating mitochondrial structure with ALS pathology are poorly understood. In this study, we found that Dynamin-related protein 1 (Drp1) was dephosphorylated in several ALS models, including those with SOD1 and TDP-43 mutations, and the dephosphorylation was mediated by the pathological induction of protein phosphatase 1 (PP1) activity in these models. Suppression of the PP1-Drp1 cascade effectively prevented ALS-related symptoms, including mitochondrial fragmentation, mitochondrial complex I impairment, axonal degeneration, and cell death, in primary neuronal culture models, iPSC-derived human MNs, and zebrafish models in vivo. These results suggest that modulation of PP1-Drp1 activity may be a therapeutic target for multiple pathological features of ALS

Escherichia coli adhesion protein FimH exacerbates colitis via CD11b+CD103- dendritic cell activation

IntroductionImmune stimulators are used to improve vaccine efficiency; however, they are accompanied by various side effects. In previous studies, we reported that the Escherichia coli adhesion protein, FimH, induces immune activity; however, we did not examine any side effects in colon inflammation.MethodsFimH was administered orally or intraperitoneally (i.p.) to mice with dextran sulfate sodium (DSS)-induced colitis, and changes in symptoms were observed. Immune cells infiltrated into the colon after the induction of colon inflammation were analyzed using a flow cytometer. Changes in Th1 and Th17 cells that induce colitis were analyzed. Further, mesenteric lymph node (mLN) dendritic cells (DCs) activated by FimH were identified and isolated to examine their ability to induce T-cell immunity.ResultsFimH oral and i.p. administration in C57BL/6 mice did not induce inflammation in the colon; however, DSS-induced colitis was exacerbated by oral and i.p. FimH administration. FimH treatment increased immune cell infiltration in the colon compared to that in DSS colitis. Th1 and Th17 cells, which are directly related to colitis, were increased in the colon by FimH; however, FimH did not directly affect the differentiation of these T cells. FimH upregulated the CD11b+CD103- DC activity in the mLNs, which produced the signature cytokines required for Th1 and Th17. In addition, isolated CD11b+CD103- DCs, after stimulation with FimH, directly induced Th1 and Th17 differentiation in a co-culture of CD4 T cells.ConclusionThis study demonstrated the side effects of FimH and indicated that the use of FimH can aggravate the disease in patients with colitis

Neutrophil-to-lymphocyte ratio as a predictor of in-hospital complications and overall mortality in Takotsubo syndrome preceded by physical triggers

Background Takotsubo syndrome (TTS) with physical triggers has worse short- and long-term clinical courses than those with emotional triggers. However, predictive factors associated with poor outcomes of TTS with physical triggers are unknown. Methods We included 231 patients identified as TTS preceded by physical triggers at two tertiary referral hospitals from 2010 to 2019. In-hospital complications (IHC)—a composite of malignant arrhythmia, need for mechanical circulatory support or mechanical ventilation, and in-hospital death—and overall mortality were retrospectively reviewed. The associations with clinical features were evaluated by multivariable logistic and Cox regression analyses. Results The mean age was 69.3 ± 11.6years, and 85 (36.8%) were male. The in-hospital complications rate was 46.8%. During a median follow-up of 883days, 96 (41.6%) had died, and overall mortality was 13.6% per patient-year. Higher neutrophil-to-lymphocyte ratio (NLR) was associated with a higher risk of IHC (area under the receiver operating characteristic curve = 0.73; positive and negative predictive value = 60.9% and 67.2% for NLR ≤ 12); odds ratio (OR) with 95% confidence interval (CI) was 1.03 (1.01–1.05), p = 0.010. Subsequently, higher NLR was also related to a greater risk of overall mortality; patients with high NLR (NLR > 12) exhibited poor long-term survival than those with low NLR (NLR ≤ 5): hazard ratio (95% CI), 3.70 (1.72–7.94) with p < 0.001. Conclusions A high NLR at initial presentation is associated with an increased risk of IHC and overall mortality in TTS preceded by physical triggers. Given that the treatment of TTS is mainly supportive, intensive monitoring with careful follow-up would be warranted in patients with high NLR.This study was funded by SNUBH Research Fund [Grant No. 14-2015-029]

Heterogeneous nuclear ribonucleoprotein A1 post-transcriptionally regulates Drp1 expression in neuroblastoma cells.

Excessive mitochondrial fission is associated with the pathogenesis of neurodegenerative diseases. Dynamin-related protein 1 (Drp1) possesses specific fission activity in the mitochondria and peroxisomes. Various post-translational modifications of Drp1 are known to modulate complex mitochondrial dynamics. However, the post-transcriptional regulation of Drp1 remains poorly understood. Here, we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) regulates Drp1 expression at the post-transcriptional level. hnRNP A1 directly interacts with Drp1 mRNA at its 3'UTR region, and enhances translation potential without affecting mRNA stability. Down-regulation of hnRNP A1 induces mitochondrial elongation by reducing Drp1 expression. Moreover, depletion of hnRNP A1 suppresses 3-NP-mediated mitochondrial fission and dysfunction. In contrast, over-expression of hnRNP A1 promotes mitochondrial fragmentation by increasing Drp1 expression. Additionally, hnRNP A1 significantly exacerbates 3-NP-induced mitochondrial dysfunction and cell death in neuroblastoma cells. Interestingly, treatment with 3-NP induces subcellular translocation of hnRNP A1 from the nucleus to the cytoplasm, which accelerates the increase in Drp1 expression in hnRNP A1 over-expressing cells. Collectively, our findings suggest that hnRNP A1 controls mitochondrial dynamics by post-transcriptional regulation of Drp1.This research was supported by a grant of the Korea–UK Collaborative Alzheimer's Disease Research Project by Ministry of Health & Welfare, Republic of Korea (A120196, HI14C1913) and was supported by the Basic Science Research Program of the National Research Foundation, Republic of Korea (2014R1A2A1A11053431). We are grateful to Wellcome Trust, Principal Research Fellowship to DCR (095317/Z/11/Z)This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.bbagrm.2015.10.01

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

De Novo Superinfection of Hepatitis B Virus in an Anti-HBs Positive Patient with Recurrent Hepatitis C Following Liver Transplantation

A 60-year-old woman with end stage liver cirrhosis caused by genotype 2 hepatitis C virus (HCV) infection received an orthotopic liver transplantation (OLT). The patient was negative for the hepatitis B surface antigen (HBsAg) and positive for the anti-hepatitis B surface antibody (anti-HBs) prior to and one and a half months following the OLT. Due to reactivation of hepatitis C, treatment with interferon-alpha and Ribavirin started two months following the OLT and resulted in a sustained virological response. We performed a liver biopsy because a biochemical response was not achieved. Surprisingly, liver pathology showed HBsAg-positive hepatocytes with a lobular hepatitis feature, which had been negative in the liver biopsy specimen obtained one and a half months post-OLT. High titers of both HBsAg and HBeAg were detected, while anti-HBs antibodies were not found. Tests for IgM anti-hepatitis B core antibody and anti-delta virus antibodies were negative. The serum HBV DNA titer was over 1×107 copies/mL. A sequencing analysis showed no mutation in the "a" determinant region, but revealed a mixture of wild and mutant strains at an overlapping region of the S and P genes (S codon 213 (Leu/Ile); P codons 221 (Phe/Tyr) and 222 (Ala/Thr)). These findings suggest that de novo hepatitis B can develop in patients with HCV infection during the post-OLT period despite the presence of protective anti-HBs

Anti-Wrinkle Effect of Magnesium Lithospermate B from Salvia miltiorrhiza BUNGE: Inhibition of MMPs via NF-kB Signaling

Skin is in direct contact with the environment and therefore undergoes aging as a consequence of environmentally induce damage. Wrinkle formation is a striking feature of intrinsic and photo-induced skin aging, which are both associated with oxidative stress and inflammatory response. The present study was undertaken to identify the mechanisms responsible for the anti-wrinkle effects of MLB, and thus, we investigated whether magnesium lithospermate B (MLB) from Salvia miltiorrhiza BUNGE associated with wrinkle formation caused by intrinsic and extrinsic skin aging using Sprague-Dawley rats aged 5 and 20 months and ultraviolet B (UVB)-irradiated human skin fibroblasts cells, respectively. The results obtained showed that the oral administration of MLB significantly upregulated the level of type I procollagen and downregulated the activities and expressions of matrix-metalloproteinases (MMPs) in rat skin. In fibroblasts, MLB suppressed the transactivation of nuclear factor-kB (NF-kB) and activator protein 1(AP-1), which are the two transcription factors responsible for MMP expression, by suppressing oxidative stress and the mitogen activated protein kinase (MAPK) pathway. Our results show that the antioxidant effect of MLB is due to the direct scavenging of reactive oxygen species (ROS) and its inhibitory effects on NF-kB-dependent inflammation genes, such as, cyclooxygenase-2 and inducible nitric oxide synthase. MLB was found to reverse both age- and UVB-related reductions in skin procollagen levels by suppressing the expressions and activities of NF-kB and AP-1-dependent MMPs by modulating ROS generation and the MAPK signaling pathway. We suggest that MLB potentially has anti-wrinkle and anti-skin aging effects