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1.  Parkin and PINK1 mutations in early-onset Parkinson’s disease: comprehensive screening in publicly available cases and control 
Journal of medical genetics  2009;46(6):375-381.
Mutations in parkin and PTEN-induced protein kinase (PINK1) represent the two most common causes of autosomal recessive parkinsonism. The possibility that heterozygous mutations in these genes also predispose to disease or lower the age of disease onset has been suggested, but currently there is insufficient data to verify this hypothesis conclusively.
To study the frequency and spectrum of parkin and PINK1 gene mutations and to investigate the role of heterozygous mutations as a risk factor for early-onset Parkinson’s disease (PD).
All exons and exon-intron boundaries of PINK1 and parkin were sequenced in 250 patients with early-onset PD and 276 normal controls. Gene dosage measurements were also performed, using high-density single-nucleotide polymorphism arrays.
In total 41 variants were found, of which 8 have not been previously described (parkin: p.A38VfsX6, P.C166Y, P.Q171X, p.D243N, p.M458L; PINK1: p.P52L, P.T420T, P.A427E). 1.60% of patients were homozygous or compound heterozygous for pathogenic mutations. Heterozygosity for pathogenic parkin or PINK1 mutations was over-represented in patients compared with healthy controls (4.00% vs. 1.81%) but the difference was not significant (p = 0.13). The mean age at disease onset was significantly lower in patients with homozygous or compound heterozygous mutations than in patients with heterozygous mutations (mean difference 11 years, 95% Cl 1.4 to 20.6, p = 0.03). There was no significant difference in the mean age at disease onset in heterozygous patients compared with patients without a mutation in parkin or PINK1 (mean difference 2 years, 95% Cl −3.7 to 7.0, p = 0.54).
Our data support a trend towards a higher frequency of heterozygosity for pathogenic parkin or PINK1 mutations in patients compared with normal controls, but this effect was small and did not reach significance in our cohort of 250 cases and 276 controls.
PMCID: PMC4767009  PMID: 19351622
2.  GWAS for executive function and processing speed suggests involvement of the CADM2 gene 
Ibrahim-Verbaas, CA | Bressler, J | Debette, S | Schuur, M | Smith, AV | Bis, JC | Davies, G | Trompet, S | Smith, JA | Wolf, C | Chibnik, LB | Liu, Y | Vitart, V | Kirin, M | Petrovic, K | Polasek, O | Zgaga, L | Fawns-Ritchie, C | Hoffmann, P | Karjalainen, J | Lahti, J | Llewellyn, DJ | Schmidt, CO | Mather, KA | Chouraki, V | Sun, Q | Resnick, SM | Rose, LM | Oldmeadow, C | Stewart, M | Smith, BH | Gudnason, V | Yang, Q | Mirza, SS | Jukema, JW | deJager, PL | Harris, TB | Liewald, DC | Amin, N | Coker, LH | Stegle, O | Lopez, OL | Schmidt, R | Teumer, A | Ford, I | Karbalai, N | Becker, JT | Jonsdottir, MK | Au, R | Fehrmann, RSN | Herms, S | Nalls, M | Zhao, W | Turner, ST | Yaffe, K | Lohman, K | van Swieten, JC | Kardia, SLR | Knopman, DS | Meeks, WM | Heiss, G | Holliday, EG | Schofield, PW | Tanaka, T | Stott, DJ | Wang, J | Ridker, P | Gow, AJ | Pattie, A | Starr, JM | Hocking, LJ | Armstrong, NJ | McLachlan, S | Shulman, JM | Pilling, LC | Eiriksdottir, G | Scott, RJ | Kochan, NA | Palotie, A | Hsieh, Y-C | Eriksson, JG | Penman, A | Gottesman, RF | Oostra, BA | Yu, L | DeStefano, AL | Beiser, A | Garcia, M | Rotter, JI | Nöthen, MM | Hofman, A | Slagboom, PE | Westendorp, RGJ | Buckley, BM | Wolf, PA | Uitterlinden, AG | Psaty, BM | Grabe, HJ | Bandinelli, S | Chasman, DI | Grodstein, F | Räikkönen, K | Lambert, J-C | Porteous, DJ | Price, JF | Sachdev, PS | Ferrucci, L | Attia, JR | Rudan, I | Hayward, C | Wright, AF | Wilson, JF | Cichon, S | Franke, L | Schmidt, H | Ding, J | de Craen, AJM | Fornage, M | Bennett, DA | Deary, IJ | Ikram, MA | Launer, LJ | Fitzpatrick, AL | Seshadri, S | van Duijn, CM | Mosley, TH
Molecular psychiatry  2015;21(2):189-197.
To identify common variants contributing to normal variation in two specific domains of cognitive functioning, we conducted a genome-wide association study (GWAS) of executive functioning and information processing speed in non-demented older adults from the CHARGE (Cohorts for Heart and Aging Research in Genomic Epidemiology) consortium. Neuropsychological testing was available for 5429–32 070 subjects of European ancestry aged 45 years or older, free of dementia and clinical stroke at the time of cognitive testing from 20 cohorts in the discovery phase. We analyzed performance on the Trail Making Test parts A and B, the Letter Digit Substitution Test (LDST), the Digit Symbol Substitution Task (DSST), semantic and phonemic fluency tests, and the Stroop Color and Word Test. Replication was sought in 1311-21860 subjects from 20 independent cohorts. A significant association was observed in the discovery cohorts for the single-nucleotide polymorphism (SNP) rs17518584 (discovery P-value = 3.12 × 10−8) and in the joint discovery and replication meta-analysis (P-value = 3.28 × 10−9 after adjustment for age, gender and education) in an intron of the gene cell adhesion molecule 2 (CADM2) for performance on the LDST/DSST. Rs17518584 is located about 170 kb upstream of the transcription start site of the major transcript for the CADM2 gene, but is within an intron of a variant transcript that includes an alternative first exon. The variant is associated with expression of CADM2 in the cingulate cortex (P-value = 4 × 10−4). The protein encoded by CADM2 is involved in glutamate signaling (P-value = 7.22 × 10−15), gamma-aminobutyric acid (GABA) transport (P-value = 1.36 × 10−11) and neuron cell-cell adhesion (P-value = 1.48 × 10−13). Our findings suggest that genetic variation in the CADM2 gene is associated with individual differences in information processing speed.
PMCID: PMC4722802  PMID: 25869804
3.  Enhanced air pollution via aerosol-boundary layer feedback in China 
Scientific Reports  2016;6:18998.
Severe air pollution episodes have been frequent in China during the recent years. While high emissions are the primary reason for increasing pollutant concentrations, the ultimate cause for the most severe pollution episodes has remained unclear. Here we show that a high concentration of particulate matter (PM) will enhance the stability of an urban boundary layer, which in turn decreases the boundary layer height and consequently cause further increases in PM concentrations. We estimate the strength of this positive feedback mechanism by combining a new theoretical framework with ambient observations. We show that the feedback remains moderate at fine PM concentrations lower than about 200 μg m−3, but that it becomes increasingly effective at higher PM loadings resulting from the combined effect of high surface PM emissions and massive secondary PM production within the boundary layer. Our analysis explains why air pollution episodes are particularly serious and severe in megacities and during the days when synoptic weather conditions stay constant.
PMCID: PMC4709519  PMID: 26753788
4.  Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification 
Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage?
The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods.
The use of more ‘steps’ of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time.
Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study.
Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem–Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification.
The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less osmotic stress resulting in better morphology, higher cell quality and improved developmental competence. This microfluidic procedure resulted in murine zygotes with a significantly smoother cell surface (P < 0.001), more spherical cellular morphology (P < 0.001), increased cytoplasmic lipid retention in vitrified and warmed bovine oocytes (P < 0.01), decreased membrane perforations and cytoplasmic leakage in CPA-exposed murine zygotes (P < 0.05) and improved developmental competence of vitrified and warmed murine zygotes (P < 0.05) than CPA exposure using the current clinically used manual pipetting method.
It is necessary to design the microfluidic device to be more user-friendly for widespread use.
The theory and approach of eliminating osmotic stress by decreasing shrinkage rate is complementary to the prevalent osmotic stress theory in cryobiology which focuses on a minimum cell volume at which the cells shrink. The auto-microfluidic protocol described here has immediate applications for improving animal and human oocyte, zygote and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sublethal damage should be broadly useful for cryobiology.
This project was funded by the National Institutes of Health and the University of Michigan Reproductive Sciences Program. The authors declare no conflicts of interest.
PMCID: PMC4262467  PMID: 25355589
cryopreservation; vitrification; microfluidics; osmotic stress
6.  Giant moving vortex mass in thick magnetic nanodots 
Scientific Reports  2015;5:13881.
Magnetic vortex is one of the simplest topologically non-trivial textures in condensed matter physics. It is the ground state of submicron magnetic elements (dots) of different shapes: cylindrical, square etc. So far, the vast majority of the vortex dynamics studies were focused on thin dots with thickness 5–50 nm and only uniform across the thickness vortex excitation modes were observed. Here we explore the fundamental vortex mode in relatively thick (50–100 nm) dots using broadband ferromagnetic resonance and show that dimensionality increase leads to qualitatively new excitation spectra. We demonstrate that the fundamental mode frequency cannot be explained without introducing a giant vortex mass, which is a result of the vortex distortion due to interaction with spin waves. The vortex mass depends on the system geometry and is non-local because of important role of the dipolar interaction. The mass is rather small for thin dots. However, its importance increases drastically with the dot thickness increasing.
PMCID: PMC4565097  PMID: 26355430
7.  The dynamic nature and territory of transcriptional machinery in the bacterial chromosome 
Our knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP) in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment and highlights the challenges in the study. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.
PMCID: PMC4440401  PMID: 26052320
RNA polymerase; bacterial nucleolus; replisome; chromosome territories; growth rate regulation; stress responses; superresolution imaging; E. coli
8.  Dendritic cell CD83 homotypic interactions regulate inflammation and promote mucosal homeostasis 
Mucosal Immunology  2014;8(2):414-428.
Dendritic cells (DCs) form an extensive network in the intestinal lamina propria, which orchestrates the mucosal immune response. Alterations in DC function can predispose to inflammatory bowel disease, although by unknown mechanisms. We show that CD83, a highly regulated DC cell surface protein, modulates the immune response to prevent colitis. Mice with a conditional knockout of CD83 in DCs develop exacerbated colitis following dextran sodium sulfate challenge, whereas mucosal overexpression of CD83 inhibits DC inflammatory response and protects against colitis. These CD83 perturbations can be modeled in vitro where we show that CD83 homotypic interaction occurs via cell–cell contact and inhibits pro-inflammatory responses. CD83 knockdown or cytoplasmic truncation abrogates the effects of homotypic binding. We demonstrate that CD83 homotypic interaction regulates DC activation via the mitogen-activated protein kinase pathway by inhibiting p38α phosphorylation. Our findings indicate that CD83 homotypic interactions regulate DC activation and promote mucosal homeostasis.
PMCID: PMC4326976  PMID: 25204675
9.  Ubiquitination by SAG regulates macrophage survival/death and immune response during infection 
Cell Death and Differentiation  2014;21(9):1388-1398.
The checkpoint between the life and death of macrophages is crucial for the host's frontline immune defense during acute phase infection. However, the mechanism as to how the immune cell equilibrates between apoptosis and immune response is unclear. Using in vitro and ex vivo approaches, we showed that macrophage survival is synchronized by SAG (sensitive to apoptosis gene), which is a key member of the ubiquitin–proteasome system (UPS). When challenged by pathogen-associated molecular patterns (PAMPs), we observed a reciprocal expression profile of pro- and antiapoptotic factors in macrophages. However, SAG knockdown disrupted this balance. Further analysis revealed that ubiquitination of Bax and SARM (sterile α- and HEAT/armadillo-motif-containing protein) by SAG-UPS confers survival advantage to infected macrophages. SAG knockdown caused the accumulation of proapoptotic Bax and SARM, imbalance of Bcl-2/Bax in the mitochondria, induction of cytosolic cytochrome c and activation of caspase-9 and -3, all of which led to disequilibrium between life and death of macrophages. In contrast, SAG-overexpressing macrophages challenged with PAMPs exhibited upregulation of protumorigenic cytokines (IL-1β, IL-6 and TNF-α), and downregulation of antitumorigenic cytokine (IL-12p40) and anti-inflammatory cytokine (IL-10). This suggests that SAG-dependent UPS is a key switch between immune defense and apoptosis or immune overactivation and tumorigenesis. Altogether, our results indicate that SAG-UPS facilitates a timely and appropriate level of immune response, prompting future development of potential immunomodulators of SAG-UPS.
PMCID: PMC4131172  PMID: 24786833
10.  MEIS2 is essential for neuroblastoma cell survival and proliferation by transcriptional control of M-phase progression 
Cell Death & Disease  2014;5(9):e1417-.
MEIS2 has an important role in development and organogenesis, and is implicated in the pathogenesis of human cancer. The molecular basis of MEIS2 action in tumorigenesis is not clear. Here, we show that MEIS2 is highly expressed in human neuroblastoma cell lines and is required for neuroblastoma cell survival and proliferation. Depletion of MEIS2 in neuroblastoma cells leads to M-phase arrest and mitotic catastrophe, whereas ectopic expression of MEIS2 markedly enhances neuroblastoma cell proliferation, anchorage-independent growth, and tumorigenicity. Gene expression profiling reveals an essential role of MEIS2 in maintaining the expression of a large number of late cell-cycle genes, including those required for DNA replication, G2-M checkpoint control and M-phase progression. Importantly, we identify MEIS2 as a transcription activator of the MuvB-BMYB-FOXM1 complex that functions as a master regulator of cell-cycle gene expression. Further, we show that FOXM1 is a direct target gene of MEIS2 and is required for MEIS2 to upregulate mitotic genes. These findings link a developmentally important gene to the control of cell proliferation and suggest that high MEIS2 expression is a molecular mechanism for high expression of mitotic genes that is frequently observed in cancers of poor prognosis.
PMCID: PMC4540202  PMID: 25210800
11.  LSD1-mediated epigenetic modification contributes to proliferation and metastasis of colon cancer 
British Journal of Cancer  2013;109(4):994-1003.
Emerging evidence has demonstrated that lysine-specific demethylase 1 (LSD1) has an important role in many pathological processes of cancer cells, such as carcinogenesis, proliferation and metastasis. In this study, we characterised the role and molecular mechanisms of LSD1 in proliferation and metastasis of colon cancer.
We evaluated the correlation of LSD1, CDH-1 and CDH-2 with invasiveness of colon cancer cells, and investigated the roles of LSD1 in proliferation, invasion and apoptosis of colon cancer cells. We further investigated the mechanisms of LSD1-mediated metastasis of colon cancer.
Lysine-specific demethylase 1 was upregulated in colon cancer tissues, and the high LSD1 expression was significantly associated with tumour-node-metastasis (TNM) stages and distant metastasis. Functionally, inhibition of LSD1 impaired proliferation and invasiveness, and induced apoptosis of colon cancer cells in vitro. The LSD1 physically interacted with the promoter of CDH-1 and decreased dimethyl histone H3 lysine 4 (H3K4) at this region, downregulated CDH-1 expression, and consequently contributed to colon cancer metastasis.
Lysine-specific demethylase 1 downregulates the expression of CDH-1 by epigenetic modification, and consequently promotes metastasis of colon cancer cells. The LSD1 antagonists might be a useful strategy to suppress metastasis of colon cancer.
PMCID: PMC3749561  PMID: 23900215
lysine-specific demethylase 1; CDH-1; CDH-2; colon cancer; metastasis; histone modifications
12.  CLIC4 regulates TGF-β dependent myofibroblast differentiation to produce a cancer stroma 
Oncogene  2013;33(7):842-850.
Cancer stroma has a profound influence on tumor development and progression. The conversion of fibroblasts to activated myofibroblasts is a hallmark of reactive tumor stroma. Among a number of factors involved in this conversion, TGF-β has emerged as a major regulator. CLIC4, an integral protein in TGF-β signaling, is highly upregulated in stroma of multiple human cancers, and overexpression of CLIC4 in stromal cells enhances the growth of cancer xenografts. In this study we show that conditioned media from tumor cell lines induces expression of both CLIC4 and the myofibroblast marker alpha smooth muscle actin (α-SMA) in stromal fibroblasts via TGF-β signaling. Genetic ablation of CLIC4 in primary fibroblasts prevents or reduces constitutive or TGF-β induced expression of α-SMA and extracellular matrix components that are markers of myofibroblasts. CLIC4 is required for the activation of p38 Map Kinase by TGF-β, a pathway that signals myofibroblast conversion in stromal cells. This requirement involves the interaction of CLIC4 with PPM1a, the selective phosphatase of activated p-38. Conditioned media from fibroblasts overexpressing CLIC4 increases tumor cell migration and invasion in a TGF-β dependent manner and promotes epithelial to mesenchymal transition indicating that high stromal CLIC4 serves to enhance tumor invasiveness and progression. Thus, CLIC4 is significantly involved in the development of a nurturing tumor microenvironment by enhancing TGF-β signaling in a positive feedback loop. Targeting CLIC4 in tumor stroma should be considered as a strategy to mitigate some of the tumor enhancing effects of the cancer stroma.
PMCID: PMC3912213  PMID: 23416981
CLIC; α-SMA; p38; microenvironment; PPM1a
To determine if sarcopenia modulates the response to a physical activity intervention in functionally limited older adults.
Secondary analysis of a randomized controlled trial.
Three academic centers.
Elders aged 70 to 89 years at risk for mobility disability who underwent dual-energy x-ray absorptiometry (DXA) for body composition at enrollment and follow-up at twelve months (N = 177).
Subjects participated in a physical activity program (PA) featuring aerobic, strength, balance, and flexibility training, or a successful aging (SA) educational program about healthy aging.
Sarcopenia as determined by measuring appendicular lean mass and adjusting for height and total body fat mass (residuals method), Short Physical Performance Battery score (SPPB), and gait speed determined on 400 meter course.
At twelve months, sarcopenic and non-sarcopenic subjects in PA tended to have higher mean SPPB scores (8.7±0.5 and 8.7±0.2 points) compared to sarcopenic and non-sarcopenic subjects in SA (8.3±0.5 and 8.4±0.2 points, p = 0.24 and 0.10), although the differences were not statistically significant. At twelve months, faster mean gait speeds were observed in PA: 0.93±0.4 and 0.95±0.03 meters/second in sarcopenic and non-sarcopenic PA subjects, and 0.89±0.4 and 0.91±0.03 meters/second in sarcopenic and non-sarcopenic SA subjects (p = 0.98 and 0.26), although not statistically significant. There was no difference between the sarcopenic and non-sarcopenic groups in intervention adherence or number of adverse events.
These data suggest that older adults with sarcopenia, who represent a vulnerable segment of the elder population, are capable of improvements in physical performance after a physical activity intervention.
PMCID: PMC4111145  PMID: 24402391
Sarcopenia; physical activity; gait speed; short physical performance battery
14.  Pericardial adipose tissue and coronary artery calcification in The Multi-Ethnic Study of Atherosclerosis (MESA) 
Obesity (Silver Spring, Md.)  2013;21(5):1056-1063.
We examined the relationship of pericardial adipose tissue (PAT) with coronary artery calcification in MESA, a large cohort in which associations by race/ethnicity can be compared. The baseline cohort comprised 6,814 Caucasian (38%), African American (28%), Chinese American (12%) and Hispanic (22%) adults aged 45–84, without known clinical cardiovascular disease. Cardiac CT was used to measure PAT (cm3) and calcification (Agatston score). We examined cross-sectional associations of PAT with the presence (score>0) and severity (continuous score if >0) of calcification using prevalence ratio (PR) (n=6,672) and linear regression (n=3,362), respectively. Main models were adjusted for age, age2, gender, race/ethnicity, field site, smoking, physical activity, alcohol and education. PAT volume (adjusted for age, height, weight and site) was greatest in Chinese males, while Black males had less PAT than all but Black females. PAT was associated with presence [PR per standard deviation (SD): 1.06 (95% CI: 1.04, 1.08)] and severity [difference in log Agatston score per SD: 0.15 (0.09, 0.21)] of calcification, but neither association varied by race/ethnicity. Adjustment for generalized adiposity attenuated but did not eliminate the associations. With further adjustment for traditional risk factors and inflammatory markers, only the association with severity remained statistically significant [PR: 1.02 (1.00, 1.04), difference: 0.10 (0.03, 0.17)]. Heterogeneity by sex was observed for presence of calcification (PR in men: 1.04; in women: 1.08; p for interaction<0.0001). Pericardial adipose tissue was associated with the presence and severity of coronary artery calcification in this cohort, but despite differences in PAT volumes and calcification across race/ethnic groups, neither association varied by race/ethnicity.
PMCID: PMC4042681  PMID: 23784910
Coronary artery calcification; pericardial fat; subclinical atherosclerosis risk factors; obesity; epidemiology
15.  The B-RafV600E inhibitor dabrafenib selectively inhibits RIP3 and alleviates acetaminophen-induced liver injury 
Cell Death & Disease  2014;5(6):e1278-.
Receptor-interacting protein (RIP)3 is a critical regulator of necroptosis and has been demonstrated to be associated with various diseases, suggesting that its inhibitors are promising in the clinic. However, there have been few RIP3 inhibitors reported as yet. B-RafV600E inhibitors are an important anticancer drug class for metastatic melanoma therapy. In this study, we found that 6 B-Raf inhibitors could inhibit RIP3 enzymatic activity in vitro. Among them, dabrafenib showed the most potent inhibition on RIP3, which was achieved by its ATP-competitive binding to the enzyme. Dabrafenib displayed highly selective inhibition on RIP3 over RIP1, RIP2 and RIP5. Moreover, only dabrafenib rescued cells from RIP3-mediated necroptosis induced by the necroptosis-induced combinations, that is, tumor necrosis factor (TNF)α, TNF-related apoptosis-inducing ligand or Fas ligand plus Smac mimetic and the caspase inhibitor z-VAD. Dabrafenib decreased the RIP3-mediated Ser358 phosphorylation of mixed lineage kinase domain-like protein (MLKL) and disrupted the interaction between RIP3 and MLKL. Notably, RIP3 inhibition of dabrafenib appeared to be independent of its B-Raf inhibition. Dabrafenib was further revealed to prevent acetaminophen-induced necrosis in normal human hepatocytes, which is considered to be mediated by RIP3. In acetaminophen-overdosed mouse models, dabrafenib was found to apparently ease the acetaminophen-caused liver damage. The results indicate that the anticancer B-RafV600E inhibitor dabrafenib is a RIP3 inhibitor, which could serve as a sharp tool for probing the RIP3 biology and as a potential preventive or therapeutic agent for RIP3-involved necroptosis-related diseases such as acetaminophen-induced liver damage.
PMCID: PMC4611716  PMID: 24901049
Diabetologia  2009;53(3):467-471.
To identify risk factors for depression and anxiety in a well-characterised cohort of subjects with type 2 diabetes mellitus.
We used baseline data from participants (n=1066, 48.7% women, aged 67.9 ± 4.2 years) from the Edinburgh Type 2 Diabetes Study. Symptoms of anxiety and depression were assessed using the Hospital Anxiety and Depression Scale (HADS). Obesity was characterised according to both overall (body mass index, fat mass) and abdominal (waist circumference) measurement. Cardiovascular disease was assessed by questionnaire, physical examination and review of medical records. Stepwise multiple linear regression was performed to identify explanatory variables related to either anxiety or depression HADS scores.
Abdominal obesity (waist circumference) and cardiovascular disease (ischaemic heart disease and ankle-brachial pressure index) were related to depression but not anxiety. Lifetime history of severe hypoglycaemia was associated with anxiety. Other cardiovascular risk factors or microvascular complications were not related to either anxiety or depressive symptoms.
Depression but not anxiety is associated with abdominal obesity and cardiovascular disease in people with type 2 diabetes mellitus. This knowledge may help to identify depressive symptoms among patients with type 2 diabetes who are at greatest risk.
PMCID: PMC3977034  PMID: 20012009
depression; anxiety; obesity; diabetes; cortisol
17.  Bacteriophage λ N protein inhibits transcription slippage by Escherichia coli RNA polymerase 
Nucleic Acids Research  2014;42(9):5823-5829.
Transcriptional slippage is a class of error in which ribonucleic acid (RNA) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (DNA) template. This phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. The bacteriophage λ N protein reduces transcriptional slippage within actively growing cells and in vitro. N appears to stabilize the RNA/DNA hybrid, particularly at the 5′ end, preventing loss of register between transcript and template. This report provides the first evidence of a protein that directly influences transcriptional slippage, and provides a clue about the molecular mechanism of transcription termination and N-mediated antitermination.
PMCID: PMC4027172  PMID: 24711367
18.  Tanshinone-1 induces tumor cell killing, enhanced by inhibition of secondary activation of signaling networks 
Cell Death & Disease  2013;4(11):e905-.
Tumor multidrug resistance (MDR) can result from overexpression of drug transporters and deregulation of cellular signaling transduction. New agents and strategies are required for overcoming MDR. Here, we report that tanshinone-1, a bioactive ingredient in traditional Chinese medicine, directly killed MDR tumor cells and their corresponding parental cells, which was potentiated by inhibition of secondary activation of signaling networks. Tanshinone-1 was slightly more potent at inducing cytotoxicity and apoptosis in MDR cells than in corresponding parental cells. Tanshinone-1-induced MDR cell killing was independent of the function and expression of drug transporters but was partially correlated with the phosphatase-dependent reduction of phospho-705-Stat3, which secondarily activated p38-, AKT-, and ERK-involved signaling networks. Cotreatments with p38, AKT, and ERK inhibitors potentiated the anti-MDR effects of tanshinone-1. Our study presents a model for MDR cell killing using a compound of natural origin. This model could lead to new therapeutic strategies for targeting signaling network(s) in MDR cancers as well as new strategies for multitarget design.
PMCID: PMC3847321  PMID: 24201804
tanshinone-1; multidrug resistance; Stat3; p38; AKT; ERK
19.  A Cross-Talk Between NFAT and NF-κB Pathways is Crucial for Nickel-Induced COX-2 Expression in Beas-2B Cells 
Current cancer drug targets  2011;11(5):548-559.
Cyclooxygenase-2 (COX-2) is a critical enzyme implicated in chronic inflammation-associated cancer development. Our studies have shown that the exposure of Beas-2B cells, a human bronchial epithelial cell line, to lung carcinogenic nickel compounds results in increased COX-2 expression. However, the signaling pathways leading to nickel-induced COX-2 expression are not well understood. In the current study, we found that the exposure of Beas-2B cells to nickel compounds resulted in the activation of both nuclear factor of activated T cell (NFAT) and nuclear factor-κB (NF-κB). The expression of COX-2 induced upon nickel exposure was inhibited by either a NFAT pharmacological inhibitor or the knockdown of NFAT3 by specific siRNA. We further found that the activation of NFAT and NF-κB was dependent on each other. Since our previous studies have shown that NF-κB activation is critical for nickel-induced COX-2 expression in Beas-2B cells exposed to nickel compounds under same experimental condition, we anticipate that there might be a cross-talk between the activation of NFAT and NF-κB for the COX-2 induction due to nickel exposure in Beas-2B cells. Furthermore, we showed that the scavenging of reactive oxygen species (ROS) by introduction of mitochondrial catalase inhibited the activation of both NFAT and NF-κB, and the induction of COX-2 due to nickel exposure. Taken together, our results defining the evidence showing a key role of the cross-talk between NFAT and NF-κB pathways in regulating nickel-induced COX-2 expression, further provide insight into the understanding of the molecular mechanisms linking nickel exposure to its lung carcinogenic effects.
PMCID: PMC3759234  PMID: 21486220
Beas-2B cells; COX-2; nickel; NFAT; NF-κB; ROS
20.  Efficient use of longitudinal CD4 counts and viral load measures in survival analysis 
Statistics in medicine  2012;31(19):2086-2097.
CD4 counts and viral loads are dynamic quantities that change with time in HIV-infected persons. Commonly used single summary measures, such as viral load set point or early CD4 count do not explicitly account for changes in viral load or CD4 counts or other features of the overall time course of these measures. However, the efficient use of all repeated measurements within each subject is often a challenge made more difficult by sparse and irregular sampling over time. Here we illustrate how functional principal component (FPC) analysis provides an effective statistical approach for exploiting the patterns in CD4 count and viral load data over time. The method is demonstrated using data from Kenyan women who acquired HIV-1 during follow-up in a high risk cohort and were subsequently followed prospectively from early infection. The FPC scores for each woman obtained by this method serve as informative summary statistics for the CD4-count and viral-load trajectories. Similar to baseline CD4 count or viral set point, the first FPC score can be interpreted as a single-value summary measure of an individual's overall CD4 count or viral load. However, unlike most single-value summaries of CD4-count or viral-load trajectories, the first FPC score summarizes the dynamics of these quantities and is seen to reveal specific features of the trajectories associated with mortality in this cohort. Moreover, FPC scores are shown to be a more powerful prognostic factor than other common summaries when used in survival analysis.
PMCID: PMC3641845  PMID: 22415871
longitudinal data; functional principal components; CD4 counts; viral loads
21.  Genome conformation capture reveals that the Escherichia coli chromosome is organized by replication and transcription 
Nucleic Acids Research  2013;41(12):6058-6071.
To fit within the confines of the cell, bacterial chromosomes are highly condensed into a structure called the nucleoid. Despite the high degree of compaction in the nucleoid, the genome remains accessible to essential biological processes, such as replication and transcription. Here, we present the first high-resolution chromosome conformation capture-based molecular analysis of the spatial organization of the Escherichia coli nucleoid during rapid growth in rich medium and following an induced amino acid starvation that promotes the stringent response. Our analyses identify the presence of origin and terminus domains in exponentially growing cells. Moreover, we observe an increased number of interactions within the origin domain and significant clustering of SeqA-binding sequences, suggesting a role for SeqA in clustering of newly replicated chromosomes. By contrast, ‘histone-like’ protein (i.e. Fis, IHF and H-NS) -binding sites did not cluster, and their role in global nucleoid organization does not manifest through the mediation of chromosomal contacts. Finally, genes that were downregulated after induction of the stringent response were spatially clustered, indicating that transcription in E. coli occurs at transcription foci.
PMCID: PMC3695519  PMID: 23632166
22.  Anti-Hepatitis C Virus Activity and Toxicity of Type III Phosphatidylinositol-4-Kinase Beta Inhibitors 
Antimicrobial Agents and Chemotherapy  2012;56(10):5149-5156.
Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.
PMCID: PMC3457382  PMID: 22825118
23.  T-cell death following immune activation is mediated by mitochondria-localized SARM 
Cell Death and Differentiation  2012;20(3):478-489.
Following acute-phase infection, activated T cells are terminated to achieve immune homeostasis, failure of which results in lymphoproliferative and autoimmune diseases. We report that sterile α- and heat armadillo-motif-containing protein (SARM), the most conserved Toll-like receptors adaptor, is proapoptotic during T-cell immune response. SARM expression is significantly reduced in natural killer (NK)/T lymphoma patients compared with healthy individuals, suggesting that decreased SARM supports NK/T-cell proliferation. T cells knocked down of SARM survived and proliferated more significantly compared with wild-type T cells following influenza infection in vivo. During activation of cytotoxic T cells, the SARM level fell before rising, correlating inversely with cell proliferation and subsequent T-cell clearance. SARM knockdown rescued T cells from both activation- and neglect-induced cell deaths. The mitochondria-localized SARM triggers intrinsic apoptosis by generating reactive oxygen species and depolarizing the mitochondrial potential. The proapoptotic function is attributable to the C-terminal sterile alpha motif and Toll/interleukin-1 receptor domains. Mechanistically, SARM mediates intrinsic apoptosis via B cell lymphoma-2 (Bcl-2) family members. SARM suppresses B cell lymphoma-extra large (Bcl-xL) and downregulates extracellular signal-regulated kinase phosphorylation, which are cell survival effectors. Overexpression of Bcl-xL and double knockout of Bcl-2 associated X protein and Bcl-2 homologous antagonist killer substantially reduced SARM-induced apoptosis. Collectively, we have shown how T-cell death following infection is mediated by SARM-induced intrinsic apoptosis, which is crucial for T-cell homeostasis.
PMCID: PMC3569988  PMID: 23175186
intrinsic T-cell death by SARM; influenza infection; adoptive transfer mouse model; neglect- and activation-induced cell death; NK/T-cell lymphoma
24.  Anomalous exchange bias at collinear/noncollinear spin interface 
Scientific Reports  2013;3:1094.
We report on the interfacial magnetic coupling in manganite bilayers of collinear ferromagnetic La0.7Sr0.3MnO3 and noncollinear multiferroic TbMnO3. Exchange bias is observed at the Néel temperature of TbMnO3 (~41 K) due to the onset of long-range antiferromagnetic ordering in the Mn spin sublattice. Interestingly, an anomalous plateau of exchange bias emerges at the ordering temperature of Tb spins (~10 K), and we ascribe this unique feature to the strong coupling between Tb and Mn spin sublattices in TbMnO3, which in turn influences the magnetic coupling across the interface. On the other hand, the enhancement of coercivity in La0.7Sr0.3MnO3/TbMnO3 shows monotonous temperature dependence. Our results illustrate a strong interfacial magnetic interaction at the La0.7Sr0.3MnO3/TbMnO3 interface, highlighting the roles of competing spin orders, magnetic frustration, and coupling between multiple spin sublattices in artificial collinear/noncollinear spin heterostructures.
PMCID: PMC3549540
25.  Free Sialic Acid Storage Disease without sialuria 
Annals of neurology  2009;65(6):753-757.
We performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on CSF and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid was elevated in CSF of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria.
PMCID: PMC3508714  PMID: 19557856
Free Sialic Acid Storage Diseases; Leukodystrophy; NMR Spectroscopy

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