Publications by authors named "Raluca Dumitru"

37 Publications

Predictors of subclinical systemic sclerosis primary heart involvement characterised by microvasculopathy and myocardial fibrosis.

Rheumatology (Oxford) 2021 Jun;60(6):2934-2945

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK.

Objectives: SSc primary heart involvement (SSc-pHI) is a significant cause of mortality. We aimed to characterize and identify predictors of subclinical SSc-pHI using cardiovascular MRI.

Methods: A total of 83 SSc patients with no history of cardiovascular disease or pulmonary arterial hypertension and 44 healthy controls (HCs) underwent 3 Tesla contrast-enhanced cardiovascular MRI, including T1 mapping and quantitative stress perfusion. High-sensitivity troponin I and N-terminal pro-brain natriuretic peptide were also measured.

Results: Cardiovascular MRI revealed a lower myocardial perfusion reserve in the SSc patients compared with HCs {median (interquartile range (IQR)] 1.9 (1.6-2.4) vs 3 (2-3.6), P < 0.001}. Late gadolinium enhancement, indicating focal fibrosis, was observed in 17/83 patients but in none of the HCs, with significantly higher extracellular volume (ECV), suggestive of diffuse fibrosis, in SSc vs HC [mean (s.d.) 31 (4) vs 25 (2), P < 0.001]. Presence of late gadolinium enhancement and higher ECV was associated with skin score [odds ratio (OR) = 1.115, P = 0.048; R2 = 0.353, P = 0.004], and ECV and myocardial perfusion reserve was associated with the presence of digital ulcers at multivariate analysis (R2 = 0.353, P < 0.001; R2 = 0.238, P = 0.011). High-sensitivity troponin I was significantly higher in patients with late gadolinium enhancement, and N-terminal pro-brain natriuretic peptide was associated with ECV (P < 0.05).

Conclusion: Subclinical SSc-pHI is characterized by myocardial microvasculopathy, diffuse and focal myocardial fibrosis but preserved myocardial contractile function. This subclinical phenotype of SSc-pHI was associated with high-sensitivity troponin I, N-terminal pro-brain natriuretic peptide, SSc disease severity and complicated peripheral vasculopathy. These data provide information regarding the underlying pathophysiological processes and provide a basis for identifying individuals at risk of SSc-pHI.
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http://dx.doi.org/10.1093/rheumatology/keaa742DOI Listing
June 2021

Cardiac magnetic resonance imaging for the detection of myocardial involvement in granulomatosis with polyangiitis.

Int J Cardiovasc Imaging 2021 Mar 14;37(3):1053-1062. Epub 2020 Oct 14.

NIHR Leeds Biomedical Research Centre and Clinical Research Facility, Leeds Teaching Hospitals NHS Trust, Leeds, UK.

The prevalence of undiagnosed cardiac involvement in granulomatosis with polyangiitis (GPA) is unknown. In this prospective study we investigated the utility of cardiovascular magnetic resonance (CMR) to identify myocardial abnormalities in GPA and their correlation with disease phenotype. Twenty-six patients with GPA and no cardiovascular disease or diabetes mellitus underwent contrast-enhanced CMR, including late gadolinium-enhancement (LGE), T1-mapping for native T1 and extra-cellular volume (ECV) quantification for assessment of myocardial fibrosis, cine imaging and tissue tagging for assessment of left ventricular (LV) function. Twenty-five healthy volunteers (HV) with comparable age, sex, BMI and arterial blood pressure served as controls. Patients with GPA had similar cardiovascular risk profile to HV. A focal, non-ischaemic LGE pattern of fibrosis was detected in 24% of patients and no controls (p = 0.010). Patients with myocardial LGE were less frequently PR3 ANCA (7% vs 93%, p = 0.007), and had involvement of the lower respiratory tract and skin. LGE scar mass was higher in patients presenting with renal involvement. Native T1 and ECV were higher in patients with GPA than HV; ECV was higher in those with relapsing disease, and native T1 was inversely associated with PR3 ANCA (β = - 0.664, p = 0.001). Peak systolic strain was slightly reduced in GPA compared to controls; LV ejection function was inversely correlated with disease duration (β = - 0.454, p = 0.026). Patients with GPA have significant myocardial abnormalities on CMR. ANCA, systemic involvement and disease severity were associated with myocardial fibrosis. CMR could be a useful tool for risk stratification of myocardial involvement in GPA.
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http://dx.doi.org/10.1007/s10554-020-02066-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969556PMC
March 2021

Cardiovascular effects of biological versus conventional synthetic disease-modifying antirheumatic drug therapy in treatment-naïve, early rheumatoid arthritis.

Ann Rheum Dis 2020 11 28;79(11):1414-1422. Epub 2020 Aug 28.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK

Objectives: To determine whether patients with early rheumatoid arthritis (ERA) have cardiovascular disease (CVD) that is modifiable with disease-modifying antirheumatic drug (DMARD) therapy, comparing first-line etanercept (ETN) + methotrexate (MTX) with MTX strategy.

Methods: Patients from a phase IV ERA trial randomised to ETN+MTX or MTX strategy±month 6 escalation to ETN+MTX, and with no CVD and maximum one traditional risk factor underwent cardiovascular magnetic resonance (CMR) at baseline, years 1 and 2. Thirty matched controls underwent CMR. Primary outcome measure was aortic distensibility (AD) between controls and ERA, and baseline to year 1 AD change in ERA. Secondary analyses between and within ERA groups performed. Additional outcome measures included left ventricular (LV) mass and myocardial extracellular volume (ECV).

Results: Eighty-one patients recruited. In ERA versus controls, respectively, baseline (geometric mean, 95% CI) AD was significantly lower (3.0×10 mm Hg (2.7-3.3) vs 4.4×10 mm Hg (3.7-5.2), p<0.001); LV mass significantly lower (78.2 g (74.0-82.7), n=81 vs 92.9 g (84.8-101.7), n=30, p<0.01); and ECV increased (27.1% (26.4-27.9), n=78 vs 24.9% (23.8-26.1), n=30, p<0.01). Across all patients, AD improved significantly from baseline to year 1 (3.0×10 mm Hg (2.7-3.4) to 3.6×10 mm Hg (3.1-4.1), respectively, p<0.01), maintained at year 2. The improvement in AD did not differ between the two treatment arms and disease activity state (Disease Activity Score with 28 joint count)-erythrocyte sedimentation rate-defined responders versus non-responders.

Conclusion: We report the first evidence of vascular and myocardial abnormalities in an ERA randomised controlled trial cohort and show improvement with DMARD therapy. The type of DMARD (first-line tumour necrosis factor-inhibitors or MTX) and clinical response to therapy did not affect CVD markers.

Trial Registration Number: ISRCTN: ISRCTN89222125; ClinicalTrials.gov: NCT01295151.
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http://dx.doi.org/10.1136/annrheumdis-2020-217653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569379PMC
November 2020

Lanthanum Ferrite Ceramic Powders: Synthesis, Characterization and Electrochemical Detection Application.

Materials (Basel) 2020 Apr 29;13(9). Epub 2020 Apr 29.

Faculty of Industrial Chemistry and Environmental Engineering, Politehnica University Timisoara, Piata Victoriei No. 2, RO-300006 Timisoara, Romania.

The perovskite-type lanthanum ferrite, LaFeO, has been prepared by thermal decomposition of obtained lanthanum ferrioxalate compound precursor, LaFe(CO)·3HO. The oxalate precursor was synthesized through the redox reaction between 1,2-ethanediol and nitrate ion and characterized by chemical analysis, infrared spectroscopy, and thermal analysis. LaFeO obtained after the calcination of the precursor for at least 550-800 °C/1 h have been investigated by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), and high-resolution transmission electron microscopy (HRTEM). A boron-doped diamond electrode (BDD) modified with LaFeO ceramic powders at 550 °C (LaFeO/BDD) by simple immersion was characterized by cyclic voltammetry and tested for the voltammetric and amperometric detection of capecitabine (CCB), which is a cytostatic drug considered as an emerging pollutant in water. The modified electrode exhibited a complex electrochemical behaviour by several redox systems in direct relation to the electrode potential range. The results obtained by cyclic voltammetry (CV), differential-pulsed voltammetry (DPV), and multiple-pulsed amperometry proved the electrocatalytic effect to capecitabine oxidation and reduction and allowed its electrochemical detection in alkaline aqueous solution.
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http://dx.doi.org/10.3390/ma13092061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7254238PMC
April 2020

Feasibility of MRI based extracellular volume fraction and partition coefficient measurements in thigh muscle.

Br J Radiol 2020 Jul 11;93(1111):20190931. Epub 2020 May 11.

Department Of Medical Physics and Engineering, Leeds Teaching Hospitals NHS Trust, Leeds, UK.

Objective: This study aimed to assess the feasibility of extracellular volume-fraction (ECV) measurement, and time to achieve contrast equilibrium (CE), in healthy muscles, and to determine whether in-flow and partial-volume errors in the femoral artery affect measurements, and if there are differences in the partition coefficient (λ) between muscles.

Methods: T1 was measured in the biceps femoris, vastus intermedius, femoral artery and aorta of 10 healthy participants. This was repeated alternately between the thigh and aorta for ≥25 min following a bolus of gadoterate meglumine. λ was calculated for each muscle/blood measurement. Time to CE was assessed semi-quantitatively.

Results: 8/10 participants achieved CE. Time to CE = 19±2 min (mean ± 95% confidence interval). Measured λ: biceps femoris/aorta = 0.210±0.034, vastus intermedius/aorta = 0.165±0.015, biceps femoris/femoral artery = 0.265±0.054, vastus intermedius/femoral artery = 0.211±0.026. There were significant differences in λ between the muscles when using the same vessel ( < 0.05), and between λ calculated in the same muscle when using different vessels ( < 0.05).

Conclusion: ECV measurements in the thigh are clinically feasible. The use of the femoral artery for the blood measurement is associated with small but significant differences in λ. ECV measurements are sensitive to differences between muscles within the healthy thigh.

Advances In Knowledge: This paper determines the time to contrast equilibrium in the healthy thigh and describes a method for measuring accurately ECV in skeletal muscle. This can aid in the diagnosis and understanding of inflammatory auto-immune diseases.
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http://dx.doi.org/10.1259/bjr.20190931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336063PMC
July 2020

Pragmatic randomised controlled trial of very early etanercept and MTX versus MTX with delayed etanercept in RA: the VEDERA trial.

Ann Rheum Dis 2020 04 29;79(4):464-471. Epub 2020 Jan 29.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK

Objectives: We sought to confirm in very early rheumatoid arthritis (ERA) a much greater superiority (30%) of first-line etanercept+methotrexate (ETN+MTX) over treat-to-target MTX (MTX-TT) than previously reported in ERA (14%); and explore whether ETN following initial MTX secures a comparable response to first-line ETN+MTX.

Methods: Pragmatic, open-label, randomised controlled trial of treatment-naïve ERA (≤12 months symptom), Disease Activity Score 28 joint (DAS28)-erythrocyte sedimentation rate (ESR) ≥3.2, rheumatoid factor (RF)+/-anticitrullinated peptide antibody (ACPA) positive or ultrasound power Doppler (PD) if RF and ACPA negative. Subjects were randomised 1:1 to ETN+MTX; or MTX-TT, escalated to ETN if week 24 DAS28-ESR ≥2.6 and intramuscular corticosteroid at protocolised time points. Primary endpoint of week 48 DAS28ESR remission with clinical and imaging secondary endpoints.

Results: We randomised 120 patients, 60 to each arm (71% female, 73% RF/84% ACPA positive, median (IQR) symptom duration 20.3 (13.1, 30.8) weeks; mean (SD) DAS28 5.1 (1.1)). Remission rates with ETN+MTX and MTX-TT, respectively, were 38% vs 33% at week 24; 52% vs 38% at week 48 (ORs 1.6, 95% CI 0.8 to 3.5, p=0.211). Greater, sustained DAS28-ESR remission observed with ETN+MTX versus MTX-TT (42% and 27%, respectively; p=0.035). PD was fully suppressed by week 48 in over 90% in each arm. Planned exploratory analysis revealed OR 2.84, 95% CI 0.8 to 9.6) of achieving remission after 24 weeks of ETN administered first line compared with administered post-MTX.

Conclusions: Compared with remission rates typically reported with first-line tumour necrosis factor inhabitor+MTX versus MTX-TT, we did not demonstrate a larger effect in very ERA. Neither strategy conferred remission in the majority of patients although ultrasound confirmed local inflammation suppression. Poorer ETN response following failure of MTX-TT is also suggested. NCT02433184.
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http://dx.doi.org/10.1136/annrheumdis-2019-216539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147179PMC
April 2020

Longitudinal dynamic functional regression.

J R Stat Soc Ser C Appl Stat 2020 Jan 12;69(1):25-46. Epub 2019 Sep 12.

North Carolina State University, Rayleigh, USA.

The paper develops a parsimonious modelling framework to study the time-varying association between scalar outcomes and functional predictors observed at many instances, in longitudinal studies. The methods enable us to reconstruct the full trajectory of the response and are applicable to Gaussian and non-Gaussian responses. The idea is to model the time-varying functional predictors by using orthogonal basis functions and to expand the time-varying regression coefficient by using the same basis. Numerical investigation through simulation studies and data analysis show excellent performance in terms of accurate prediction and efficient computations, when compared with existing alternatives. The methods are inspired and applied to an animal science application, where of interest is to study the association between the feed intake of lactating sows and the minute-by-minute temperature throughout the 21 days of their lactation period. R code and an R illustration are provided.
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http://dx.doi.org/10.1111/rssc.12376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953745PMC
January 2020

Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity.

Elife 2019 11 4;8. Epub 2019 Nov 4.

Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, United States.

Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.
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http://dx.doi.org/10.7554/eLife.47789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927689PMC
November 2019

Self-assembled zinc oxide hierarchical structures with enhanced antibacterial properties from stacked chain-like zinc oxalate compounds.

J Colloid Interface Sci 2019 Sep 18;552:258-270. Epub 2019 May 18.

"Ilie Murgulescu" Institute of Physical Chemistry, Romanian Academy, Spl. Independentei 202, 060021 Bucharest, Romania. Electronic address:

Single ZnO crystallites assembled into porous hierarchical structures have been prepared by topotactic thermal decomposition of in situ obtained zinc oxalate precursors, whose synthesis involves a redox reaction between 1,2-ethanediol and nitrate ion. For the first time it was demonstrated that post-synthesis protocols of the precursors (e.g. ultrasound irradiation, hydrolytic decomposition) master the hydrogen bonds formed between oxalate chains, allowing that way the adjustment of materials properties (morphology, porosity and optical) and a rational introduction of different dopants (Eu/Er). The ZnO surface reactivity is confirmed by the significant biocidal activity of the obtained materials against Gram-positive and Gram-negative planktonic and biofilm-embedded cells, superior to those reported in the literature for other ZnO-based materials or antibiotics, associated also with a good biocompatibility.
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http://dx.doi.org/10.1016/j.jcis.2019.05.051DOI Listing
September 2019

Incidental significant arrhythmia in scleroderma associates with cardiac magnetic resonance measure of fibrosis and hs-TnI and NT-proBNP.

Rheumatology (Oxford) 2019 07;58(7):1221-1226

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK.

Objectives: To screen for significant arrhythmias with an implantable loop recorder (ILR) in patients with SSc and no known cardiovascular disease, and identify associated disease phenotype, blood and cardiovascular magnetic resonance (CMR) biomarkers.

Methods: Twenty patients with SSc with no history of primary SSc heart disease, traditional cardiovascular disease, diabetes or maximum one traditional cardiovascular risk factor underwent clinical assessment, contrast-enhanced CMR and ILR insertion.

Results: ILR data were available for 19 patients: 63% female, mean (s.d.) age of 53 (12) years, 32% diffuse SSc. Eight patients had significant arrhythmias over 3 years: one complete heart block, two non-sustained ventricular tachycardia [all three dcSSc, two anti-topoisomerase antibodies (Scl70) positive, three interstitial lung disease and two previous digital ulceration] and five atrial arrhythmias of which four were with limited SSc. These required interventions with one permanent pacemaker implantation, four anti-arrhythmic pharmacotherapy, one anticoagulation.Patients with significant arrhythmia had higher baseline high-sensitivity troponin I and N-terminal pro-brain natriuretic peptide [mean difference (95% CI) 117 (-11, 245) and 92 (-30, 215) ng/l, respectively], and CMR-extracellular volume [mean (s.d.) 32 (2) vs 29 (4)%]. Late gadolinium enhancement was observed in five patients, only one with significant arrhythmia.

Conclusion: This first ILR study identified potentially life-threatening arrhythmias in asymptomatic SSc patients attributable to a primary SSc heart disease. Disease phenotype, CMR-extracellular volume (indicating diffuse fibrosis) and cardiac biomarkers may identify at-risk patients that would benefit from ILR screening. Future studies can inform a risk model and provide insights into SSc-associated arrhythmia pathogenesis.
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http://dx.doi.org/10.1093/rheumatology/key430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587914PMC
July 2019

European multicentre study validates enhanced liver fibrosis test as biomarker of fibrosis in systemic sclerosis.

Rheumatology (Oxford) 2019 02;58(2):254-259

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, UK.

Objectives: To validate enhanced liver fibrosis (ELF) test and its components-amino-terminal propeptide of procollagen type III (PIIINP), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and HA-as biomarkers of fibrosis in SSc in an independent, international, multicentre cohort.

Methods: Two hundred and fifty-four SSc patients from six Rheumatology Centres were included. Sera were collected and stored according to EUSTAR biobanking recommendations and analysed through automated high throughput diagnostics. Statistical analysis was performed with SPSS software.

Results: Two hundred and forty-seven SSc patients (mean age 55.7 ± 13.9 years, 202 F) were analysed. ELF score, TIMP-1 and PIIINP levels were higher in males (P = 0.0197, P = 0.0107, P = 0.0108 respectively) and in dcSSc (P = 0.001, P = 0.0008, P < 0.0001 respectively). ELF score and the single markers significantly correlated with modified Rodnan skin score (r = 0.37, P < 0.0001), disease activity and severity (P < 0.0001 for all markers, except for HA P = 0.0001) and inversely with forced vital capacity, (FVC) % (TIMP-1, r = -0.21, P = 0.0012; PIIINP, r = -0.26, P = 0.0001), TLC% (ELF score, r = -0.20, P = 0.0036; TIMP-1, r = -0.32, P < 0.0001; PIIINP, r = -0.28, P < 0.0001), diffusion capacity of the lung for carbon monoxide (DLCO) % (P < 0.0001 for all markers, except for HA P = 0.0115). Multivariate analysis indicated that age (P < 0.001), modified Rodnan skin score (P < 0.001) and DLCO% (P = 0.005) were independently associated with ELF score.

Conclusion: Between the first and this validation studies, the value of the ELF score as independent marker of skin and lung involvement in SSc is confirmed in 457 patients. A longitudinal study is on-going to identify an SSc specific algorithm with predictive value for skin and lung progression.
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http://dx.doi.org/10.1093/rheumatology/key271DOI Listing
February 2019

Inheritance of OCT4 predetermines fate choice in human embryonic stem cells.

Mol Syst Biol 2018 09 3;14(9):e8140. Epub 2018 Sep 3.

Department of Genetics, University of North Carolina, Chapel Hill, Chapel Hill, NC, USA

It is well known that clonal cells can make different fate decisions, but it is unclear whether these decisions are determined during, or before, a cell's own lifetime. Here, we engineered an endogenous fluorescent reporter for the pluripotency factor OCT4 to study the timing of differentiation decisions in human embryonic stem cells. By tracking single-cell OCT4 levels over multiple cell cycle generations, we found that the decision to differentiate is largely determined before the differentiation stimulus is presented and can be predicted by a cell's preexisting OCT4 signaling patterns. We further quantified how maternal OCT4 levels were transmitted to, and distributed between, daughter cells. As mother cells underwent division, newly established OCT4 levels in daughter cells rapidly became more predictive of final OCT4 expression status. These results imply that the choice between developmental cell fates can be largely predetermined at the time of cell birth through inheritance of a pluripotency factor.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120590PMC
http://dx.doi.org/10.15252/msb.20178140DOI Listing
September 2018

Intra-cavity stem cell therapy inhibits tumor progression in a novel murine model of medulloblastoma surgical resection.

PLoS One 2018 10;13(7):e0198596. Epub 2018 Jul 10.

Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Background: Cytotoxic neural stem cells (NSCs) have emerged as a promising treatment for Medulloblastoma (MB), the most common malignant primary pediatric brain tumor. The lack of accurate pre-clinical models incorporating surgical resection and tumor recurrence limits advancement in post-surgical MB treatments. Using cell lines from two of the 5 distinct MB molecular sub-groups, in this study, we developed an image-guided mouse model of MB surgical resection and investigate intra-cavity NSC therapy for post-operative MB.

Methods: Using D283 and Daoy human MB cells engineered to express multi-modality optical reporters, we created the first image-guided resection model of orthotopic MB. Brain-derived NSCs and novel induced NSCs (iNSCs) generated from pediatric skin were engineered to express the pro-drug/enzyme therapy thymidine kinase/ganciclovir, seeded into the post-operative cavity, and used to investigate intra-cavity therapy for post-surgical MB.

Results: We found that surgery reduced MB volumes by 92%, and the rate of post-operative MB regrowth increased 3-fold compared to pre-resection growth. Real-time imaging showed NSCs rapidly homed to MB, migrating 1.6-fold faster and 2-fold farther in the presence of tumors, and co-localized with MB present in the contra-lateral hemisphere. Seeding of cytotoxic NSCs into the post-operative surgical cavity decreased MB volumes 15-fold and extended median survival 133%. As an initial step towards novel autologous therapy in human MB patients, we found skin-derived iNSCs homed to MB cells, while intra-cavity iNSC therapy suppressed post-surgical tumor growth and prolonged survival of MB-bearing mice by 123%.

Conclusions: We report a novel image-guided model of MB resection/recurrence and provide new evidence of cytotoxic NSCs/iNSCs delivered into the surgical cavity effectively target residual MB foci.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198596PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038981PMC
December 2018

Association of Oral-Health Related Quality of Life and General Health Assessment in Patients with Rheumatoid Arthritis.

Oral Health Prev Dent 2018;16(3):271-280

Purpose: To determine the impact of oral health related quality of life (OHRQoL) on general health in patients suffering from rheumatoid arthritis (RA).

Materials And Methods: Ninety-one patients with RA (mean age 52.82 ± 11 years, 75.82% female, 20.87% smokers) and 30 systemically healthy patients (control) were evaluated for their OHRQoL by means of the Geriatric Oral Health Assessment Index (GOHAI) and the Oral Health Impact Profile (OHIP)-14 questionnaires. Self-perceived RA status was assessed using the Routine Assessment of Patient Index Data 3 (RAPID3).

Results: The mean SC-GOHAI score was 3.69 ± 2.47 for RA subjects and 1.36 ± 2.69 in the control group. Statistically significant differences were seen between RA and control groups (p < 0.05). RA patients with and without periodontitis (PA) exhibited similar SC-GOHAI (Simple Count GOHAI) scores (p = 0.980). No statistically significant differences were observed between any of the groups, either for the OHIP 14-extent or for the OHIP 14-prevalence. RAPID3 scores showed that the majority of the RA patients (65.93%) had high disease severity (RAPID3 >12, mean RAPID3 score 14.39 ± 5.14). Statistically significantly higher values were recorded for general health assessment (PTGE, p = 0.009) and fatigue (FT, p = 0.004) in RA with PA as compared to those without. SC-GOHAI with values between 5 and 8 was statistically significantly associated with high severity health impairment (RAPID3 >12, p = 0.014, OR: 8.64).

Conclusion: Within their limits, the present findings indicate that: a) moderate OHRQoL as assessed by GOHAI may contribute to high severity impairment of health in RA patients, and b) the GOHAI questionnaire may represent a more adequate tool than OHIP-14 for assessing OHRQoL in patients suffering from RA.
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http://dx.doi.org/10.3290/j.ohpd.a39912DOI Listing
October 2018

Effects of non-surgical periodontal therapy on periodontal laboratory and clinical data as well as on disease activity in patients with rheumatoid arthritis.

Clin Oral Investig 2019 Jan 27;23(1):141-151. Epub 2018 Mar 27.

Department of Periodontology, School of Dental Medicine, University of Bern, Freiburgstrasse 7, 3010, Bern, Switzerland.

Objectives: To compare the effect of non-surgical periodontal therapy on clinical and inflammatory parameters in patients with moderate to severe chronic periodontitis (CP) and rheumatoid arthritis (RA) (RA-CP) with that in CP patients without RA.

Material And Methods: Eighteen patients with RA-CP and 18 systemically healthy patients with CP were treated with scaling and root planing (SRP) within 24 h. At baseline, and at 3 and 6 months after SRP, clinical periodontal parameters, inflammatory markers, and microorganisms in subgingival biofilm were assessed. In addition, disease activity markers of RA (DAS28, CRP, ESR) and specific antibodies (RF) were monitored in the RA-CP group.

Results: In both groups, non-surgical therapy yielded to statistically significant improvements in all investigated clinical periodontal variables; in RA patients, a statistically significant decrease in serum-CRP was seen at 3 months. At all time-points, levels of inflammatory markers in GCF were higher in RA-CP than in CP patients. Counts of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola decreased statistically significantly in CP but not in the RA-CP group. Changes of DAS28 correlated positively with those of P. gingivalis and negatively with the plaque index.

Conclusions: Within their limits, the present data suggest that (a) non-surgical periodontal therapy improves periodontal conditions in CP patients with and without RA and (b) in patients with RA, eradication of P. gingivalis in conjunction with a high level oral hygiene may transiently decrease disease activity of RA.

Clinical Relevance: In patients with RA and CP, non-surgical periodontal therapy is a relevant modality not only to improve the periodontal condition but also to decrease RA activity.
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http://dx.doi.org/10.1007/s00784-018-2420-3DOI Listing
January 2019

Rapid DNA replication origin licensing protects stem cell pluripotency.

Elife 2017 11 17;6. Epub 2017 Nov 17.

Department of Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, United States.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.
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http://dx.doi.org/10.7554/eLife.30473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5720591PMC
November 2017

Messenger RNAs localized to distal projections of human stem cell derived neurons.

Sci Rep 2017 04 4;7(1):611. Epub 2017 Apr 4.

UNC/NC State Joint Department of Biomedical Engineering, UNC-Chapel Hill, Chapel Hill, NC, 27599, USA.

The identification of mRNAs in distal projections of model organisms has led to the discovery of multiple proteins that are locally synthesized for functional roles such as axon guidance, injury signaling and regeneration. The extent to which local protein synthesis is conserved in human neurons is unknown. Here we used compartmentalized microfluidic chambers to characterize the transcriptome of distal projections of human embryonic stem cells differentiated using a protocol which enriched for glutamatergic neurons (hESC-neurons). Using gene expression analysis, we identified mRNAs proportionally enriched in these projections, representing a functionally unique local transcriptome as compared to the human neuronal transcriptome inclusive of somata. Further, we found that the most abundant mRNAs within these hESC-neuron projections were functionally similar to the axonal transcriptome of rat cortical neurons. We confirmed the presence of two well characterized axonal mRNAs in model organisms, β-actin and GAP43, within hESC-neuron projections using multiplexed single molecule RNA-FISH. Additionally, we report the novel finding that oxytocin mRNA localized to these human projections and confirmed its localization using RNA-FISH. This new evaluation of mRNA within human projections provides an important resource for studying local mRNA translation and has the potential to reveal both conserved and unique translation dependent mechanisms.
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http://dx.doi.org/10.1038/s41598-017-00676-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428799PMC
April 2017

Consensus best practice pathway of the UK Systemic Sclerosis Study group: management of cardiac disease in systemic sclerosis.

Rheumatology (Oxford) 2017 06;56(6):912-921

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds.

Objective: Cardiac disease in SSc can manifest in various ways and is associated with a poor prognosis. There is little evidence on how best to detect and manage cardiac disease in SSc. Our objective was to produce an expert consensus best practice pathway for the management of cardiac disease in SSc.

Methods: The UK Systemic Sclerosis Study Group set up several working groups to develop a number of consensus best practice pathways for the management of SSc-specific complications, including cardiac disease. A multidisciplinary task force was convened. The guidelines were partly informed by a comprehensive literature review.

Results: A best practice pathway for cardiac disease (with a focus on primary cardiac disease) in SSc is presented, including approaches for early detection and standard pharmacological and device therapies. Due to the benefits, shared care and a multidisciplinary approach are recommended. A future research agenda has been formulated in response to the relative lack of understanding of the natural history of primary cardiac disease that was highlighted by the initiative.

Conclusion: The physician should be alert to the possibility of cardiac disease in SSc; it is best managed within a multidisciplinary team including both rheumatologists and cardiologists. This pathway provides a reference for all physicians managing patients with SSc.
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http://dx.doi.org/10.1093/rheumatology/kew488DOI Listing
June 2017

Tumor-homing cytotoxic human induced neural stem cells for cancer therapy.

Sci Transl Med 2017 02;9(375)

Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Engineered neural stem cells (NSCs) are a promising approach to treating glioblastoma (GBM). The ideal NSC drug carrier for clinical use should be easily isolated and autologous to avoid immune rejection. We transdifferentiated (TD) human fibroblasts into tumor-homing early-stage induced NSCs (h-iNSC), engineered them to express optical reporters and different therapeutic gene products, and assessed the tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSC in patient-derived GBM models of surgical and nonsurgical disease. Molecular and functional analysis revealed that our single-factor SOX2 TD strategy converted human skin fibroblasts into h-iNSC that were nestin and expressed pathways associated with tumor-homing migration in 4 days. Time-lapse motion analysis showed that h-iNSC rapidly migrated to human GBM cells and penetrated human GBM spheroids, a process inhibited by blockade of CXCR4. Serial imaging showed that h-iNSC delivery of the proapoptotic agent tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) reduced the size of solid human GBM xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days. Additionally, h-iNSC thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSC-TK) reduced the size of patient-derived GBM xenografts 20-fold and extended survival from 32 to 62 days. Mimicking clinical NSC therapy, h-iNSC-TK therapy delivered into the postoperative surgical resection cavity delayed the regrowth of residual GBMs threefold and prolonged survival from 46 to 60 days. These results suggest that TD of human skin into h-iNSC is a platform for creating tumor-homing cytotoxic cell therapies for cancer, where the potential to avoid carrier rejection could maximize treatment durability in human trials.
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http://dx.doi.org/10.1126/scitranslmed.aah6510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719790PMC
February 2017

Widespread Chromatin Accessibility at Repetitive Elements Links Stem Cells with Human Cancer.

Cell Rep 2016 11;17(6):1607-1620

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599, USA; Department of Genetics, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pediatrics, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Chromatin regulation is critical for differentiation and disease. However, features linking the chromatin environment of stem cells with disease remain largely unknown. We explored chromatin accessibility in embryonic and multipotent stem cells and unexpectedly identified widespread chromatin accessibility at repetitive elements. Integrating genomic and biochemical approaches, we demonstrate that these sites of increased accessibility are associated with well-positioned nucleosomes marked by distinct histone modifications. Differentiation is accompanied by chromatin remodeling at repetitive elements associated with altered expression of genes in relevant developmental pathways. Remarkably, we found that the chromatin environment of Ewing sarcoma, a mesenchymally derived tumor, is shared with primary mesenchymal stem cells (MSCs). Accessibility at repetitive elements in MSCs offers a permissive environment that is exploited by the critical oncogene responsible for this cancer. Our data demonstrate that stem cells harbor a unique chromatin landscape characterized by accessibility at repetitive elements, a feature associated with differentiation and oncogenesis.
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http://dx.doi.org/10.1016/j.celrep.2016.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5267842PMC
November 2016

LPS-TLR4 Pathway Mediates Ductular Cell Expansion in Alcoholic Hepatitis.

Sci Rep 2016 10 18;6:35610. Epub 2016 Oct 18.

Division of Gastroenterology and Hepatology, Departments of Medicine and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there are no effective therapies. Patients with AH show impaired hepatocyte proliferation, expansion of inefficient ductular cells and high lipopolysaccharide (LPS) levels. It is unknown whether LPS mediates ductular cell expansion. We performed transcriptome studies and identified keratin 23 (KRT23) as a new ductular cell marker. KRT23 expression correlated with mortality and LPS serum levels. LPS-TLR4 pathway role in ductular cell expansion was assessed in human and mouse progenitor cells, liver slices and liver injured TLR4 KO mice. In AH patients, ductular cell expansion correlated with portal hypertension and collagen expression. Functional studies in ductular cells showed that KRT23 regulates collagen expression. These results support a role for LPS-TLR4 pathway in promoting ductular reaction in AH. Maneuvers aimed at decreasing LPS serum levels in AH patients could have beneficial effects by preventing ductular reaction development.
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http://dx.doi.org/10.1038/srep35610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067590PMC
October 2016

A prospective, single-centre, randomised study evaluating the clinical, imaging and immunological depth of remission achieved by very early versus delayed Etanercept in patients with Rheumatoid Arthritis (VEDERA).

BMC Musculoskelet Disord 2016 Feb 5;17:61. Epub 2016 Feb 5.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK.

Background: Rheumatoid arthritis (RA) is a chronic inflammatory arthritis, with significant impact on quality of life and functional status. Whilst biologic disease modifying anti-rheumatic drugs (bDMARD) such as tumour necrosis factor-inhibitor (TNFi) agents have revolutionised outcomes in RA, early diagnosis with immediate conventional therapy, titrated in a treat to target approach is also associated with high remission rates. The main aim of the VEDERA study (Very Early versus Delayed Etanercept in Rheumatoid Arthritis) is to assess the depth of remission, sustainability of remission and immunological normalisation induced by very early TNFi with etanercept (ETN) or standard of care +/- delayed ETN.

Methods/design: VEDERA is a pragmatic, phase IV single-centre open-label randomised superiority trial of 120 patients with early, treatment-naive RA. Patients will be randomised 1:1 to first-line ETN and methotrexate (MTX) or MTX with additional synthetic disease modifying anti-rheumatic drugs (sDMARDs) according to a treat to target (TT) protocol with further step up to ETN and MTX after 24 weeks if remission is not achieved. Participants will have regular disease activity assessments and imaging evaluation including musculoskeletal ultrasound and MRI. The main objective of this study is to assess the proportion of patients with early RA that achieve clinical remission at 48 weeks, following either treatment strategy. In addition, the participants are invited to take part in a cardio-vascular sub-study (Coronary Artery Disease in RA, CADERA), which aims to identify the incidence of cardiovascular abnormalities in early RA.

Discussion: The hypothesis underlining this study is that very early treatment with first-line ETN increases the proportion of patients with rheumatoid arthritis achieving clinical remission, in comparison to conventional therapy.

Trial Registration: NCT02433184 , 23/04/2015.
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http://dx.doi.org/10.1186/s12891-016-0915-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743173PMC
February 2016

Therapeutically engineered induced neural stem cells are tumour-homing and inhibit progression of glioblastoma.

Nat Commun 2016 Feb 2;7:10593. Epub 2016 Feb 2.

Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

Transdifferentiation (TD) is a recent advancement in somatic cell reprogramming. The direct conversion of TD eliminates the pluripotent intermediate state to create cells that are ideal for personalized cell therapy. Here we provide evidence that TD-derived induced neural stem cells (iNSCs) are an efficacious therapeutic strategy for brain cancer. We find that iNSCs genetically engineered with optical reporters and tumouricidal gene products retain the capacity to differentiate and induced apoptosis in co-cultured human glioblastoma cells. Time-lapse imaging shows that iNSCs are tumouritropic, homing rapidly to co-cultured glioblastoma cells and migrating extensively to distant tumour foci in the murine brain. Multimodality imaging reveals that iNSC delivery of the anticancer molecule TRAIL decreases the growth of established solid and diffuse patient-derived orthotopic glioblastoma xenografts 230- and 20-fold, respectively, while significantly prolonging the median mouse survival. These findings establish a strategy for creating autologous cell-based therapies to treat patients with aggressive forms of brain cancer.
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http://dx.doi.org/10.1038/ncomms10593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740908PMC
February 2016

Transferable neuronal mini-cultures to accelerate screening in primary and induced pluripotent stem cell-derived neurons.

Sci Rep 2015 Feb 10;5:8353. Epub 2015 Feb 10.

1] UNC/NCSU Joint Department of Biomedical Engineering [2] UNC Neuroscience Center [3] Carolina Institute for Developmental Disabilities.

The effort and cost of obtaining neurons for large-scale screens has limited drug discovery in neuroscience. To overcome these obstacles, we fabricated arrays of releasable polystyrene micro-rafts to generate thousands of uniform, mobile neuron mini-cultures. These mini-cultures sustain synaptically-active neurons which can be easily transferred, thus increasing screening throughput by >30-fold. Compared to conventional methods, micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. We validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics. This affordable high-throughput approach has the potential to transform drug discovery in neuroscience.
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http://dx.doi.org/10.1038/srep08353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4322355PMC
February 2015

Human embryonic stem cells have constitutively active Bax at the Golgi and are primed to undergo rapid apoptosis.

Mol Cell 2012 Jun 3;46(5):573-83. Epub 2012 May 3.

Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

Human embryonic stem (hES) cells activate a rapid apoptotic response after DNA damage but the underlying mechanisms are unknown. A critical mediator of apoptosis is Bax, which is reported to become active and translocate to the mitochondria only after apoptotic stimuli. Here we show that undifferentiated hES cells constitutively maintain Bax in its active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having preactivated Bax. After DNA damage, active Bax rapidly translocated to the mitochondria by a p53-dependent mechanism. Interestingly, upon differentiation, Bax was no longer active, and cells were not acutely sensitive to DNA damage. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development.
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http://dx.doi.org/10.1016/j.molcel.2012.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372694PMC
June 2012

Cnot1, Cnot2, and Cnot3 maintain mouse and human ESC identity and inhibit extraembryonic differentiation.

Stem Cells 2012 May;30(5):910-22

Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, RTP, North Carolina, USA.

Embryonic stem cell (ESC) identity and self-renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here, we show that three members of the Ccr4-Not complex, Cnot1, Cnot2, and Cnot3, play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts, these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs, Cnot1, Cnot2, and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self-renewal pathways or core transcription factors. Instead, they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly, these Cnot genes are also necessary for the maintenance of human ESCs, and silencing them mainly lead to TE and primitive endoderm differentiation. Together, our results indicate that Cnot1, Cnot2, and Cnot3 represent a novel component of the core self-renewal and pluripotency circuitry conserved in mouse and human ESCs.
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http://dx.doi.org/10.1002/stem.1070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3787717PMC
May 2012

Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

PLoS One 2011 17;6(11):e27520. Epub 2011 Nov 17.

Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, United States of America.

Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0027520PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219675PMC
May 2012

Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentous-growth induction.

Eukaryot Cell 2008 Jun 18;7(6):980-7. Epub 2008 Apr 18.

School of Biological Sciences, University of Nebraska, Lincoln, NE 68588-0666, USA.

Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to respond to farnesol. Treatment of C. albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1-regulated hypha-specific genes, HWP1 and RBT1. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Farnesol did not affect EFG1 (a transcription regulator of filament development), NRG1, or RFG1 mRNA levels, demonstrating specific gene regulation in response to farnesol. Furthermore, the tup1/tup1 and nrg1/nrg1 mutants produced 17- and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Our data are consistent with the role of Tup1 as a crucial component of the response to farnesol in C. albicans.
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http://dx.doi.org/10.1128/EC.00357-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2446655PMC
June 2008