Bone size is an important determinant of bone strength and is under strong genetic control.
To identify quantitative trait loci (QTL) for areal bone size variation, a large‐scale genomewide linkage scan was carried out in 451 Caucasian families.
Participants and methods
Of 4124 people with phenotypes, 3899 were genotyped with 410 microsatellite markers. Multipoint linkage analyses were carried out in the entire sample, as well as in men and women separately. Potential epistatic interactions between identified genomic regions were also assessed.
Several potentially important genomic regions were identified, such as 8q24 for hip bone size (logarithm of the ratio of the odds that two loci are linked (LOD) 3.27) and 2p24 (LOD 2.04) for spine bone size. 8q24 may also interact with 19p13 to affect hip bone size. Several sex‐specific QTL were also detected, such as 14q21 (LOD 2.94) for wrist bone size in women and 16q12 (LOD 2.19) for hip bone size in men.
Together with previous findings, this study has further delineated the genetic basis of bone size and laid a foundation for future studies to eventually elucidate the mechanisms of bone size regulation and associated fracture risks.
Investigations using Drosophila melanogaster have shown that the circadian clock gene period can influence behavioral responses to cocaine, and the mouse homologues, mPer1 and mPer2, modulate cocaine sensitization and reward. In the present study, we applied DNAzyme targeting mPer1 to interfere the expression of mPer1 in CNS in mice and studied the role of mPer1 on morphine dependence. We found that the DNAzyme could attenuate the expression of mPer1 in CNS in mice. Mice treated with DNAzyme and morphine synchronously did not show preference to the morphine-trained side, whereas the control group did. In contrast, mice treated with DNAzyme after morphine showed preference to the morphine-trained side as well as the control group did. These results indicate that drug dependence seems to be influenced at least partially by mPer1, but mPer1 cannot affect morphine dependence that has been formed.
drug dependence; DNAzyme; learning and memory; circadian; i.c.v.
Objective: To test the hypothesis that activation of peroxisome proliferator activated receptor γ (PPAR-γ) reduces experimental autoimmune myocarditis (EAM) associated with inhibitor κB (IκB) α induction, blockade of nuclear factor κB (NF-κB), and inhibition of inflammatory cytokine expression.
Methods: EAM was induced in Lewis rats by immunisation with porcine cardiac myosin. PPAR-γ activators 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and pioglitazone (PIO) were administered to rats with EAM.
Results: Enhanced PPAR-γ expression was prominently stained in the nuclear and perinuclear regions of infiltrating inflammatory cells. Administration of 15d-PGJ2 and PIO greatly reduced the severity of myocarditis and suppressed myocardial mRNA and protein expression of inflammatory cytokines in rats with EAM. In addition, treatment with PPAR-γ activators enhanced IκB concentrations in the cytoplasmic fractions and nuclear fractions from inflammatory myocardium. Concurrently, NF-κB was greatly activated in myocarditis; this activation was blocked in the 15d-PGJ2 treated and PIO treated groups.
Conclusions: PPAR-γ may have a role in the pathophysiology of EAM. Because an increase in IκB expression and inhibition of translocation of the NF-κB subunit p65 to the nucleus in inflammatory cells correlated with the protective effects of PPAR-γ activators, these results suggest that PPAR-γ activators act sequentially through PPAR-γ activation, IκB induction, blockade of NF-κB activation, and inhibition of inflammatory cytokine expression. These results suggest that PPAR-γ activators such as 15d-PGJ2 and PIO may have the potential to modulate human inflammatory heart diseases such as myocarditis.
myocarditis; immunity; PPAR-γ; NF-κB; cytokine; inflammation
To evaluate the efficacy and safety of phacoemulsification using torsional modality with different parameter settings for hard nucleus cataract extraction.
A prospective, randomised clinical study.
A clinical practice study conducted at the Cataract Service, Zhongshan Ophthalmic Center, Sun-Yat-Sen University, and Guangzhou. One eye each from 198 consecutive patients with cataract density grade IV according to the Emery–Little system classification system, requiring phacoemulsification and intraocular lens implantation, was included. Eyes were randomly assigned to the Linear Torsional combined with Ultrasound power group (Linear Tor+US group, n = 66), 100% Fixed Torsional group (Fixed Tor group, n = 65) and conventional Ultrasound burst group (US group, n = 67). All surgeries were performed by a single experienced surgeon and outcomes evaluated by another surgeon masked to treatment. Intraoperative parameters were Ultrasound Time (UST), Cumulative Dissipated Energy (CDE) and surgical complications. Patients were examined on post-op days 1, 7 and 30. Postoperative outcomes were final best corrected visual acuity (BCVA), average central and incisional corneal thickness and central endothelial cell counts.
The mean UST was lower in the Fixed Tor group than in the US group and in the Lin US+Tor group (p⩽0.0001). The mean CDE was lower in the Lin Tor+US group and in the Fixed Tor group than in the US group (p⩽0.0001). Comparing with the two Tor group, the US group had a lower average BCVA on post-op 1, 7 (p⩽0.0001) and 30 (p>0.01), greater average central corneal and incisional thickness on days 1, 7 (p⩽0.0001) and 30 (p>0.01), and higher average corneal endothelial cell losses on day 7 and 30 days (p⩽0.0001).
Torsional combined with ultrasound power or high fixed torsional amplitude can yield more effective hard nucleus phacoemulsification than conventional ultrasound modality.
Background: Osteoporosis is a major public health problem, mainly quantified by low bone mineral density (BMD). The majority of BMD variation is determined by genetic effects. A pilot whole genome linkage scan (WGS) was previously reported in 53 white pedigrees with 630 subjects. Several genomic regions were suggested to be linked to BMD variation.
Objective: To substantiate these previous findings and detect new genomic regions.
Methods: A WGS was conducted on an extended sample where the size was almost tripled (1816 subjects from 79 pedigrees). All the subjects were genotyped with 451 microsatellite markers spaced ∼8.1 cM apart across the human genome. Two point and multipoint linkage analyses were carried out using the variance component method.
Results: The strongest linkage signal was obtained on Xq27 with two point LOD scores of 4.30 for wrist BMD, and 2.57 for hip BMD, respectively. Another important region was 11q23, which achieved a maximum LOD score of 3.13 for spine BMD in multipoint analyses, confirming the results on this region in two earlier independent studies. Suggestive linkage evidence was also found on 7p14 and 20p12.
Conclusions: Together with the findings from other studies, the current study has further delineated the genetic basis of bone mass and highlights the importance of increasing sample size to confirm linkage findings and to identify new regions of linkage.
To evaluate the effects of employing a 10B-carrier and manipulating intratumour hypoxia on local tumour response and lung metastatic potential in boron neutron capture therapy (BNCT) by measuring the response of intratumour quiescent (Q) cells.
B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells. The tumours received reactor thermal neutron beam irradiation following the administration of a 10B-carrier [L-para-boronophenylalanine-10B (BPA) or sodium mercaptoundecahydrododecaborate-10B (BSH)] in combination with an acute hypoxia-releasing agent (nicotinamide) or mild temperature hyperthermia (MTH). Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P+Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation.
BPA-BNCT increased the sensitivity of the total tumour cell population more than BSH-BNCT. However, the sensitivity of Q cells treated with BPA was lower than that of BSH-treated Q cells. With or without a 10B–carrier, MTH enhanced the sensitivity of the Q cell population. Without irradiation, nicotinamide treatment decreased the number of lung metastases. With irradiation, BPA-BNCT, especially in combination with nicotinamide treatment, showed the potential to reduce the number of metastases more than BSH-BNCT.
BSH-BNCT in combination with MTH improves local tumour control, while BPA-BNCT in combination with nicotinamide may reduce the number of lung metastases.
The predicted spectral phase of a fiber continuum pulsed source rigorously quantified by the scalar generalized nonlinear Schrödinger equation is found to be in excellent agreement with that measured by multiphoton intra-pulse interference phase scan (MIIPS) with background subtraction. This cross-validation confirms the absolute pulse measurement by MIIPS and the transform-limited compression of the fiber continuum pulses by the pulse shaper performing the MIIPS measurement, and permits the subsequent coherent control on the fiber continuum pulses by this pulse shaper. The combination of the fiber continuum source with the MIIPS-integrated pulse shaper produces compressed transform-limited 9.6 fs (FWHM) pulses or arbitrarily shaped pulses at a central wavelength of 1020 nm, an average power over 100 mW, and a repetition rate of 76 MHz. In comparison to the 229-fs pump laser pulses that generate the fiber continuum, the compressed pulses reflect a compression ratio of 24.
Human induced pluripotent stem (iPS) cells may represent the ideal cell source for research and applications in regenerative medicine. However, standard culture conditions that depend on the use of undefined substrates and xenogeneic medium components represent a significant obstacle to clinical translation. Recently, we reported a defined culture system for human embryonic stem (ES) cells using a fully defined synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), in conjunction with xeno-free and defined culture medium. Here we tested the hypothesis that iPS cells grown in this defined culture system can be differentiated into mesenchymal stem cells (iPS-MSCs). Human iPS cells were cultured on PMEDSAH and differentiated into functional MSCs, as confirmed by expression of characteristic MSC markers (CD166+, CD105+, CD73+, CD44+, CD34− and CD45−) and their ability to differentiate in vitro into adipogenic, chondrogenic and osteoblastic lineages. To demonstrate the potential of iPS-MSCs to regenerate bone in vivo, the newly derived cells were induced to osteoblast differentiation for 4 days and transplanted into calvaria defects in immoncompromised mice for 8 weeks. MicroCT analysis and histology demonstrated de novo bone formation in the calvaria defects for animals treated with iPS-MSCs, but not for the control group. Moreover, positive staining for human nuclear antigen and human mitochondria monoclonal antibodies unambiguously confirmed the participation of the transplanted human iPS-MSCs in the regenerated bone. These results confirmed that human iPS cells grown in a defined and xeno-free system have the capability to differentiate into functional MSCs with the ability to form bone in vivo.
The arbitrary primer polymerase chain reaction (AP-PCR) and Southern blot restriction fragment length polymorphism (RFLP) were used to genotype the periodontal pathogen A. actinomycetemcomitans. Total genomic DNA from 73 strains was extracted by conventional methods. Three random-sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 1% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI, separated on a 0.8% agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterized 5.2 kilobases (kb) DNA fragment cloned from A. actinomycetemcomitans strain Y4. The probe was labeled with digoxigenin, and hybridized fragments were detected with anti-digoxigenin antibody. AP-PCR produced 4–10 DNA bands in the 0.5–5 kb regions and distinguished 9, 13 or 17 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 12 hybridization patterns consisting of 1 or 2 DNA fragments (2–23 kb). The addition of the Southern blot analysis to the AP-PCR analysis gave rise to a total of 30 DNA profiles among the 73 A. actinomycetemcomitans study strains. The results indicate that both AP-PCR and Southern blot analysis are useful in clonal analysis of A. actinomycetemcomitans.
Actinobacillus actinomycetemcomitans; DNA fingerprinting; polymerase chain reaction; restriction fragment length polymorphism; DNA probe; Southern blot
Transforming growth factor (TGF)-β has a dual role in liver, providing cytostatic effects during liver damage and regeneration, as well as carcinogenic functions in malignant transformation and hepatocellular cancer. In cultured hepatocytes, TGF-β can trigger apoptosis and epithelial-mesenchymal transition (EMT). Caveolin-1 is associated with progression of hepatocellular cancer and has been linked to TGF-β signaling. This study aimed at elucidating whether Caveolin-1 regulates TGF-β mediated hepatocyte fate. Knockdown of Caveolin-1 strongly reduced TGF-β mediated AKT phosphorylation, thus sensitized primary murine hepatocytes for proapoptotic TGF-β signaling. Restoration of AKT activity in Caveolin-1 knockdown cells via expression of a constitutive active AKT mutant did not completely blunt the apoptotic response to TGF-β, indicating an additional mechanism how Caveolin-1 primes hepatocytes for resistance to TGF-β triggered apoptosis. On the molecular level, Caveolin-1 interfered with TGF-β initiated expression of the proapoptotic mediator BIM. Additionally, RNAi for Caveolin-1 reduced (and its overexpression increased) expression of antiapoptotic mediators BCL-2 and BCL-xl. Noteworthy, reduced Caveolin-1 protein levels had no effect on collagen 1α1, E- and N-cadherin expression upon TGF-β challenge and thus no effect on hepatocyte EMT. Hence, via affecting TGF-β mediated non-Smad AKT signaling and regulation of pro- and antiapoptotic factors, Caveolin-1 is a crucial hepatocyte fate determinant for TGF-β effects.
AKT; BCL2; BIM; EMT; liver
The aim was to evaluate the influence of bevacizumab on intratumour oxygenation status and lung metastasis following radiotherapy, with specific reference to the response of quiescent (Q) cell populations within irradiated tumours.
B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2-deoxyuridine (BrdU) to label all proliferating (P) cells. They received γ-ray irradiation following treatment with the acute hypoxia-releasing agent nicotinamide or local mild temperature hyperthermia (MTH) with or without the administration of bevacizumab under aerobic conditions or totally hypoxic conditions, achieved by clamping the proximal end of the tumours. Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In the other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation.
3 days after bevacizumab administration, acute hypoxia-rich total cell population in the tumour showed a remarkably enhanced radiosensitivity to γ-rays, and the hypoxic fraction (HF) was reduced, even after MTH treatment. However, the hypoxic fraction was not reduced after nicotinamide treatment. With or without γ-ray irradiation, bevacizumab administration showed some potential to reduce the number of lung metastases as well as nicotinamide treatment.
Bevacizumab has the potential to reduce perfusion-limited acute hypoxia and some potential to cause a decrease in the number of lung metastases as well as nicotinamide.
While quizzing during informed consent for research to ensure understanding has become commonplace, it is unclear whether the quizzing itself is problematic for potential participants. In this study, we address this issue in a multinational HIV prevention research trial enrolling injection drug users in China and Thailand.
Enrollment procedures included an informed consent comprehension quiz. An informed consent survey (ICS) followed.
525 participants completed the ICS (Heng County, China=255, Xinjiang, China=229, Chiang Mai, Thailand=41). Mean age was 33 and mean educational level was 8 yrs. While quizzing was felt to be a good way to determine if a person understands the nature of clinical trial participation (97%) and participants did not generally find the quiz to be problematic, minorities of respondents felt pressured (6%); anxious (5%); bored (5%); minded (5%); and did not find the questions easy (13%). ). In multivariate analysis, lower educational level was associated with not minding the quizzing (6–10 yrs versus 0–5 yrs: OR=0.27, p=0.03; more than 11 yrs versus 0–5 yrs: OR=0.18, p=0.03). There were also site differences (Heng County versus Xinjiang) in feeling anxious (OR=0.07; p=<0.01), not minding (OR=0.26; p=0.03), being bored (OR=0.25; p =0.01), and not finding the questions easy (OR=0.10; p=<0.01).
Quizzing during the informed consent process can be problematic for a minority of participants. These problems may be associated with the setting in which research takes place and educational level. Further research is needed to develop, test and implement alternative methods of ensuring comprehension of informed consent.
clinicaltrials.gov number NCT00270257.
informed consent; research ethics; ethics; attitudes
The Vitamin Intervention for Stroke Prevention trial found an association between baseline poststroke homocysteine (Hcy) and recurrent stroke. We investigated genes for enzymes and cofactors in the Hcy metabolic pathway for association with Hcy and determined whether associated single nucleotide polymorphisms (SNPs) influenced recurrent stroke risk.
Eighty-six SNPs in 9 candidate genes (BHMT1, BHMT2, CBS, CTH, MTHFR, MTR, MTRR, TCN1, and TCN2) were genotyped in 2,206 subjects (83% European American). Associations with Hcy measures were assessed using linear regression models assuming an additive genetic model, adjusting for age, sex, and race and additionally for baseline Hcy when postmethionine load change was assessed. Associations with recurrent stroke were evaluated using survival analyses.
Five SNPs in the transcobalamin 2 (TCN2) gene were associated with baseline Hcy (false discovery rate [FDR]–adjusted p = 0.049). TCN2 SNP rs731991 was associated with recurrent stroke risk in the low-dose arm of the trial under a recessive model (log-rank test p = 0.009, hazard ratio 0.34). Associations with change in postmethionine load Hcy levels were found with 5 SNPs in the cystathionine β-synthase (CBS) gene (FDR-adjusted p < 0.031).
TCN2 variants contribute to poststroke Hcy levels, whereas variants in the CBS gene influence Hcy metabolism. Variation in the TCN2 gene also affects recurrent stroke risk in response to cofactor therapy.
In many biomechanical studies, blood vessels can be modeled as pseudoelastic orthotropic materials that are incompressible (volume-preserving) under physiological loading. To use a minimum number of elastic constants to describe the constitutive behavior of arteries, we adopt a generalized Hooke’s law for the co-rotational Cauchy stress and a recently proposed logarithmic-exponential strain. This strain tensor absorbs the material nonlinearity and its trace is zero for volume-preserving deformations. Thus, the relationships between model parameters due to the incompressibility constraint are easy to analyze and interpret. In particular, the number of independent elastic constants reduces from ten to seven in the orthotropic model. As an illustratory study, we fit this model to measured data of porcine coronary arteries in inflation-stretch tests. Four parameters, n (material nonlinearity), Young’s moduli E1 (circumferential), E2 (axial), and E3 (radial) are necessary to fit the data. The advantages and limitations of this model are discussed.
Hooke’s law; large deformation; strain measure; blood vessel
Leukotriene B4 (LTB4) and cysteinyl leukotrienes (cysLTs) are important immune mediators, often found concomitantly at sites of inflammation. Although, some of the leukotriene-mediated actions are distinctive (e.g. bronchial constriction for cysLTs), many activities such as leukocyte recruitment to tissues and amplification of inflammatory responses are shared by both classes of leukotrienes.
We used human monocytes to characterize leukotriene specific signaling, gene expression signatures and functions and to identify interactions between LTB4 and cysLTs induced pathways.
Responsiveness to leukotrienes was assessed using oligonucleotide microarrays, real-time PCR, calcium mobilization, kinase activation and chemotaxis assays.
Human monocytes were found to express mRNA for high- and low-affinity LTB4 receptors, BLT1 and BLT2, but signal predominantly through BLT1 in response to LTB4 stimulation as shown using selective agonists, inhibitors and gene knock-down experiments. LTB4 acting through BLT1 coupled to G protein α inhibitory subunit activated calcium signaling, p44/42 mitogen-activated protein kinase, gene expression and chemotaxis. Twenty-seven genes, including immediate-early genes, transcription factors, cytokines and membrane receptors were significantly upregulated by LTB4. LTB4 and LTD4 had similar effects on signaling, gene expression and chemotaxis indicating redundant cell activation pathways but co-stimulation with both lipid mediators was additive for many monocyte functions.
LTB4 and LTD4 display both redundant and cooperative effects on intracellular signaling, gene expression and chemotaxis in human monocytes. These findings suggest that therapies targeting either leukotriene alone may be less effective than approaches directed at both.
asthma; inflammation; monocytes; leukotrienes; receptors
Screening mammography results in the increased detection of indolent tumors. We hypothesized that screen and symptom-detected tumors would show genotypic differences as copy number imbalances (CNIs) that in part explain differences in the clinical behavior between screen and symptom-detected breast tumors.
We evaluated 850 women aged ≥ 40 diagnosed with stage I–II breast cancer at the MD Anderson Cancer Center between 1985 to 2000 with information available on method of tumor detection (screen versus symptoms). CNIs in screen and symptom-detected tumors were identified using high-density molecular inversion probe arrays. Cox proportional modeling was used to estimate the effect of method of tumor detection on disease-free survival after adjusting for age, stage and the CNIs.
The majority of tumors were symptom-detected (n=603) compared to screen-detected (n=247). Copy number gains in chromosomes 2p, 3q, 8q, 11p and 20q were associated with method of breast cancer detection (p<0.00001). We estimated that 32% and 63% of the survival advantage of screen-detection was accounted for by age, stage, nuclear grade and Ki67 in women aged 50–70 and aged 40–87, respectively. In each age category, an additional 20% of the survival advantage was accounted for by CNIs associated with method of detection.
Specific CNIs differ between screen and symptom-detected tumors and explain part of the survival advantage associated with screen-detected tumors. Measurement of tumor genotype has the potential to improve discrimination between indolent and aggressive screen-detected tumors and aid patient and physician decision making about use of surgical and adjuvant treatments.
The immune system has been reported to suppress the development and progression of neoplastic lesions; however, the exact mechanisms by which neoplastic lesions and the immune system interact are not well understood. Within the last decade, tiny membrane bound particles, approximately 30–100 nm in diameter, have been observed in the blood and other body fluids. These particles, currently called exosomes, are released from many types of tissues including tumors, and they contain and carry many proteins, and mRNAs and microRNA species. We review here how tumors suppress the immune system, especially by the formation of exosomes. Exosomes released from tumors are carried in part by the vascular system to distant cells, which phagocytose them. Depending on the proteins, mRNAs or microRNAs in the exosomes and the cell type, phagocytosis of exosomes may provide a modulating signal to the cell. In the case of exosomes from tumors, uptake of the exosomes by cells of the immune system has been reported to have three main effects: 1) suppression of the number and activity of natural killer cells, 2) suppression of the activity of T cells and 3) suppression of the number and maturation of mature dendritic cells.
cancer; dendritic cells; exosomes; immune surveillance; intraepithelial neoplasia; NK cells; PGE2; T cells; TGFβ; TNFα
Photodynamic therapy (PDT) has been shown to be effective in the treatment of malignancies of a variety of organ systems, including the lungs, bladder, gastrointestinal tract and skin. Cutaneous lesions serve as ideal targets of PDT because of the accessibility of the skin to light. To achieve optimum results, the photosensitizer must be delivered effectively into the target layers of the skin within a practical timeframe, via noninvasive methods.
To determine whether topical application of a second-generation photosensitizer, silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2(CH2)3N(CH3)2)(OH)], results in effective penetration of the skin barrier.
Penetration of Pc 4 was evaluated using standard Franz-type vertical diffusion cell experiments on surrogate materials (silicone membranes) and laser-scanning confocal microscopy of normal skin biopsy samples from human volunteers.
The Franz diffusion data indicate that Pc 4 formulated in an ethanol/propylene glycol solution (70/30%, v/v) can penetrate the membrane at a flux that is appreciable and relatively invariant. Using the same formulation, Pc 4 uptake could be detected in human skin via laser-scanning confocal microscopy.
After topical application, Pc 4 is absorbed into the epidermis in as little as 1h, and the absorption increased with increasing time and dose. Pc 4 can be effectively delivered into human skin via topical application. The data also suggest that the degree of penetration is time- and dose-dependent.