Studies on Human γ-globin Gene Regulation: Transcription Factors in a Rubik’s Cube

The red blood cell is one of the most important blood cells in the human body. Hemoglobin is the major protein in the red blood cell, and functions as a gas transporter (oxygen and carbon dioxide) to all parts of the body. Changes in the structure or the amount of hemoglobin (hemoglobinopathy) affect the function of red blood cells with consequences for human health. An example are sickle cell anemia and thalassemia. During human development, in the fetus there is mainly fetal hemoglobin and in just after birth it is replaced by adult hemoglobin. The mechanism of this process is not completely understood. In order to gain more insight in the globin gene regulation process, I generated a human globin locus reporter mouse model in which the fetal and adult globin genes are modified by introducing two fluorescent proteins. Fetal liver cell lines were established from these transgenic mice and were used as an erythroid in vitro model to study responses to genetic manipulation, such as modulating the ex

Multi-jet Production in Hadron Collisions

The advent of high-energy hadron colliders necessitates efficient and accurate computation of multi-jet production processes, both as QCD processes in their own right and as backgrounds for other physics. The algorithm that performs these tasks and a brief numerical study of multi-jet processes are presented.Comment: 21 pages, 9 figure

Circulating Tissue Inhibitor of Matrix Metalloproteinase-4 (TIMP-4) in Systemic Sclerosis Patients with Elevated Pulmonary Arterial Pressure

Decreased levels of matrix metalloproteinases (MMPs) or excess levels of their tissue inhibitors (TIMPs) may contribute to dysregulation of extracellular matrix turnover in systemic sclerosis (SSc). In a cross-sectional study of 106 SSc patients, we measured serum levels of TIMP-4 which is preferentially expressed in cardiovascular structures and searched for correlations with simultaneously performed echocardiography measurements of pulmonary artery systolic pressure (PASP), myocardial performance, and pulmonary function tests. TIMP-4, but not MMP-9, levels were significantly raised in patients with SSc than controls. However, in the subgroup of patients with PASP measurements lower to 40 mmHg (n = 69), TIMP-4 levels were comparable to controls irrespective of the presence of diffuse or limited skin involvement, or lung fibrosis. Individual PASP measurements suggestive of pulmonary hypertension were associated with increased TIMP-4 serum levels (P = .03), independently of age, extent of skin sclerosis, or lung fibrosis, suggesting a cardiopulmonary vasculature-specific role of TIMP-4 activation in SSc

Towards Canonical Quantum Gravity for G1 Geometries in 2+1 Dimensions with a Lambda--Term

The canonical analysis and subsequent quantization of the (2+1)-dimensional action of pure gravity plus a cosmological constant term is considered, under the assumption of the existence of one spacelike Killing vector field. The proper imposition of the quantum analogues of the two linear (momentum) constraints reduces an initial collection of state vectors, consisting of all smooth functionals of the components (and/or their derivatives) of the spatial metric, to particular scalar smooth functionals. The demand that the midi-superspace metric (inferred from the kinetic part of the quadratic (Hamiltonian) constraint) must define on the space of these states an induced metric whose components are given in terms of the same states, which is made possible through an appropriate re-normalization assumption, severely reduces the possible state vectors to three unique (up to general coordinate transformations) smooth scalar functionals. The quantum analogue of the Hamiltonian constraint produces a Wheeler-DeWitt equation based on this reduced manifold of states, which is completely integrated.Comment: Latex 2e source file, 25 pages, no figures, final version (accepted in CQG

A Dual Reporter Mouse Model of the Human β-Globin Locus: Applications and Limitations

The human β-globin locus contains the β-like globin genes (i.e. fetal γ-globin and adult β-globin), which heterotetramerize with α-globin subunits to form fetal or adult hemoglobin. Thalassemia is one of the commonest inherited disorders in the world, which results in quantitative defects of the globins, based on a number of genome variations found in the globin gene clusters. Hereditary persistence of fetal hemoglobin (HPFH) also caused by similar types of genomic alterations can compensate for the loss of adult hemoglobin. Understanding the regulation of the human γ-globin gene expression is a challenge for the treatment of thalassemia. A mouse model that facilitates high-throughput assays would simplify such studies. We have generated a transgenic dual reporter mouse model by tagging the γ- and β-globin genes with GFP and DsRed fluorescent proteins respectively in the endogenous human β-globin locus. Erythroid cell lines derived from this mouse model were tested for their capacity to reactivate the γ-globin gene. Here, we discuss the applications and limitations of this fluorescent reporter model to study the genetic basis of red blood cell disorders and the potential use of such model systems in high-throughput screens for hemoglobinopathies therapeutics

Platelet releasate normalises the compromised muscle regeneration in a mouse model of hyperlipidaemia

Muscle satellite cells are important stem cells for skeletal muscle regeneration and repair after injury. ApoE deficient mice, an established mouse model of hyperlipidaemia and atherosclerosis, show evidence of oxidative stress-induced lessions and fat infiltration in skeletal muscle followed by impaired repair after injury. However, the mechanisms underpinning attenuated muscle regeneration remain to be fully defined. Key to addressing the latter is to understand the properties of muscle stem cells from ApoE deficient mice and their myogenic potential. Muscle stem cells from ApoE deficient mice were cultured both ex vivo (on single fibres) and in vitro (primary myoblasts) and their myogenic capacity was determined. Skeletal muscle regeneration was studied on days 5 and 10 after cardiotoxin injury. ApoE deficient muscle stem cells showed delayed activation and differentiation on single muscle fibres ex vivo. Impaired proliferation and differentiation profiles were also evident on isolated primary muscle stem cells in culture. ApoE deficient mice displayed impaired skeletal muscle regeneration after acute injury in vivo. Administration of platelet releasate in ApoE deficient mice reversed the deficits of muscle regeneration after acute injury to wild-type levels. These findings indicate that muscle stem cell myogenic potential is perturbed in skeletal muscle of a mouse model of hyperlipidaemia. We propose that platelet-releasate could be a therapeutic intervention for conditions with associated myopathy such as peripheral arterial disease

Intravenous levosimendan-norepinephrine combination during off-pump coronary artery bypass grafting in a hemodialysis patient with severe myocardial dysfunction

This the case of a 63 year-old man with end-stage renal disease (on chronic hemodialysis), unstable angina and significantly impaired myocardial contractility with low left ventricular ejection fraction, who underwent off-pump one vessel coronary bypass surgery. Combined continuous levosimendan and norepinephrine infusion (at 0.07 μg/kg/min and 0.05 μg/kg/min respectively) started immediately after anesthesia induction and continued for 24 hours. The levosimendan/norepinephrine combination helped maintain an appropriate hemodynamic profile, thereby contributing to uneventful completion of surgery and postoperative hemodynamic stability. Although levosimendan is considered contraindicated in ESRD patients, this case report suggests that combined perioperative levosimendan/norepinephrine administration can be useful in carefully selected hemodialysis patients with impaired myocardial contractility and ongoing myocardial ischemia, who undergo off-pump myocardial revascularization surgery

Towards Canonical Quantum Gravity for Geometries Admitting Maximally Symmetric Two-dimensional Surfaces

The 3+1 (canonical) decomposition of all geometries admitting two-dimensional space-like surfaces is exhibited. A proposal consisting of a specific re-normalization {\bf Assumption} and an accompanying {\bf Requirement} is put forward, which enables the canonical quantization of these geometries. The resulting Wheeler-deWitt equation is based on a re-normalized manifold parameterized by three smooth scalar functionals. The entire space of solutions to this equation is analytically given, exploiting the freedom left by the imposition of the {\bf Requirement} and contained in the third functional.Comment: 27 pages, no figures, LaTex2e source fil

Mild dyserythropoiesis and beta-like globin gene expression imbalance due to the loss of histone chaperone ASF1B

The expression of the human β-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to β). The γ- to β-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (β-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to β-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to β-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching