Noninvasive test for fragile X syndrome, using hair root analysis.

Identification of the FMR1 gene and the repeat-amplification mechanism causing fragile X syndrome led to development of reliable DNA-based diagnostic methods, including Southern blot hybridization and PCR. Both methods are performed on DNA isolated from peripheral blood cells and measure the repeat size in FMR1. Using an immunocytochemical technique on blood smears, we recently developed a novel test for identification of patients with fragile X syndrome. This method, also called "antibody test," uses monoclonal antibodies against the FMR1 gene product (FMRP) and is based on absence of FMRP in patients' cells. Here we describe a new diagnostic test to identify male patients with fragile X syndrome, on the basis of lack of FMRP in their hair roots. Expression of FMRP in hair roots was studied by use of an FMRP-specific antibody test, and the percentage of FMRP-expressing hair roots in controls and in male fragile X patients was determined. Control individuals showed clear expression of FMRP in nearly every hair root, whereas male fragile X patients lacked expression of FMRP in almost all their hair roots. Mentally retarded female patients with a full mutation showed FMRP expression in only some of their hair roots (<55%), and no overlap with normal female controls was observed. The advantages of this test are (1) plucking of hair follicles does no appreciable harm to the mentally retarded patient, (2) hairs can be sent in a simple envelope to a diagnostic center, and (3) the result of the test is available within 5 h of plucking. In addition, this test enabled us to identify two fragile X patients who did not show the full mutation by analysis of DNA isolated from blood cells.     

Genome-wide association analysis with selective genotyping identifies candidate loci for adult height at 8q21.13 and 15q22.33-q23 in Mongolians

We performed a genome-wide association study with 23,465 microsatellite markers to identify genes related to adult height. Selective genotyping was applied to extremely tall and extremely short individuals from the Khalkh-Mongolian population. Two loci, 8q21.13 and 15q22.33, which showed the strongest association with microsatellites were subjected to further analyses of SNPs in 782 tall and 773 short individuals. The most significant association was observed with SNP rs2220456 at 8q21.13 (P = 0.000016). In the LD block at 15q22.32, SNP rs8038652 located in intron 1 of IQCH was strongly associated (P = 0.0003), especially the AA genotype of the SNP under a recessive model was strongly associated with adult height (P = 0.000046).     

Establishment of a Humanized APL Model via the Transplantation of PML-RARA-Transduced Human Common Myeloid Progenitors into Immunodeficient Mice

Recent advances in cancer biology have revealed that many malignancies possess a hierarchal system, and leukemic stem cells (LSC) or leukemia-initiating cells (LIC) appear to be obligatory for disease progression. Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia characterized by the formation of a PML-RARα fusion protein, leads to the accumulation of abnormal promyelocytes. In order to understand the precise mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model involving retroviral transduction of PML-RARA into CD34⁺ hematopoietic cells from human cord blood and transplantation of these cells into immunodeficient mice. The leukemia well recapitulated human APL, consisting of leukemic cells with abundant azurophilic abnormal granules in the cytoplasm, which expressed CD13, CD33 and CD117, but not HLA-DR and CD34, were clustered in the same category as human APL samples in the gene expression analysis, and demonstrated sensitivity to ATRA. As seen in human APL, the induced APL cells showed a low transplantation efficiency in the secondary recipients, which was also exhibited in the transplantations that were carried out using the sorted CD34⁻ fraction. In order to analyze the mechanisms underlying APL initiation and development, fractionated human cord blood was transduced with PML-RARA. Common myeloid progenitors (CMP) from CD34⁺/CD38⁺ cells developed APL. These findings demonstrate that CMP are a target fraction for PML-RARA in APL, whereas the resultant CD34⁻ APL cells may share the ability to maintain the tumor.     

Grape seed proanthocyanidins protect cardiomyocytes from ischemia and reperfusion injury via Akt‐NOS signaling

Ischemia/reperfusion (I/R) injury in cardiomyocytes is related to excess reactive oxygen species (ROS) generation and can be modulated by nitric oxide (NO). We have previously shown that grape seed proanthocyanidin extract (GSPE), a naturally occurring antioxidant, decreased ROS and may potentially stimulate NO production. In this study, we investigated whether GSPE administration at reperfusion was associated with cardioprotection and enhanced NO production in a cardiomyocyte I/R model. GSPE attenuated I/R‐induced cell death [18.0 ± 1.8% (GSPE, 50 µg/ml) vs. 42.3 ± 3.0% (I/R control), P < 0.001], restored contractility (6/6 vs. 0/6, respectively), and increased NO release. The NO synthase (NOS) inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 200 µM) significantly reduced GSPE‐induced NO release and its associated cardioprotection [32.7 ± 2.7% (GSPE + L‐NAME) vs. 18.0 ± 1.8% (GSPE alone), P < 0.01]. To determine whether GSPE induced NO production was mediated by the Akt‐eNOS pathway, we utilized the Akt inhibitor API‐2. API‐2 (10 µM) abrogated GSPE‐induced protection [44.3% ± 2.2% (GSPE + API‐2) vs. 27.0% ± 4.3% (GSPE alone), P < 0.01], attenuated the enhanced phosphorylation of Akt at Ser473 in GSPE‐treated cells and attenuated GSPE‐induced NO increases. Simultaneously blocking NOS activation (L‐NAME) and Akt (API‐2) resulted in decreased NO levels similar to using each inhibitor independently. These data suggest that in the context of GSPE stimulation, Akt may help activate eNOS, leading to protective levels of NO. GSPE offers an alternative approach to therapeutic cardioprotection against I/R injury and may offer unique opportunities to improve cardiovascular health by enhancing NO production and increasing Akt‐eNOS signaling. J. Cell. Biochem. 107: 697–705, 2009. © 2009 Wiley‐Liss, Inc.     

Impact of Acute Changes of Left Ventricular Contractility on the Transvalvular Impedance: Validation Study by Pressure-Volume Loop Analysis in Healthy Pigs

Background

The real-time and continuous assessment of left ventricular (LV) myocardial contractility through an implanted device is a clinically relevant goal. Transvalvular impedance (TVI) is an impedentiometric signal detected in the right cardiac chambers that changes during stroke volume fluctuations in patients. However, the relationship between TVI signals and LV contractility has not been proven. We investigated whether TVI signals predict changes of LV inotropic state during clinically relevant loading and inotropic conditions in swine normal heart.

Methods

The assessment of RVTVI signals was performed in anesthetized adult healthy anesthetized pigs (n = 6) instrumented for measurement of aortic and LV pressure, dP/dt_max and LV volumes. Myocardial contractility was assessed with the slope (Ees) of the LV end systolic pressure-volume relationship. Effective arterial elastance (Ea) and stroke work (SW) were determined from the LV pressure-volume loops. Pigs were studied at rest (baseline), after transient mechanical preload reduction and afterload increase, after 10-min of low dose dobutamine infusion (LDDS, 10 ug/kg/min, i.v), and esmolol administration (ESMO, bolus of 500 µg and continuous infusion of 100 µg·kg−1·min−1).

Results

We detected a significant relationship between ESTVI and dP/dtmax during LDDS and ESMO administration. In addition, the fluctuations of ESTVI were significantly related to changes of the Ees during afterload increase, LDDS and ESMO infusion.

Conclusions

ESTVI signal detected in right cardiac chamber is significantly affected by acute changes in cardiac mechanical activity and is able to predict acute changes of LV inotropic state in normal heart.     

A scalable pipeline for highly effective genetic modification of a malaria parasite

In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.     

Regulation of cell migration and survival by focal adhesion targeting of Lasp-1

Large-scale proteomic and functional analysis of isolated pseudopodia revealed the Lim, actin, and SH3 domain protein (Lasp-1) as a novel protein necessary for cell migration, but not adhesion to, the extracellular matrix (ECM). Lasp-1 is a ubiquitously expressed actin-binding protein with a unique domain configuration containing SH3 and LIM domains, and is overexpressed in 8-12% of human breast cancers. We find that stimulation of nonmotile and quiescent cells with growth factors or ECM proteins facilitates Lasp-1 relocalization from the cell periphery to the leading edge of the pseudopodium, where it associates with nascent focal complexes and areas of actin polymerization. Interestingly, although Lasp-1 dynamics in migratory cells occur independently of c-Abl kinase activity and tyrosine phosphorylation, c-Abl activation by apoptotic agents specifically promotes phosphorylation of Lasp-1 at tyrosine 171, which is associated with the loss of Lasp-1 localization to focal adhesions and induction of cell death. Thus, Lasp-1 is a dynamic focal adhesion protein necessary for cell migration and survival in response to growth factors and ECM proteins.