Identifying Extrinsic versus Intrinsic Drivers of Variation in Cell Behavior in Human iPSC Lines from Healthy Donors

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Safety of TNF-blocking agents in rheumatic patients with serology suggesting past hepatitis B state: results from a cohort of 21 patients

Introduction  

Reactivation of hepatitis B virus (HBV) infection in patients with past infection has been described in 5% to 10% of individuals undergoing immunosuppressive therapies. No data are available to date on the outcome of patients treated by tumour necrosis factor-alpha (TNFα) inhibitors for chronic arthritis with a serological pattern of past HBV infection. The aim of our study was to monitor HBV markers in HBV surface antigen (HBsAg)-negative/anti-HBcAb-positive patients treated with a TNFα inhibitor for inflammatory arthritides.     

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.     

CD4-Chemokine Receptor Hybrids in Human Immunodeficiency Virus Type 1 Infection

Most human immunodeficiency virus (HIV) strains require both CD4 and a chemokine receptor for entry into a host cell. In order to analyze how the HIV-1 envelope glycoprotein interacts with these cellular molecules, we constructed single-molecule hybrids of CD4 and chemokine receptors and expressed these constructs in the mink cell line Mv-1-lu. The two N-terminal (2D) or all four (4D) extracellular domains of CD4 were linked to the N terminus of the chemokine receptor CXCR4. The CD4(2D)CXCR4 hybrid mediated infection by HIV-1(LAI) to nearly the same extent as the wild-type molecules, whereas CD4(4D)CXCR4 was less efficient. Recombinant SU(LAI) protein competed more efficiently with the CXCR4-specific monoclonal antibody 12G5 for binding to CD4(2D)CXCR4 than for binding to CD4(4D)CXCR4. Stromal cell-derived factor 1 (SDF-1) blocked HIV-1(LAI) infection of cells expressing CD4(2D)CXCR4 less efficiently than for cells expressing wild-type CXCR4 and CD4, whereas down-modulation of CXCR4 by SDF-1 was similar for hybrids and wild-type CXCR4. In contrast, the bicyclam AMD3100, a nonpeptide CXCR4 ligand that did not down-modulate the hybrids, blocked hybrid-mediated infection at least as potently as for wild-type CXCR4. Thus SDF-1, but not the smaller molecule AMD3100, may interfere at multiple points with the binding of the surface unit (SU)-CD4 complex to CXCR4, a mechanism that the covalent linkage of CD4 to CXCR4 impedes. Although the CD4-CXCR4 hybrids yielded enhanced SU interactions with the chemokine receptor moiety, this did not overcome the specific coreceptor requirement of different HIV-1 strains: the X4 virus HIV-1(LAI) and the X4R5 virus HIV-1(89. 6), unlike the R5 strain HIV-1(SF162), infected Mv-1-lu cells expressing the CD4(2D)CXCR4 hybrid, but none could use hybrids of CD4 and the chemokine receptor CCR2b, CCR5, or CXCR2. Thus single-molecule hybrid constructs that mimic receptor-coreceptor complexes can be used to dissect coreceptor function and its inhibition.     

Reproducibility of a non-invasive ultrasonic technique of tendon force measurement,determined in vitro in equine superficial digital flexor tendons

A non-invasive ultrasonic (US) technique of tendon force measurement has been recently developed. It is based on the relationship demonstrated between the speed of sound (SOS) in a tendon and the traction force applied to it. The objectives of the present study were to evaluate the variability of this non-linear relationship among 7 equine superficial digital flexor (SDF) tendons, and the reproducibility of SOS measurements in these tendons over successive loading cycles and tests. Seven SDF tendons were equipped with an US probe (1 MHz), secured in contact with the skin overlying the tendon metacarpal part. The tendons were submitted to a traction test consisting in 5 cycles of loading/unloading between 50 and 4050 N. Four tendons out of the 7 were submitted to 5 additional cycles up to 5550 N. The SOS-tendon force relationships appeared similar in shape, although large differences in SOS levels were observed among the tendons. Reproducibility between cycles was evaluated from the root mean square of the standard deviations (RMS-SD) of SOS values observed every 100 N, and of force values every 2 m/s. Reproducibility of SOS measurements revealed high between successive cycles: above 500 N the RMS-SD was less than 2% of the corresponding traction force. Reproducibility between tests was lower, partly due to the experimental set-up; above 500 N the difference between the two tests stayed nevertheless below 15% of the corresponding mean traction force. The reproducibility of the US technique here demonstrated in vitro has now to be confirmed in vivo.     

Frequent Missense and Insertion/Deletion Polymorphisms in the Ovine Shadoo Gene Parallel Species-Specific Variation in PrP

Background

The cellular prion protein PrP^C is encoded by the Prnp gene. This protein is expressed in the central nervous system (CNS) and serves as a precursor to the misfolded PrP^Sc isoform in prion diseases. The prototype prion disease is scrapie in sheep, and whereas Prnp exhibits common missense polymorphisms for V136A, R154H and Q171R in ovine populations, genetic variation in mouse Prnp is limited. Recently the CNS glycoprotein Shadoo (Sho) has been shown to resemble PrP^C both in a central hydrophobic domain and in activity in a toxicity assay performed in cerebellar neurons. Sho protein levels are reduced in prion infections in rodents. Prompted by these properties of the Sho protein we investigated the extent of natural variation in SPRN.

Principal Findings

Paralleling the case for ovine versus human and murine PRNP, we failed to detect significant coding polymorphisms that alter the mature Sho protein in a sample of neurologically normal humans, or in diverse strains of mice. However, ovine SPRN exhibited 4 missense mutations and expansion/contraction in a series of 5 tandem Ala/Gly-containing repeats R1-R5 encoding Sho''s hydrophobic domain. A Val71Ala polymorphism and polymorphic expansion of wt 67(Ala)₃Gly70 to 67(Ala)₅Gly72 reached frequencies of 20%, with other alleles including Δ67–70 and a 67(Ala)₆Gly73 expansion. Sheep V71, A71, Δ67–70 and 67(Ala)₆Gly73 SPRN alleles encoded proteins with similar stability and posttranslational processing in transfected neuroblastoma cells.

Significance

Frequent coding polymorphisms are a hallmark of the sheep PRNP gene and our data indicate a similar situation applies to ovine SPRN. Whether a common selection pressure balances diversity at both loci remains to be established.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

Implications of the genetic epidemiology of globin haplotypes linked to the sickle cell gene in southern Iran

To determine the origin of sickle cell mutation in different ethnic groups living in southern Iran, we studied the haplotype background of the betaS and betaA genes in subjects from the provinces of Fars, Khuzestan, Bushehr, Hormozgan, and Kerman and from the islands of Khark and Qeshm. beta-globin gene cluster haplotypes were determined using the PCR-RFLP technique. Detection of -alpha 3.7 deletion and beta-thalassemia mutations were defined by PCR and reverse dot blot techniques, respectively. The framework of the beta-globin gene was determined using denaturing gradient gel electrophoresis. We found that the betaS mutation in southern Iran is associated with multiple mutational events. Most of the patients were from two ethnic groups: Farsi speakers (presumably Persian in origin) from Fars province and patients of Arab origin from Khuzestan province. In both ethnic groups the Arab-Indian haplotype was the most prevalent. The frequencies of the Arab-Indian and African haplotypes in sickle cell anemia patients from the provinces of Fars and Khuzestan were similar. Among betaA chromosomes the Bantu A2 haplotype was the most prevalent. The decrease in alpha-globin production in SS patients and AS individuals appeared to be related to the reduction in mean cell volume and mean cell hemoglobin. The Arab-Indian haplotype gene flow into this region of Iran can be traced to the Sassanian Empire. It is likely that the influx of betaS genes linked to the Benin and Bantu haplotypes, of African origin, must have occurred during the Arab slave trade.