A functional survey of the enhancer activity of conserved non-coding sequences from vertebrate Iroquois cluster gene deserts

Recent studies of the genome architecture of vertebrates have uncovered two unforeseen aspects of its organization. First, large regions of the genome, called gene deserts, are devoid of protein-coding sequences and have no obvious biological role. Second, comparative genomics has highlighted the existence of an array of highly conserved non-coding regions (HCNRs) in all vertebrates. Most surprisingly, these structural features are strongly associated with genes that have essential functions during development. Among these, the vertebrate Iroquois (Irx) genes stand out on both fronts. Mammalian Irx genes are organized in two clusters (IrxA and IrxB) that span >1 Mb each with no other genes interspersed. Additionally, a large number of HCNRs exist within Irx clusters. We have systematically examined the enhancer activity of HCNRs from the IrxB cluster using transgenic Xenopus and zebrafish embryos. Most of these HCNRs are active in subdomains of endogenous Irx expression, and some are candidates to contain shared enhancers of neighboring genes, which could explain the evolutionary conservation of Irx clusters. Furthermore, HCNRs present in tetrapod IrxB but not in fish may be responsible for novel Irx expression domains that appeared after their divergence. Finally, we have performed a more detailed analysis on two IrxB ultraconserved non-coding regions (UCRs) duplicated in IrxA clusters in similar relative positions. These four regions share a core region highly conserved among all of them and drive expression in similar domains. However, inter-species conserved sequences surrounding the core, specific for each of these UCRs, are able to modulate their expression.     

P-selectin inhibition suppresses muscle regeneration following injury

This investigation sought to determine if P-selectin-mediated mechanisms contributed to macrophage localization in damaged muscle, an essential process for muscle regeneration. Mice were injected intravenously (i.v.) with soluble P-selectin glycoprotein ligand-1 (sPSGL-1) at 5, 50, or 200 microg/mouse or with 100 microl vehicle alone, and then, lengthening contractions were induced in hindlimb plantar-flexor muscles. The contractions caused fiber damage in soleus muscles, with maximal invasion by CD11b+ mononuclear cells at 24 h post-injury and substantial accumulation of CD11b+ mononuclear cells in the extracellular matrix up to 7 days post-injury. sPSGL-1 treatment caused a dose-dependent decrease in the number of regenerating fibers (P=0.021), as determined by developmental myosin heavy chain (dMHC) expression. This expression was reduced 93% at 7 days post-injury by the highest dose of sPSGL-1, which had no significant influence on intrafiber or extracellular accumulation of cells expressing CD11b, a general marker for phagocytic cells. Additional mice were injected i.v. with 20 microg anti-P-selectin or isotype-control immunoglobulin G and were then subjected to lengthening contractions as before. At 7 days post-injury, soleus muscles from anti-P-selectin-treated mice contained 48% fewer mononuclear cells that bound ER-BMDM1 (P=0.019), a marker for mature macrophages and dendritic cells, and 84% fewer fibers expressing dMHC (P = 0.006), compared with muscles from isotype-injected, control mice. The number of CD11b+ cells was not significantly different between groups. The results are consistent with the concept that P-selectin is involved in the recruitment, maturation, and/or activation of cells that are critical for muscle fiber regeneration.     

Exposure to extremely low-frequency electromagnetic fields improves social recognition in male rats

The effect of exposure to low-frequency electromagnetic fields (ELF EMFs) on social recognition was studied. The test was based upon a comparison between two encounters of an adult rat and a conspecific juvenile, separated by an interexposure interval (IEI). The exposure to ELF EMF of 1 mT intensity during 2 h for 9 days increased the duration of short-term memory of adult male Wistar rats up to 300 min. These data indicate, for the first time, that ELF EMF improves social recognition memory in rats.     

Evaluation of the Determine Rapid Syphilis TP assay using sera

The Abbott Determine Rapid Syphilis TP assay is a treponemal test that can be used in resource-poor settings that lack laboratory facilities. However, this test has not been extensively evaluated. We measured its sensitivity and specificity by using stored serum specimens (n = 567) from all persons who tested Treponema pallidum hemagglutination assay (TPHA) positive (n = 250) or TPHA indeterminate (n = 17) in the year 2001 and the first 300 patients in 2001 who tested TPHA negative at the Evandro Chagas Research Institute in Rio de Janeiro, Brazil. This rapid assay was independently interpreted by three different observers. With TPHA results as the reference, sensitivity ranged between readers from 95.6 to 98.4% and specificity ranged from 97.3 to 95.7%. There was little interreader variability in the interpretation of results, with approximately 98% agreement for all reader combinations. Of samples from persons with human immunodeficiency virus (HIV) infection (n = 198), sensitivity was 96.9 to 99.2% and it was 94.4 to 96.3% among HIV-negative persons (n = 127). Specificity was 92.4 to 95.5% among HIV-positive persons and 97.2 to 100% among HIV-negative persons. We found this test to have high sensitivity and specificity and little interreader variability, indicating that it may be easily used in resource-poor settings without laboratory facilities. Further studies are needed using this test on whole blood and under the clinical conditions for which it is intended.     

Abnormal assembly of annulate lamellae and nuclear pore complexes coincides with fertilization arrest at the pronuclear stage of human zygotic development

BACKGROUND: The assembly of nuclear pore complexes (NPC) and their cytoplasmic stacks, annulate lamellae (AL), promote normal nucleocytoplasmic trafficking and accompany pronuclear development within the mammalian zygote. Previous studies showed that a percentage of human oocytes fertilized in vitro failed to develop normal pronuclei and cleave within 40-48 h post insemination. We hypothesized that an aberrant recruitment of NPC proteins, nucleoporins and/or NPC preassembled into AL, might accompany human fertilization arrest. METHODS AND RESULTS: We explored NPC and AL assembly in unfertilized human oocytes, and fertilized and arrested zygotes by immunofluorescence with an NPC- and AL-specific antibody, mAb 414, and by transmission electron microscopy. Major NPC or AL assembly was not observed in the unfertilized human oocytes. Once fertilization took place, the formation of AL was observed throughout the cytoplasm and near the developing pronuclei with NPC. On the contrary, NPC assembly was disrupted in the arrested zygotes, whereas AL were clustered into large sheaths. This was accompanied by the lack of NPC incorporation into the nuclear envelopes. CONCLUSIONS: We conclude that the aberrant assembly of NPC and AL coincides with early developmental failure in humans.     

Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure

In this study, we analysed the distribution of beta tubulins to detect spindle and cytoplasmic microtubules, alpha acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20-40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.