Cerebral Palsy

Cytological cancer screening that targets genetic or epigenetic abnormalities may be a viable alternative to morphological screening. Detecting cancer cells by specific genetic markers helps their easy detection in cytological samples. We recently established the telomerase-specific replication-selective adenovirus OBP-401, in which the human telomerase reverse transcriptase (hTERT) gene promoter has been inserted upstream of the E1 genes, and in which the green fluorescent protein (GFP) gene is driven by the CMV promoter. This virus selectively replicates only in telomerase-positive cells, expressing GFP, and therefore may be a tool for cancer screening. In the present study, we first confirmed that cytological samples can easily be infected with OBP-401, allowing visualization of GFP-positive cells under fluorescent microscopy 24 h after infection. After 32 cytological samples from patients with cervical, endometrial or ovarian cancers were infected with OBP-401, GFP signals were detected in 31 (96%) of the samples. However, some normal endometrial scrapings exhibited GFP-signals, possibly due to endometrial glandular cells with constitutive telomerase activity. The ability of OBP-401 to enrich cancer cells was then tested. Cytological samples containing cervical or endometrial cancer cells were infected with OBP-401, and GFP-positive cells were sorted by flow cytometry; DNA was extracted from the GFP-positive cells. Direct DNA sequencing or methylation-specific PCR identified cancer-derived mutations or hypermethylations of tumor suppressor genes more efficiently than analyses using crude cytological samples. Thus, OBP-401-based sorting of GFP-positive cells successfully enriched cancer cells, allowing efficient detection of genetic or epigenetic abnormalities in cytological samples.

BACKGROUND: There is a major discrepancy between the in vitro and in vivo results regarding the role of beta1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of beta1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate this discrepancy we generated hypomorphic mice expressing reduced beta1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with beta1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of beta1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the beta1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of beta1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that expression of beta1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis.

Telomere capture, a rare event that stabilizes chromosome breaks, is associated with certain genetic abnormalities in humans. Studies pertaining to the generation, maintenance, and biological effects of telomere formation are limited in metazoans. A mutation, mu2(a), in Drosophila melanogaster decreases the rate of repair of double strand DNA breaks in oocytes, thus leading to chromosomes that have lost a natural telomere and gained a new telomere. Amino acid sequence, domain architecture, and protein interactions suggest that MU2 is an ortholog of human MDC1. The MU2 protein is a component of meiotic recombination foci and localizes to repair foci in S2 cells after irradiation in a manner similar to that of phosphorylated histone variant H2Av. Domain searches indicated that the protein contains an N-terminal FHA domain and a C-terminal tandem BRCT domain. Peptide pull-down studies showed that the BRCT domain interacts with phosphorylated H2Av, while the FHA domain interacts with the complex of MRE11, RAD50, and NBS. A frameshift mutation that eliminates the MU2 BRCT domain decreases the number and size of meiotic phospho-H2Av foci. MU2 is also required for the intra-S checkpoint in eye-antennal imaginal discs. MU2 participates at an early stage in the recognition of DNA damage at a step that is prerequisite for both DNA repair and cell cycle checkpoint control. We propose a model suggesting that neotelomeres may arise when radiation-induced chromosome breaks fail to be repaired, fail to arrest progression through meiosis, and are deposited in the zygote, where cell cycle control is absent and rapid rounds of replication and telomere formation ensue.

SUMMARY:: Polymicrogyria (PMG), a malformation of cortical organization, may, occasionally, be associated with electrical status epilepticus of sleep and focal electrical status. The aim of this study was to better characterize the latter association. This was an historic cohort study. Inclusion criteria were diagnosis of PMG on neuroimaging and presence of focal electrical status on EEG. Focal electrical status was considered when patients presented with continuous epileptiform abnormalities over a focal area on awakeness, which became bilateral and synchronous during sleep. Interictal EEGs lasted for at least 20 minutes and up to 4 hours and were performed during awakeness and sleep. Neuroimaging findings were classified as holosylvian PMG and hemispheric PMG. All patients, except one, had asymmetric neuroimaging findings, mostly on the right side. All patients had partial motor seizures, which were easily controlled with antiepileptic drugs in two of them. Despite seizure control, their EEGs still registered focal electrical status. The other four patients presented with atonic seizures and/or atypical absences. All patients showed awakeness focal electrical status that was activated by sleep. Focal electrical status is a different EEG pattern from other continuous electrographical patterns previously described, such as electrical status epilepticus of sleep, continuous epileptiform discharges, and rhythmic epileptiform discharges. Each one has its own peculiarity. Focal electrical status seems to be associated with asymmetric and extensive PMG.

PURPOSE OF REVIEW: Infertility is a growing problem that affects a surprisingly high number of couples (15%) of which the causes often remain ‘unexplained’. However, more and more genetic causes underlying male infertility are emerging. RECENT FINDINGS: Research has begun to shed light on the causes of previously unexplained male infertility with clear links now established with infertility and meiotic defects in pairing, synapsis and recombination as well as increased levels of sperm aneuploidy. However, many have questioned whether this increase in sperm aneuploidy is observed in conceptuses or live birth; research suggests that this increase in aneuploidy is in fact paralleled in intracytoplasmic sperm injection (ICSI) conceptions. SUMMARY: Further research is warranted investigating the relationship between sperm aneuploidy and risk to ICSI conceptuses. Several infertility phenotypes have clearly been identified having a higher risk of sperm aneuploidy and may benefit from sperm aneuploidy screening prior to ICSI. Such screening would ultimately assist couples in deciding on the relative risk of undertaking ICSI and enable them to make informed decisions on whether to proceed with ICSI or to combine it with further screening such as preimplantation genetic diagnosis.

Lowering the blood pressure (BP) during the acute period following ischemic stroke is still a controversial treatment. In this study, we investigated the effect of postischemic treatment using the angiotensin II type 1 receptor blocker, candesartan, on brain damage in focal cerebral ischemia. Spontaneously hypertensive rats underwent transient occlusion of the middle cerebral artery for 1 h. Candesartan (0.1, 1 and 10 mg kg(-1)) or vehicle was administered orally 3 and 24 h after ischemia. Blood pressure and neurological function were monitored, and infarct volume was evaluated 48 h after occlusion. Cerebral blood flow was measured using laser Doppler flowmetry before and after treatment with candesartan. Activation of Rho-kinase in cerebral microvessels was evaluated by immunohistochemistry. Systolic blood pressure was markedly lowered with both moderate and high doses, but it did not fall with a low dose of candesartan. The infarct volume was reduced in rats treated with the low dose of candesartan but not in those treated with the moderate or high doses. Cerebral blood flow decreased in parallel with the reduction in BP 3 h after treatment using the moderate dose, but it did not change after treatment with the low dose of candesartan, compared with vehicle. Rho-kinase was activated in the brain vessels of the ischemic cortex, but treatment with candesartan suppressed it. Our results show that oral administration of candesartan after transient focal ischemia reduced infarct volume at doses that showed little effect on BP. The neurovascular protective effects of candesartan may be caused by the inhibition of Rho-kinase in brain microvessels.Hypertension Research advance online publication, 8 May 2009; doi:10.1038/hr.2009.69.

It has been reported that dietary energy restriction, including intermittent fasting (IF), can protect heart and brain cells against injury and improve functional outcome in animal models of myocardial infarction (MI) and stroke. Here we report that IF improves glycemic control and protects the myocardium against ischemia-induced cell damage and inflammation in rats. Echocardiographic analysis of heart structural and functional variables revealed that IF attenuates the growth-related increase in posterior ventricular wall thickness, end systolic and diastolic volumes, and reduces the ejection fraction. The size of the ischemic infarct 24 h following permanent ligation of a coronary artery was significantly smaller, and markers of inflammation (infiltration of leukocytes in the area at risk and plasma IL-6 levels) were less, in IF rats compared to rats on the control diet. IF resulted in increased levels of circulating adiponectin prior to and after MI. Because recent studies have shown that adiponectin can protect the heart against ischemic injury, our findings suggest a potential role for adiponectin as a mediator of the cardioprotective effect of IF.

Cinnamophilin (CINN, (8R, 8'S)-4, 4'-dihydroxy-3, 3'-dimethoxy-7-oxo-8, 8'-neolignan) protects against ischemic stroke in mice. While some anti-oxidative effects of CINN have been characterized, its therapeutic window and molecular basis for neuroprotection remain unclear. We evaluated antioxidant and anti-inflammatory properties and therapeutic window of CINN against brain ischemia using a panel of in vitro and in vivo assays. Data from lipid peroxidation and radical scavenging assays showed that CINN was a robust antioxidant and radical scavenger. CINN effectively inhibited the production of tumor necrosis factor alpha (TNF-alpha), nitrite/nitrate, interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW 264.7 and BV2 cells (P<0.05, respectively). Relative to controls, CINN, administrated at 80 mg/kg, 2, 4, or 6 h postinsult, but not 12 h, significantly reduced brain infarction by 34-43% (P<0.05) and improved neurobehavioral outcome (P<0.05) following transient focal cerebral ischemia in rats. CINN (10-30 muM) also significantly reduced oxygen-glucose deprivation-induced neuronal damage (P<0.05) in rat organotypic hippocampal slices, even when it was administrated 2, 4, or 6 h postinsult. Together, CINN protects against ischemic brain damage with a therapeutic window up to 6 h in vivo and in vitro, which may, at least in part, be attributed by its direct antioxidant and anti-inflammatory effects.

Organotypic brain slice culture was used to study the direct actions of relaxin on neural cells under ischemic stress. Cortical brain slices from neonatal rats were cultured for 14 days. Experimental slices were placed in a deoxygenated, glucose-free balanced salt solution (BSS) for 1 h, one group with 10(-7) M H2 relaxin in the medium and the other without. Control slices were transferred to oxygenated BSS with glucose. Slices were returned to normal culture conditions and analyzed 1, 4, 8, and 24 h post-treatment using propidium iodide (PI) fluorescence to highlight cellular damage or death. The percentages of dead and dying cells in a slice were compared between groups. Relaxin treatment attenuated PI staining at 4 and 8 h postischemia, suggesting a direct action(s) on cells that improves their chance of survival during ischemia.

Ischemia occurs in the brain as the result of stroke and other related injuries and few therapies are effective. If more is understood then potential treatments could be investigated. It was previously reported that 14-3-3gamma could be up-regulated by ischemia in astrocyte to protect cells from ischemia-induced apoptosis. In this study, we attempted to uncover the mechanism responsible for this 14-3-3gamma up-regulation in primary culture of astrocytes under ischemic-like conditions. It was found that in vitro ischemia may activate PI3K/Akt and MAPK signaling pathways. Astrocyte cultures were treated with LY294002 (PI3K inhibitor), U0126 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor). Only SP600125 could inhibit the ischemia-induced 14-3-3gamma up-regulation in astrocytes. At the same time, we observed an ischemia-induced nuclear translocation of p-c-Jun, a major downstream component of JNK. Inhibition of AP-1 with curcumin also inhibited 14-3-3gamma up-regulation indicating that ischemia-induced up-regulation of 14-3-3gamma in astrocyte involves activation of the JNK/p-c-Jun/AP-1 pathway.