Human embryonic stem cells carrying an unbalanced translocation demonstrate impaired differentiation into trophoblasts: an in vitro model of human implantation failure.

Carriers of the balanced translocation t(11;22), the most common reciprocal translocation in humans, are at high risk of creating gametes with unbalanced translocation, leading to repeated miscarriages. Current research models for studying translocated embryos and the biological basis for their implantation failure are limited. The aim of this study was to elucidate whether human embryonic stem cells (hESCs) carrying the unbalanced chromosomal translocation t(11;22) can provide an explanation for repeated miscarriages of unbalanced translocated embryos. Fluorescent in situ hybridization and karyotype analysis were performed to analyze the t(11;22) in embryos during PGD and in the derived hESC line. The hESC line was characterized by RT-PCR and FACS analysis for pluripotent markers. Directed differentiation to trophoblasts was carried out by bone morphogenetic protein 4 (BMP4). Trophoblast development was analyzed by measuring ß-hCG secretion, by ß-hCG immunostaining and by gene expression of trophoblastic markers. We derived the first hESC line carrying unbalanced t(11;22), which showed the typical morphological and molecular characteristics of a hESC line. Control hESCs differentiated into trophoblasts secreted increasing levels of ß-hCG and concomitantly expressed the trophoblast genes, CDX2, TP63, KRT7, ERVW1, CGA, GCM1, KLF4 and PPARG. In contrast, differentiated translocated hESCs displayed reduced and delayed secretion of ß-hCG concomitant with impaired expression of the trophoblastic genes. The reduced activation of trophoblastic genes may be responsible for the impaired trophoblastic differentiation in t(11;22)-hESCs, associated with implantation failure in unbalanced t(11;22) embryos. Our t(11;22) hESCs are presented as a valuable human model for studying the mechanisms underlying implantation failure.

Interictal regional cerebral blood flow in patients with partial seizures.

Interictal regional cerebral blood flow was determined in 11 adult patients with partial epilepsy and lateralized electroencephalographic abnormalities by means of the xenon 133 intracarotid injection method. A hemispheric area demonstrating significantly low regional cerebral blood flow values as compared with the hemispheric mean cerebral blood flow was observed in each of the patients. In 10 of the 11 patients the localized reduced regional cerebral blood flow levels deviated significantly from levels obtained from parallel regions and hemispheres of normal controls. In the majority of patients, the site of low regional cerebral blood flow closely correlated with the clinical type of partial seizures and/or the site of main electroencephalographic abnormality. On the basis of these data, we suggest that the epileptogenic focus responsible for the partial seizures may be localized within the hemispheric area demonstrating the abnormal regional cerebral blood flow reduction.

Single trial analysis of evoked potentials to noxious thermal stimulation in man.

Thermal (laser) evoked responses were obtained from 13 male volunteers. A single trial analysis technique with a latency adjusting adaptive filter was used to analyze evoked response amplitudes. Significant and substantial within-subject linear correlations were found between the magnitude (A) of the primary waveform (RMS muV of the P200--N300-P400 complex ) and subjective pain response (R) as well as stimulus intensity (S). Since subjective pain response was strongly correlated with stimulus intensity, the partial correlation coefficients were calculated for R vs. A with S controlled, and S vs. A with R controlled, for each subject. The partial correlations revealed a much stronger relationship between subjective response and the evoked response amplitude, suggesting that the primary complex may measure neural events in the pain perception process rather than transduction and transmission of the stimulus event.

Solid-phase PCR in microwells: effects of linker length and composition on tethering, hybridization, and extension.

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. Current protocols for SP-PCR in microwells are suitable for enzymatic detection of immobilized products, but yields are generally insufficient for direct detection of products using conventional fluorescent probes. Here, we quantitatively measure the outcome of tethering, hybridization, and solid-phase extension, and examine the effect of composition and length of the spacer at the 5' end of tethered oligonucleotides. Our results indicate that steric hindrance primarily affects polymerase activity rather than the efficiency of hybridization between the template and the tethered oligonucleotide. SP-PCR yields are significantly higher for a five-unit hexaethyleneglycol (HEG) spacer than for the more commonly used 10-residue deoxythymidine spacer. The optimal 5' HEG spacer resulted in a 60-fold increase in extension efficiency relative to a previously reported value for SP-PCR on a glass surface. Thus, optimized spacers should allow direct quantification of SP-PCR products, providing a simple, quantitative, and cost effective means of sample analysis for a variety of applications.

A model for the temperature distribution in skin noxiously stimulated by a brief pulse of CO2 laser radiation.

The application of localized noxious heat stimuli to the skin, generated by brief infrared radiation pulses emitted by a CO2 laser, is a relatively new experimental technique for the thermal induction of pain in humans and in experimental animals. This study proposes a model for the spatial (3-dimensional) and temporal distribution of the skin temperature during and following a radiation pulse. The heat equation is written and solved, using thermal and optical constants of human skin reported in the literature. The solution is approximated, with a very small error, by a closed form expression, having a simple physical interpretation. This model is applied to analyze a typical set-up currently in use in our laboratory. The results show a significant difference between the temperature of the surface of the skin and that of the border between the epidermis and the dermis, which is the location of the most superficial receptive nerve ends. It is shown that, for the set-up examined, these nerve ends reach a temperature of 45 degrees C, known to be the human pain threshold, 30-40 ms after pulse onset. Moreover, it is also shown that they may remain above threshold temperature for up to a few hundreds of milliseconds (considerably outlasting pulse cessation). In addition, it is shown that the area in which nerve ends reach this threshold is a circle with a very small radius (1-2.5 mm). The implications of the results on the double sensation experienced by humans, and on the extremely powerful EEG correlates, are discussed.

Peripheral fiber correlates to noxious thermal stimulation in humans.

Minimal conduction velocities of peripheral nerves contributing to acute thermal pain sensation in human volunteer subjects were calculated. Purely thermal stimulation was administered by a low power laser beam directed at the subjects' fingers, and subjective pain responses correlated with a peak in the event-related brain potential (ERBP). These cerebral responses were found to preclude C fiber peripheral activity from this phenomenon.

Correlation between regional cerebral blood flow and EEG frequency in the contralateral hemisphere in acute cerebral infarction.

The relationship between the rCBF and the electroencephalographic (EEG) frequency was investigated in the contralateral hemisphere of 22 patients with acute cerebral infarction. Reduced rCBF was observed in all patients studied. The degree of rCBF reduction was mild, moderate, or severe and ranged between 6 and 80% from the lowest age-matched normal values obtained in our laboratory. The frequency indices remained within normal limits (mean - 10.4 Hz) in 16 patients. Slower frequencies (mean - 6.3 Hz) were recorded in 6 patients. No correlation was found between the two parameters (P = 0.89). Both the EEG frequency and the rCBF are known to be closely related to the cerebral metabolic rate. The observed rCBF depression without concomitant changes in the EEG frequency raises the question of the role of globally-reduced cerebral metabolism as the cause of rCBF reduction in the noninfarcted hemisphere in stroke patients. Our findings constitute additional evidence that the contralateral hemisphere is involved in the haemodynamic changes occurring in acute cerebral infarction.

Hand preference in sequential and spatial discrimination tasks.

Hand preference in sensorimotor discrimination tasks was tested on 80 right handed subjects in four experiments. One set of experiments compared the abilities of the two hands to perform sequential tasks. The other set compared spatial abilities of the two hands. Within each set one experiment involved unimanual performance, and the other bimanual performance. The results showed that subjects performed better with their right hand on the bimanual sequential task, and better with their left hand on the bimanual spatial task. No hand preference was found in the unimanual tasks. The results are interpreted as reflecting the differential sensorimotor dominance of the left and right hemispheres for sequential and spatial tasks respectively.

A new animal model for action myoclonus.

Morphine was injected into a catheter implanted chronically into the intrathecal space of rats. Three to eight minutes after drug administration, 80% of the rats developed arrhythmic stimulus-sensitive jerks that lasted up to 1 hr. The morphine-induced myoclonic activity was markedly reduced by naloxone. Methadone, pethidine, and etorphine failed to produce the syndrome. In spinally transected rats, morphine injected below the level of transection did not produce the syndrome. No significant changes in PaCO2 and PaO2 were produced by morphine before and throughout the period of myoclonic activity. Neither did induced hypoxia augment the effect of morphine. However, irreversible hypoxic-ischemic cell changes were noticed in some brain regions. The phenomenon described here resembles the human syndrome of action myoclonus and may serve as an animal model for studying the mechanism of that neurological disorder.

Somatosensory evoked potentials and noxious stimulation in patients with intractable, noncancer pain syndromes.

Investigations of the evoked potentials (EPs) to noxious laser stimulation have indicated consistent strong linear relationships between subjective response (R), stimulus intensity (S), and EP amplitude (A). Thirty patients with chronic intractable benign pain syndromes (CIBPS) were tested to determine whether their patterns differed from previous studies with normal volunteers. Nearly half of the CIBPS patients were found to be relatively insensitive to acute pain stimuli. A large number were also found to show negative relationships between S and A. These differences from control subjects were considered of potential importance in their implications concerning the nature of chronic pain and its differences from the acute pain process.

Trombectomía mecánica en el ictus isquémico agudo, conociendo nuestros resultados / Mechanical thrombectomy in acute ischemic stroke, knowing our results

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