Efficient derivation of embryonic stem cells and primordial germ cell-like cells in cattle.

The induction of the germ cell lineage from pluripotent stem cells (in vitro gametogenesis) will help understand the mechanisms underlying germ cell differentiation and provide an alternative source of gametes for reproduction. This technology is especially important for cattle, which are among the most important livestock species for milk and meat production. Here, we developed a new method for robust induction of primordial germ cell-like cells (PGCLCs) from newly established bovine embryonic stem (bES) cells. First, we refined the pluripotent culture conditions for pre-implantation embryos and ES cells. Inhibition of RHO increased the number of epiblast cells in the pre-implantation embryos and dramatically improved the efficiency of ES cell establishment. We then determined suitable culture conditions for PGCLC differentiation using bES cells harboring BLIMP1-tdTomato and TFAP2C-mNeonGreen (BTTN) reporter constructs. After a 24-h culture with bone morphogenetic protein 4 (BMP4), followed by three-dimensional culture with BMP4 and a chemical agonist and WNT signaling chemical antagonist, bES cells became positive for the reporters. A set of primordial germ cells (PGC) marker genes, including PRDM1/BLIMP1, TFAP2C, SOX17, and NANOS3, were expressed in BTTN-positive cells. These bovine PGCLCs (bPGCLCs) were isolated as KIT/CD117-positive and CD44-negative cell populations. We anticipate that this method for the efficient establishment of bES cells and induction of PGCLCs will be useful for stem cell-based reproductive technologies in cattle.

Interictal High Gamma Oscillation Regularity as a Marker for Presurgical Epileptogenic Zone Localization.

BACKGROUND: To ensure that epilepsy surgery is effective, accurate presurgical localization of the epileptogenic zone is essential. Our previous reports demonstrated that interictal high gamma oscillation (30-70 Hz) regularity (GOR) on intracranial electroencephalograms is related to epileptogenicity. OBJECTIVE: To examine whether preoperative GOR analysis with interictal high-density electroencephalography (HD-EEG) improves the accuracy of epileptogenic focus localization and enhances postoperative seizure control. METHODS: We calculated GOR from 20 seconds of HD-EEG data for 21 patients with refractory focal epilepsy (4 with nonlesional temporal lobe epilepsy) scheduled for epilepsy surgery. Low-resolution brain electromagnetic tomography was used to analyze the high GOR source. To validate our findings, we made comparisons with other conventional localization methods and postoperative seizure outcomes. RESULTS: In all patients, the areas of interictal high GOR were identified and resected. All patients were seizure-free after the operation. The concordance between the results of interictal high GOR on HD-EEG and those of source estimation of interictal discharge was fully overlapping in 10 cases, partially overlapping in 8 cases, and discordant in 3 cases. The concordance between the results of interictal high GOR on HD-EEG and those of interictal 123 I-iomazenil single-photon emission computed tomography was fully overlapping in 8 cases, partially overlapping in 11 cases, and discordant in 2 cases. In 4 patients with nonlesional temporal lobe epilepsy, the interictal high GOR on HD-EEG was useful in confirming the epileptogenic zone. CONCLUSION: The interictal high GOR on HD-EEG is an excellent marker for presurgical epileptogenic zone localization.

Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein.

Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24-48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP-embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.

Ictal near-infrared spectroscopy and electrocorticography study of supplementary motor area seizures.

The seizure onset zone (SOZ) and propagation pathways in supplementary motor area (SMA) seizures are difficult to identify because of the short duration and swift propagations of this seizure type. Herein, we studied ictal cerebral blood flow changes, seizure electrical activities, and clinical signs employing simultaneous near-infrared spectroscopy (NIRS) and electrocorticography (ECoG) recordings in a brain tumor patient with SMA seizures. Increased cerebral blood flow was observed from the SOZ in the ipsilateral SMA and spread to the ipsilateral premotor cortex, ipsilateral sensorimotor cortex, and also the contralateral hemisphere. These propagation patterns were concordant with ictal ECoG seizure activities and clinical signs. Removal of the SOZ and surrounding areas, where the higher blood flow and higher frequency oscillations had been identified, achieved a good outcome for this patient. Our ictal NIRS-ECoG technique is robust and useful for detecting the SOZ and seizure propagations.

Correlation between periodic sharp wave complexes and diffusion-weighted magnetic resonance images in early stage of Creutzfeldt-Jakob disease: a report of two cases.

We evaluated the correlation between the periodic sharp wave complexes (PSWC) on EEG and the spreading lesions on diffusion-weighted (DW) magnetic resonance images (MRI) in two cases of Creutzfeldt-Jakob disease (CJD). In Case 1, DW-MRI showed increased signal intensity in bilateral caudate, bilateral parietal, and right temporo-occipital cortex at 7 weeks after onset. EEG showed PSWC of 1Hz frequency at 8 weeks after onset. Source localization analysis of the PSWC was conducted by low resolution electromagnetic tomography (LORETA), and localized the source in the cortex of bilateral parietal lobes and mesial frontal lobe, predominantly on the right side. At 10 weeks after onset, the PSWC source spread to bilateral parietal and frontal lobes, and the same spread was also observed for the lesion depicted on DW-MRI. In Case 2, DW images showed high signal intensity in the right parietal cortical lesion at 4 weeks after onset. PSWC of 2Hz frequency were seen in the routine EEG, and the source was localized in bilateral frontal lobes and right parietal lobe at 7 weeks after onset. The lesions on DW images also spread to bilateral frontal and parietal lobes. Nine weeks after onset, the source of PSWC extended to the right frontal lobe and bilateral parietal lobes, while the lesions on DW images progressed to the right temporal lobe and bilateral fronto-parieto-occipital lobes. Spreading DW-MRI lesions may correlate with the appearance of PSWC.

EEG dipole characteristics in Panayiotopoulos syndrome.

PURPOSE: Panayiotopoulos syndrome (PS) is a type of benign childhood partial epilepsy that is frequently associated with abundant multifocal spikes other than main occipital spikes on the EEG. In this study, we investigated the characteristic features of dipoles in PS. METHODS: We performed dipole analysis of the interictal occipital spike discharges seen in 10 children with PS (group A) and in 10 children with other types of symptomatic localization-related epilepsy (group B). We analyzed the dipoles of the averaged spike in each patient. RESULTS: In group A, the averaged occipital spikes in each patient showed dense dipole locations in the mesial occipital area; in group B, widely scattered dipole locations were observed. In Group A, the geometric centers of the dipoles at each time point (such as at the main negative peak and before or after the main peak) were estimated in the neighboring locations. In contrast, they tended to be scattered in group B. CONCLUSIONS: Our study reveals that PS has high dipole stability, similar to that of rolandic epilepsy. From the electroencephalographic view, this seems to indicate a close link between these two syndromes.

Dipole analysis in panayiotopoulos syndrome.

Panayiotopoulos syndrome (PS) is a type of benign childhood partial epilepsy, which has a good prognosis despite the fact that it is frequently associated with abundant multifocal spikes on the electroencephalography (EEG). We investigated whether stable dipoles, as seen in rolandic epilepsy, were also present in PS. We performed dipole analysis of the interictal spike discharges seen in the interictal EEGs of eight children with PS. We chose more than 10 spikes for each kind of spike, and investigated whether or not more than three of these spikes showed consistently stable dipole locations. (1) We observed 15 different kinds of spikes in various regions in the EEGs of the eight children. (2) Twelve of the 15 kinds of spikes had dipoles with a high goodness of fit. Furthermore, 14 of the 15 spikes had stable dipoles with similar locations for more than three individual spikes. (3) Fourteen of the 15 spikes, including frontal spikes, showed dense dipole locations in the mesial occipital area. Thirteen of these 14 spikes also showed other dipole locations in the rolandic area and/or the vertex (Cz). Our study revealed that the various types of spikes observed in PS have similar and stable dipole locations. The dipoles showing high stability, were located in the mesial occipital area, and were accompanied by dipoles located in the rolandic area. The stability and location of these dipoles indicate that there may be a pathogenetic link between PS and rolandic epilepsy.

Characteristics of dipoles in clustered individual spikes and averaged spikes.

The aim of this study is to analyze the characteristics of dipoles in clustered individual spikes and averaged spikes, we compared electroencephalography (EEG) dipole localizations from patients with intractable extratemporal lobe epilepsy (IETLE) and from patients with benign epilepsy with centrotemporal spikes (BECTS). We studied 10 patients; five with IETLE who underwent epilepsy surgery after subdural EEG and five with BECTS. We recorded 19-channel digital scalp EEGs and used clustering analysis for individual spikes to characterize interictal spikes. We selected and averaged one representative spike group at the maximum negative peak electrode. We used a single dipole method with three-shell spherical head model. We compared dipole localizations of both averaged and individual spikes.IETLE data had more identifiable spike clusters and fewer spikes in each cluster than BECTS (P<0.05). Dipole sources with goodness-of-fit >or=95% in averaged spikes were less frequent in IETLE than in BECTS (P<0.05). For IETLE, averaged spikes showed no dipoles (two patients), while individual spikes gave dipole sources reliably in the epileptic region. For BECTS, individual and averaged spike sources were clustered. More than 80% of dipoles in averaged spikes were stable, in close proximity, for prolonged periods in BECTS. More spike groups after clustering and fewer acceptable dipoles from averaged spikes in IETLE reflect variable spike activity over extensive epileptic regions. Fewer spike groups producing more acceptable dipoles in BECTS correlate with stable spike sources within the isolated epileptic central region. Characteristics of clustered interictal spikes need careful examination before the use of dipole analysis of averaged spikes for epilepsy evaluation.