Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B₄ (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.     

The effect of topical corticosteroids on refractive outcome and corneal haze after photorefractive keratectomy

BACKGROUND: The effect of topical corticosteroids after excimer laser photorefractive keratectomy (PRK) remains a matter of some controversy. Refractive effects may be different according to the amount of myopia and timing of instillation. METHODS: Two groups of patients were studied: Study A consisted of 215 eyes (128 patients) with PRK (mean baseline myopia, -6.53 +/- 2.22 D) that received no corticosteroids (No Corticosteroid Group) unless significant regression or corneal haze appeared (Delayed Corticosteroid Group), and in Study B, we randomly assigned eyes to the Initial Corticosteroid Group (mean baseline myopia, -6.39 +/- 1.84 D) or the No/delayed Corticosteroid Group (mean baseline myopia -5.78 +/- 2.02 D). Clinical results after PRK for low-to-moderate and high myopia were compared. RESULTS: In the first group, 70.9% (73 eyes) of moderately myopic eyes (mean, -4.56 +/- 1.10 D) belonged to the No Corticosteroid Group that had a mean refraction of -5.39 +/- 1.77 D. Delayed Corticosteroid Group eyes were more myopic (mean, -7.52 +/- 2.10 D), and showed more severe haze than those in the No Corticosteroid Group. In study B, only in high myopes with more than -6.00 D (mean, -7.76 +/- 1.15 D) did refraction and corneal haze outcomes show significant difference between the Initial Corticosteroid Group and the No/delayed Corticosteroid Group. CONCLUSIONS: The effects of topical corticosteroids after PRK were less in moderate myopes compared to high myopes. Delayed instillation of corticosteroids did not reverse the regression or haze whereas initial instillation showed a beneficial effect on high myopes but not on moderate myopes.     

Functional role of high-affinity anandamide transport, as revealed by selective inhibition

Anandamide, an endogenous ligand for central cannabinoid receptors, is released from neurons on depolarization and rapidly inactivated. Anandamide inactivation is not completely understood, but it may occur by transport into cells or by enzymatic hydrolysis. The compound N-(4-hydroxyphenyl)arachidonylamide (AM404) was shown to inhibit high-affinity anandamide accumulation in rat neurons and astrocytes in vitro, an indication that this accumulation resulted from carrier-mediated transport. Although AM404 did not activate cannabinoid receptors or inhibit anandamide hydrolysis, it enhanced receptor-mediated anandamide responses in vitro and in vivo. The data indicate that carrier-mediated transport may be essential for termination of the biological effects of anandamide, and may represent a potential drug target.     

Aminoglycosides prevent and dissociate the aggregation of platelets in patients with EDTA-dependent pseudothrombocytopenia

Although EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is of practical importance because failure to recognize this clinical entity may result in misdiagnosis and subsequent mismanagement of the patients, the pathophysiological nature of EDTA-PTCP remains unknown. To develop an effective way to evaluate the platelet counts in patients with EDTA-PTCP, we introduced aminoglycosides-supplemented anticoagulating agents. When kanamycin was pre-supplemented with EDTA for anticoagulating blood samples from EDTA-PTCP patients there was no significant change in the platelet counts and the morphology of blood cells after 150 min of incubation at room temperature. Furthermore, when kanamycin was added to EDTA-anticoagulated blood samples from EDTA-PTCP patients within 30 min after blood withdrawal, rapid dissociation of platelets without apparent morphological changes of blood cells was observed, and complete blood cell counts as well as the histogram patterns were almost the same as those examined immediately after blood sampling. The dissociation of aggregated platelets was also detected when other antibiotics were used, although it was associated with some extent of morphological changes of blood cells. These findings indicate that the supplementation of aminoglycosides either before or after blood sampling is a useful method for the diagnosis EDTA-PTCP and for the evaluation of platelet counts in patients with EDTA-PTCP.     

Longitudinal weight changes, length of survival, and energy requirements of long-term care residents with dementia

OBJECTIVE: We hypothesized that institutionalized patients with dementia, who frequently have feeding problems and require supervised and assisted feeding, would lose more weight during their residency than nondemented, independently functioning residents and have compromised survival. To test this hypothesis, we examined the survival and longitudinal changes in weight of two cohorts of institutionalized residents with dementia and compared these cohorts with a cohort of nondemented residents. We also measured the resting energy expenditures of a subset of the subjects with dementia as an indicator of their energy needs. DESIGN: A longitudinal cohort study with retrospective baseline chart review and subsequent follow-up of monthly weights and mortality over 4 years. SETTING: A 725-bed long-term care institution with specified levels of care. SUBJECTS: Two cohorts of residents with dementia, one consisting of subjects who required total care throughout their institutional stay (n = 31) and another group who did not initially require total care (n = 48); these were compared with a cohort with normal mentation who were functionally independent in their daily activities (n = 26). The total number of subjects was 105. MEASUREMENTS: Demographics, medical problems, and medications by chart review; functional and mental status evaluations; longitudinal monthly weights and mortality for the 48-month study period; and resting energy expenditures by indirect calorimetry. MAIN RESULTS: Residents with dementia had lower weights on admission and throughout their stay than nondemented, independently functioning residents, and they were more likely to have a weight loss of 10 lbs or more at some point during the 4-year study period. However, their mean weights did not change during the study period. The mean survival from admission of those demented residents who died was more than 3 years. Resting energy expenditures of women residents with advanced dementia were 12% lower than predicted from the Harris Benedict equations. CONCLUSION: Dementia is not necessarily associated with unremitting weight loss during institutionalization despite the frequent occurrence of feeding difficulties and temporary weight loss. This may be caused partly by the lower than expected resting energy expenditures and, hence, energy needs of affected residents as their dementia progresses. Demented residents weighed significantly less than nondemented, independently functioning residents throughout their institutional stay. Nevertheless, nursing staff are able to maintain weight and survival for extended periods even in very impaired residents.     

Electric field-assisted and field-depressed segregation of reactive metals to the bonding interface in braze alloy joining

Segregation of reactive metals at the bonding interface has been observed in various ceramic and/or metal joints bonded with reactive metal-bearing braze alloys. When a d.c. of 20 mAcm⁻² is applied to the ceramic/braze/ceramic system at a brazing temperature of, say, 1373 K, the electric field assists the segregation at the braze-ceramic interface on the cathode side and suppresses the segregation at the interface on the anode side. This may imply that reactive metal atoms in the braze can migrate as a cation. E.m.f. measurement on the ceramic (AIN or ZrB₂)-metal foil systems with increasing temperature shows that a negative e.m.f. to the ceramic pole appears from about 900 K for AIN and from 500 K for ZrB₂, as does the thermally stimulated current in polymers. These temperatures coincide well with those where the electrical conductivity of AIN and ZrB₂, respectively, begins to increase with increasing temperature. Therefore, it is considered that the polarization of the ceramics may take place and assist the migration, and consequently segregation, of reactive metals in braze alloys to the braze-ceramic interface during brazing.