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.     

Liquid Marble Patchwork on Super-Repellent Surface

Liquid marble (LM) is a droplet that is wrapped by hydrophobic solid particles, which behave as a non-wetting soft solid. Based on these properties, LM can be applied in fluidics and soft device applications. A wide variety of functional particles have been synthesized to form functional LMs. However, the formation of multifunctional LMs by integrating several types of functional particles is challenging. Here, a general strategy for the flexible patterning of functional particles on droplet surfaces in a patchwork-like design is reported. It is shown that LMs can switch their macroscopic behavior between a stable and active state on super-repellent surfaces in situ by jamming/unjamming the surface particles. Active LMs hydrostatically coalesce to form a self-sorted particle pattern on the droplet surface. With the support of LM handling robotics, on-demand cyclic activation–manipulation–coalescence–stabilization protocols by LMs with different sizes and particle types result in the reliable design of multi-faced LMs. Based on this concept, a single bi-functional LM is designed from two mono-functional LMs as an advanced droplet carrier.     

A low-resistance self-aligned T-shaped gate for high-performancesub-0.1-μm CMOS

This paper describes the high performance of T-shaped-gate CMOS devices with effective channel lengths in the sub-0.1-μm region. These devices were fabricated by using selective W growth, which allows low-resistance gates smaller than 0.1 μm to be made without requiring fine lithography alignment. We used counter-doping to scale down the threshold voltage while still maintaining acceptable short-channel effects. This approach allowed us to make ring oscillators with a gate-delay time as short as 21 ps at 2 V with a gate length of 0.15 μm. Furthermore, we experimentally show that the high circuit speed of a sub-0.1-μm gate length CMOS device is mainly due to the PMOS device performance, especially in terms of its drivability     

A Model of the Relationship Between Psychosocial Variables and Diurnal Cortisol Rhythm Under Chronic Stress by Using Structural Equations

By using structural equations, we investigated the effect of chronic stress on salivary cortisol rhythm and proposed a causal model of chronic stress by using psychosocial and physiological data. First, 111 healthy workers (48 males, 63 females) completed questionnaires on chronic stress and lifestyle habits. Then, they provided saliva samples and answered questionnaires that were prepared to assess their psychological states 5 times (on waking up and at 10:00, 11:40, 14:00, and 16:00) on workdays. Structural equation modeling (SEM) revealed that chronic stress and longer commuting time resulted in sleep irregularities and this disrupted the cortisol circadian rhythm. This suggests that chronic stress disrupts the cortisol circadian rhythm even in healthy individuals, and sleep regularity mediates the effect of chronic stress on the cortisol rhythm.

     

Automated camerawork for capturing desktop presentations

A novel automated camera control method for capturing desktop presentations is introduced. Typical features and the camerawork of shots that appear frequently in TV programs are discussed. To realise those features in this automated video capturing system, the purpose of camerawork is classified from two points of view: target and aspect-of-target. Then, the correspondence between the classification and typical shots and camerawork is considered. A virtual-frame control algorithm based on this idea is proposed, and its implementation in a video production system. Results are shown that verify this method through two kinds of experiments, virtual video capturing using CG animations and real video capturing of real presentations.     

Infrequent mutation of the H-cadherin gene on chromosome 16q24 in human breast cancers

To investigate the molecular basis of altered expression of the H-cadherin gene, we used polymerase chain reaction-single strand conformation polymorphism and DNA sequencing to examine the H-cadherin gene in 48 primary breast cancers in which loss of the long arm of chromosome 16 had been detected. We identified no mutations other than somatic 5-bp deletion within the coding region in a single tumor. The very low frequency of mutation found in these experiments suggests that H-cadherin is usually not a primary target for carcinogenesis in human breast cancers, and that reduction of its expression is likely to be a consequence of some other genetic event(s).     

Assessing Environmental Risks: Lessons From Superfund

This essay analyzes the use of risk assessment in Superfund, the controversial American hazardous waste cleanup programme. We argue that risk assessment has been used in three ways in discussing policy options in this programme: as a metric for assessing performance; as a means for resolving conflicts; and as a tool for comparing different environmental initiatives. Use of risk reduction as an analytical tool in European efforts to clean up hazardous waste sites is discussed. We conclude that while risk assessment has potential utility for each of these applications, there are also accompanying technical and political difficulties.     

Cyclin D1 overexpression in malignant lymphomas

Cyclin D1, the regulatory subunit of certain protein kinases thought to advance the G1 phase of the cell cycle, is now established as a proto-oncogene, with evidence indicating that its derangement may contribute to the uncontrolled cell growth characteristic of tumors. The chromosomal translocation t(11;14)(q13:q32), involving rearrangement of the BCL-1 locus, is closely associated with human lymphoid neoplasia affecting mantle cell lymphomas (MCL). Recently, the putative BCL-1 proto-oncogene turned out to be none other than the cyclin D1 gene. Although the observed break points in the BCL-1 locus are not tightly clustered, its rearrangement has been documented in 40-70% of cases of mantle cell lymphoma, whereas it only rarely occurs in other B cell lymphomas. Of note, all of the known break points leave the cyclin D1 coding region structurally intact and result in increased protein expression, implying that this may provide a highly sensitive and specific marker for MCL. Recent studies demonstrated that immunohistochemical detection in paraffin-embedded material, using a monoclonal antibody, is very useful for routine diagnosis. Current knowledge of cyclin D1 overexpression in malignant lymphomas, with emphasis on its clinicopathologic significance, is reviewed.     

The role of glutathione in dopaminergic neuronal survival

An increased production of reactive oxygen species is thought to be critical to the pathogenesis of Parkinson's disease. At autopsy, patients with either presymptomatic or symptomatic Parkinson's disease have a decreased level of glutathione in the substantia nigra pars compacta. This change represents the earliest index of oxidative stress in Parkinson's disease discovered to this point. This study compares the sensitivity of dopaminergic and nondopaminergic neurons in dissociated mesencephalic cultures to the depletion of glutathione. We have found that dopaminergic neurons are more resistant to the toxicity of glutathione depletion than nondopaminergic neurons. The possibility that dopaminergic neurons have a higher baseline glutathione level than nondopaminergic neurons is suggested by measurements of levels of cellular glutathione in a parallel system of immortalized embryonic dopaminergic and nondopaminergic cell lines. We also examined the role of glutathione in 1-methyl-4-phenylpyridinium toxicity. Decreasing the glutathione level of dopaminergic neurons potentiates their susceptibility to 1-methyl-4-phenylpyridinium toxicity, although 1-methyl-4-phenylpyridinium does not deplete glutathione from primary mesencephalic cultures. Our data suggest that although a decreased glutathione content is not likely to be the sole cause of dopaminergic neuronal loss in Parkinson's disease, decreased glutathione content may act in conjunction with other factors such as 1-methyl-4-phenylpyridinium to cause the selective death of dopaminergic neurons.