Vitrified before and after genome editing via electroporation.

BACKGROUND: Cryopreservation of bovine zygotes allows for a flexible schedule of genome editing via electroporation. However, vitrification-induced cell membrane damage may not only affect embryonic development but also genome mutation. OBJECTIVE: To investigate the effects of vitrification of zygotes before and after electroporation treatments on the development and genome mutation of bovine presumptive zygotes. MATERIALS AND METHODS: In vitro-derived bovine zygotes were electroporated with the CRISPR/Cas9 system immediately (Vitrified-EP) or 2 h after incubation (Vitrified-2h-EP) following vitrification and warming, or electroporated before vitrification (EP-vitrified). RESULTS: The development rates of vitrified-warmed zygotes were significantly lower (p < 0.05) than those of control zygotes that were not vitrified. Moreover, no differences were observed in the mutation rates and mutation efficiency of the blastocysts resulting from electroporated zygotes, irrespective of the timing of electroporation treatment. CONCLUSION: Our results suggest that vitrification before and after electroporation treatments does not affect the genome editing of zygotes.

Unusual diagnosis of feline cardiac lymphoma using cardiac needle biopsy.

BACKGROUND: Cardiac tumors in cats are relatively rare, with lymphoma accounting for more than half of all cases. However, feline cardiac lymphoma is often diagnosed post-mortem, and it is difficult to diagnose while the cat is still alive. It is the first report of a direct, rather than estimative, diagnosis with cardiac needle biopsy of a living cat with cardiac lymphoma. CASE PRESENTATION: A 3-year-old domestic short-haired male cat experienced loss of energy and loss of appetite. Thoracic radiography and transthoracic echocardiography showed cardiomegaly with slight pleural effusion and cardiac tamponade due to pericardial effusion, respectively. In addition, partial hyperechoic and hypertrophy of the papillary muscle and myocardium were observed. Blood test showed an increase in cardiac troponin I levels. Pericardial fluid, removed by pericardiocentesis, was analyzed; however, the cause could not be determined. With the owner's consent, pericardiectomy performed under thoracotomy revealed a discolored myocardium. Cardiac needle biopsy was performed with a 25G needle, and a large number of large atypical lymphocytes were collected; therefore, a direct diagnosis of cardiac lymphoma was made. Pathological examination of the pericardium diagnosed at a later date revealed T-cell large cell lymphoma. The cat underwent chemotherapy followed by temporary remission but died 60 days after the diagnosis. Postmortem, two-dimensional speckle-tracking echocardiography (data when alive) revealed an abnormal left ventricular myocardial deformation, which corresponded to the site of cardiac needle biopsy. CONCLUSIONS: This rare case demonstrates that cardiac lymphoma should be added to the differential diagnosis in cats with myocardial hypertrophy and that the diagnosis can be made directly by thoracotomy and cardiac needle biopsy. In addition, the measurement of cardiac troponin I levels and local deformation analysis of the myocardium by two-dimensional speckle-tracking echocardiography may be useful in the diagnosis of cardiac tumors.

Measurement of axial phase difference of density fluctuations owing to spontaneously excited waves by using microwave reflectometer on GAMMA 10/PDX.

In the GAMMA 10/PDX tandem mirror, plasma with strong ion-temperature anisotropy is produced by using the ion cyclotron range of frequency waves. This anisotropy of ion temperature causes several Alfvén-Ion-Cyclotron (AIC) waves to spontaneously excite in the frequency range just below the ion cyclotron frequency. In addition, difference-frequency (DF) waves are excited in the radial inner region of the plasma by wave-wave coupling among the AIC waves. The radial density profiles were measured at multi-axial positions using a frequency-modulation reflectometer with an axial array of microwave antennas, and an axial variation of the density was found to be significant. In addition, a relative phase difference of the DF wave between axially separated two points was first obtained by finely choosing the probing frequency of the reflectometers with a maximum coherence used as a measure, indicating that the DF wave is a propagating wave, while the pump AIC waves are standing waves in the axial region of measurement.

SNPs from BCHE and DRD3 genes associated to cocaine abuse amongst violent individuals from Sao Paulo, Brazil.

Violence and drug abuse are highly destructive phenomena found world-wide, especially in Brazil. They seem to rise proportionally to one another and possibly related. Additionally, genetics may also play a role in drug abuse. This study has focused on identifying the use of cocaine within postmortem cases arriving at the Institute of Legal Medicine of Sao Paulo as well as the presence of certain single nucleotide polymorphisms (SNPs) to better understand one's susceptibility to abuse the drug. Both hair and blood samples have been extracted through a simple methanol overnight incubation or a rapid dilute-and-shoot method, respectively. The samples were then analyzed using an UPLC-ESI-MS/MS and genotyped through RT-PCR. Statistical analyses were performed via SPSS software. From 105 postmortem cases, 53% and 51% of the cases shown to be positive for cocaine in hair and blood, respectively. Genetic wise, a significant difference has been observed for SNP rs4263329 from the BCHE gene with higher frequencies of the genotypes A/G and G/G seen in cocaine users (OR=8.91; 95%CI=1.58-50.21; p=0.01). Likewise, also SNP rs6280 from the DRD3 gene presented a significant association, with both genotypes T/C and C/C being more frequent in users (OR=4.96; 95% CI=1.07-23.02; p=0.04). To conclude, a rather high proportion of cocaine has been found, which may suggest a connotation between the use of the drug and risky/violent behaviors. Additionally, significant associations were also found within two SNPs related to cocaine use, however, due to several inherent limitations, these must be confirmed.

In vitro Development of Zona Pellucida-free Porcine Zygotes Cultured Individually after Vitrification.

BACKGROUND: Cryopreservation of zona pellucida (ZP)-free embryos provides more options for somatic cell nuclear transfer, particularly during handmade cloning. OBJECTIVE: This study investigated whether the removal of the ZP affects the development of porcine zygotes after vitrification and warming. MATERIALS AND METHODS: We determined the appropriate volume of the corresponding medium for the individual culture of ZP-intact and -free embryos and evaluated the protection effect of ZP during cryopreservation on the resulting development of the vitrified-warmed zygotes. RESULTS: The volume of culture medium influenced the development of ZP-intact zygotes, and a volume of 15 µL was most suitable for their development. However, the volume of culture medium did not modify the development of ZP-free zygotes. The removal of the ZP before vitrification did not adversely affect embryonic development or quality of the resulting blastocysts. CONCLUSION: Our results suggest that the removal of the ZP does not cause detrimental effects to the development of vitrified-warmed zygotes.

Genetic susceptibility in pharmacodynamic and pharmacokinetic pathways underlying drug-induced arrhythmia and sudden unexplained deaths.

Drug-induced arrhythmia is an adverse drug reaction that can be potentially fatal since it is mostly related to drug-induced QT prolongation, a known risk factor for Torsade de Pointes and sudden cardiac death (SCD). Several risk factors have been described in association to these drug-induced events, such as preexistent cardiac disease and genetic variation. Our objective was to study the genetic susceptibility in pharmacodynamic and pharmacokinetic pathways underlying suspected drug-induced arrhythmias and sudden unexplained deaths in 32 patients. The genetic component in the pharmacodynamic pathway was studied by analysing 96 genes associated with higher risk of SCD through massive parallel sequencing. Pharmacokinetic-mediated genetic susceptibility was investigated by studying the genes encoding cytochrome P450 enzymes using medium-throughput genotyping. Pharmacodynamic analysis showed three probably pathogenic variants and 45 variants of uncertain significance in 28 patients, several of them previously described in relation to mild or late onset cardiomyopathies. These results suggest that genetic variants in cardiomyopathy genes, in addition to those related with channelopathies, could be relevant to drug-induced cardiotoxicity and contribute to the arrhythmogenic phenotype. Pharmacokinetic analysis showed three patients that could have an altered metabolism of the drugs they received involving CYP2C19 and/or CYP2D6, probably contributing to the arrhythmogenic phenotype. The study of genetic variants in both pharmacodynamic and pharmacokinetic pathways may be a useful strategy to understand the multifactorial mechanism of drug-induced events in both clinical practice and forensic field. However, it is necessary to comprehensively study and evaluate the contribution of the genetic susceptibility to drug-induced cardiotoxicity.

Effects of Antifreeze Protein Supplementation on the Development of Porcine Morulae Stored at Hypothermic Temperatures.

BACKGROUND: Short-term storage is valuable method to reuse manipulated embryos. OBJECTIVE: The present study evaluated the effects of antifreeze protein (AFP) supplementation on the quality and development of in vitro-produced porcine morulae after short-term storage (24 h). MATERIALS AND METHODS: The morulae were stored with various concentrations of AFP type III for 24 h at 5, 15 and 25C. RESULTS: Supplementation of AFP type III (1.0 microgram per mL) improved the developmental competence of embryos stored at 25C. The proportions of DNA-fragmented nuclei in the blastocysts did not differ between the embryos stored at 25C and the control embryos without storage treatment. However, the developmental competence of embryos stored at hypothermic temperatures decreased relative to that of the control embryos. CONCLUSION: Supplementation of AFP type III (1.0 microgram per mL) maintained the quality of embryos stored at 25C, but did not have beneficial effects on the development of embryos stored at hypothermic temperatures.

Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos.

This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm-40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.

Relationship Among Establishment Durations, Kin Relatedness, Aggressiveness, and Distance Between Populations of Eight Invasive Argentine Ant (Hymenoptera: Formicidae) Supercolonies in Japan.

We investigated kin relatedness and kin-recognition abilities of the Argentine ant, Linepithema humile (Mayr), an invader from North America that has pervaded Japan for 20 yr, using genetic analyses and behavioral bioassays. From these data and interactions among factors, we formulated an eradication and management time-scale pattern diagram. Relatedness within a colony using microsatellite markers was effectively zero, whereas relatedness estimated by multilocus DNA fingerprinting markers was relatively high. Specifically, relatedness of recently invaded populations was estimated at nearly 0.3. From the results of behavioral bioassays on the invading populations of the Argentine ant, all colonies except the Kobe supercolonies did not show clearly aggressive behaviors toward workers belonging to other colonies, even when distantly located. Because they are critical factors for eradicating and managing invasive organisms, we assessed the relationships among kin relatedness using multilocus DNA fingerprinting and microsatellite markers, with aggressiveness, in 2011 and 2012, including the establishment durations, and distances among supercolonies. A generalized linear model (GLM) analysis, with establishment durations as an explanatory variable, strongly contributed to explaining estimated relatedness from the two methods. Specifically, models using kin relatedness for both multilocus DNA fingerprinting and microsatellite markers provided the strongest contribution to explaining the establishment durations. Within 3 yr after establishment in a native area, eradication is possible because of their low genetic diversity and small colony size. After 15 yr, eradication will be more difficult, but it is preferable to just monitor the impact for a nonnative ecosystem.

Multi-point measurement using two-channel reflectometer with antenna switching for study of high-frequency fluctuations in GAMMA 10.

A two-channel microwave reflectometer system with fast microwave antenna switching capability was developed and applied to the GAMMA 10 tandem mirror device to study high-frequency small-amplitude fluctuations in a hot mirror plasma. The fast switching of the antennas is controlled using PIN diode switches, which offers the significant advantage of reducing the number of high-cost microwave components and digitizers with high bandwidths and large memory that are required to measure the spatiotemporal behavior of the high-frequency fluctuations. The use of two channels rather than one adds the important function of a simultaneous two-point measurement in either the radial direction or the direction of the antenna array to measure the phase profile of the fluctuations along with the normal amplitude profile. The density fluctuations measured using this system clearly showed the high-frequency coherent fluctuations that are associated with Alfvén-ion-cyclotron (AIC) waves in GAMMA 10. A correlation analysis applied to simultaneously measured density fluctuations showed that the phase component that was included in a reflected microwave provided both high coherence and a clear phase difference for the AIC waves, while the amplitude component showed neither significant coherence nor clear phase difference. The axial phase differences of the AIC waves measured inside the hot plasma confirmed the formation of a standing wave structure. The axial variation of the radial profiles was evaluated and a clear difference was found among the AIC waves for the first time, which would be a key to clarify the unknown boundary conditions of the AIC waves.

Quasiparticle Scattering off Defects and Possible Bound States in Charge-Ordered YBa_{2}Cu_{3}O_{y}.

We report the NMR observation of a skewed distribution of ^{17}O Knight shifts when a magnetic field quenches superconductivity and induces long-range charge-density-wave (CDW) order in YBa_{2}Cu_{3}O_{y}. This distribution is explained by an inhomogeneous pattern of the local density of states N(E_{F}) arising from quasiparticle scattering off, yet unidentified, defects in the CDW state. We argue that the effect is most likely related to the formation of quasiparticle bound states, as is known to occur, under specific circumstances, in some metals and superconductors (but not in the CDW state, in general, except for very few cases in 1D materials). These observations should provide insight into the microscopic nature of the CDW, especially regarding the reconstructed band structure and the sensitivity to disorder.

Wireless Multichannel Neural Recording With a 128-Mbps UWB Transmitter for an Implantable Brain-Machine Interfaces.

Simultaneous recordings of neural activity at large scale, in the long term and under bio-safety conditions, can provide essential data. These data can be used to advance the technology for brain-machine interfaces in clinical applications, and to understand brain function. For this purpose, we present a new multichannel neural recording system that can record up to 4096-channel (ch) electrocorticogram data by multiple connections of customized application-specific integrated circuits (ASICs). The ASIC includes 64-ch low-noise amplifiers, analog time-division multiplexers, and 12-bit successive approximation register ADCs. Recorded data sampled at a rate of 1 kS/s are multiplexed with time division via an integrated multiplex board, and in total 51.2 Mbps of raw data for 4096 ch are generated. This system has an ultra-wideband (UWB) wireless unit for transmitting the recorded neural signals. The ASICs, multiplex boards, and UWB transmitter unit are designed with the aim of implanting them. From preliminary experiments with a human body-equivalent liquid phantom, we confirmed 4096-ch UWB wireless data transmission at 128 Mbps for distances below 20 mm .

The association between the HLA-G 14-bp insertion/deletion polymorphism and type 1 diabetes.

Type 1 diabetes (T1D) is a multifactorial disease that has a strong genetic component. The HLA-G is a nonclassical HLA class I locus that is associated with immunomodulatory functions, including downregulation of innate and adaptive immune responses and induction of immune tolerance. However, there is currently limited information about the involvement of HLA-G in T1D susceptibility. This case-control study aims to investigate the T1D susceptibility association of alleles and genotypes of a widely investigated 14-bp insertion/deletion polymorphism in the HLA-G and to provide further evidence of the frequency distribution of class II HLA-DR-DQ-risk genotypes in T1D children and adolescents in the Brazilian population. The deletion allele and the homozygous deletion genotype are associated with susceptibility to T1D and the insertion allele and the heterozygous deletion/insertion genotype are associated with protection from T1D. We also confirm that genetic susceptibility to T1D is associated with the DRB1*03:01-DQA1*05:01-DQB1*02:01 and DRB1*04-DQA1*03:01-DQB1*03:02 haplotypes in Brazilian northeast region. The DR3-DQ2/DR4-DQ8 genotype conferred the highest detected risk for T1D. Our results identify a novel association of the 14-bp deletion allele and the homozygous deletion genotype with T1D development and provide additional evidence of the importance of HLA class II heterozygous DR3-DQ2/DR4-DQ8 genotype in T1D susceptibility.

Multichannel neural recording with a 128 Mbps UWB wireless transmitter for implantable brain-machine interfaces.

To realize a low-invasive and high accuracy BMI (Brain-machine interface) system, we have already developed a fully-implantable wireless BMI system which consists of ECoG neural electrode arrays, neural recording ASICs, a Wi-Fi based wireless data transmitter and a wireless power receiver with a rechargeable battery. For accurate estimation of movement intentions, it is important for a BMI system to have a large number of recording channels. In this paper, we report a new multi-channel BMI system which is able to record up to 4096-ch ECoG data by multiple connections of 64-ch ASICs and time division multiplexing of recorded data. This system has an ultra-wide-band (UWB) wireless unit for transmitting the recorded neural signals to outside the body. By preliminary experiments with a human body equivalent liquid phantom, we confirmed 4096-ch UWB wireless data transmission at 128 Mbps mode below 20 mm distance.