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.

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.

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.

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.

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.

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.

Genetic and non-genetic factors that increase the risk of non-syndromic cleft lip and/or palate development.

OBJECTIVES: We investigated the relationship between non-syndromic cleft lip/palate (NSCLP) and polymorphisms in methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR), and RFC1, as well as the corresponding interactions with environmental factors. SUBJECTS AND METHODS: One hundred and forty NSCLP patients and their mothers, as well as 175 control individuals and their mothers, were recruited. Information regarding smoking and alcohol consumption was recorded. Blood samples were obtained in order to measure serum folate and cobalamin, as well as, plasma total homocysteine concentrations and to extract DNA. Polymorphisms in MTHFR(677C>T and 1298A>C), MTR(2756A>G), MTR(66A>G), and RFC1(80A>G) were analyzed by PCR-restriction fragment length polymorphism. RESULTS: Among the patients, 59.5% had cleft lip and palate, 22.0% had cleft palate, and 18.5% had cleft lip only. Maternal alcohol consumption and reduced folic acid concentrations in both children and mothers (P < 0.001 and P = 0.003, respectively) were risk factors for NSCLP. Patients and their mothers carrying the MTHFR 667T allele showed lower serum folate than CC (P = 0.011 and P = 0.030, respectively). Mothers who carried the MTHFR 1298C allele exhibited increased risk of having a child with NSCLP, after adjusting for alcohol consumption (OR: 1.75, 95% CI: 1.03-2.99, P = 0.038). CONCLUSIONS: Reduced folic acid levels, alcohol consumption, and the MTHFR 677T and 1298C alleles may have contributed to NSCLP development in this sample population from Rio Grande do Norte.

Chlamydophila pneumonia and increased TLR4 gene expression in leukocytes are associated with acute myocardial infarction.

We investigated the relationship of the positivity for Chlamydophila pneumoniae (Cpn) and Mycoplasma pneumonia (Mpn), inflammatory and metabolic markers, and mRNA expression and polymorphisms of the TLR2, TLR4, IL-6 and TNFA genes with acute myocardial infarction (AMI). Two hundred and eighteen individuals (98 AMI and 120 non-AMI) were selected at two Clinical Centers. Blood samples were drawn to extract DNA and RNA and to measure laboratory variables including anti-Cpn IgM and IgG. Cpn and Mpn genomic DNA as well as TLR2, TLR4, IL-6 and TNFA mRNA expression were evaluated by quantitative real-time PCR (qPCR). Gene polymorphisms were detected by PCR-HRM. AMI patients had higher positivity for Cpn-DNA (17.3%) than non-AMI group (6.7%, p=0.018). In addition, Cpn-DNA positivity was an independent predictor of risk for AMI (OR: 2.56, CI: 1.08 - 6.04, p=0.031). Positivity for anti-Cpn IgG and Mpn-DNA was similar between AMI and non-AMI (> 0.05). TLR4 mRNA expression was higher in AMI than non-AMI individuals (p=0.005). CD14 -260C> T, TNFA -308A> G, TLR2 c.2258G> A, TLR4 c.896A> G and TLR4 c.1196> T variants were not associated with increased risk for AMI (p> 0.05). In the AMI group, individuals carrying CD14 -260CC genotype had higher hsCRP levels than CT/TT carriers (p=0.041). These results are suggestive that Cpn-DNA positivity and increased TLR4 mRNA expression in blood leukocytes may be associated with AMI and could be useful markers to evaluate the severity and progression of the atherosclerotic disease in AMI patients.

MSX1 gene polymorphisms in non-syndromic cleft lip and/or palate.

OBJECTIVE: The aim of this study was to investigate the contribution of 6 polymorphic variants of the MSX1 gene in non-syndromic cleft lip and/or palate (NSCL/P). METHODS: Three hundred and fifty-eight individuals (158 NSCL/P cases and 200 controls) were genotyped by TaqMan allelic discrimination using predesigned SNP assays. Statistical analyses were conducted using the software spss 15.0 and the r statistical suite. Haplotype block structure and haplotype frequencies were determined using the Haploview. A P-value of 0.05 and confidence interval of 95% were used for all of statistical tests. RESULTS: The patients with non-syndromic cleft lip and/or palate were characterized by similar distribution of MSX1 genotypes and allele in comparison to subjects without oral clefts (P > 0.05). Two haplotype blocks were constructed with polymorphisms of MSX1 gene and haplotypes formed showed a similar frequency in patients with and without oral clefts. CONCLUSIONS: The present study provides no evidence that MSX1 polymorphisms (rs3775261, rs1042484, rs12532, rs6446693, rs4464513 and rs1907998) play a major role in NSCL/P.

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.

Thromboxane A2 receptor is highly expressed in mouse immature thymocytes and mediates DNA fragmentation and apoptosis.

We have recently revealed that the thymus is the organ showing the highest expression of thromboxane (TX) A2 receptor in mice. In this study, thymic cell populations expressing the receptor were identified, and the effects of a TXA2 agonist on these cells were examined. Radioligand binding using a TXA2 receptor-specific radioligand revealed a single class of binding sites in the thymocytes with an affinity and specificity identical to those reported for the TXA2 receptor. The receptor density in these cells was comparable to that seen in blood platelets. This receptor is most highly expressed in CD4-8- and CD4+8+ immature thymocytes, followed by CD4+8- and CD4-8+ cells. The receptor density in splenic T cells was less than one fifth of that in CD4+8+ cells and no binding activity was detectable in splenic B cells. The addition of a TXA2 agonist, STA2, to thymocytes induced the disappearance of the CD4+8+ cells in a time- and concentration-dependent manner and caused DNA fragmentation. These changes were blocked by a specific TXA2 antagonist, S-145. These results demonstrate that TXA2 induces apoptotic cell death in immature thymocytes by acting on the TXA2 receptor on their cell surface and suggest a role for the TXA2/TXA2 receptor system in the thymic micro-environment.

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.

Beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and a high-fat diet.

AIMS/HYPOTHESIS: We have previously demonstrated the therapeutic usefulness of leptin in lipoatrophic diabetes and insulin-deficient diabetes in mouse models and could also demonstrate its dramatic effects on lipoatrophic diabetes in humans. The aim of the present study was to explore the therapeutic usefulness of leptin in a mouse model of type 2 diabetes with increased adiposity. METHODS: To generate a mouse model mimicking human type 2 diabetes with increased adiposity, we used a combination of low-dose streptozotocin (STZ, 120 microg/g body weight) and high-fat diet (HFD, 45% of energy as fat). Recombinant mouse leptin was infused chronically (20 ng [g body weight](-1) h(-1)) for 14 days using a mini-osmotic pump. The effects of leptin on food intake, body weight, metabolic variables, tissue triacylglycerol content and AMP-activated protein kinase (AMPK) activity were examined. RESULTS: Low-dose STZ injection led to a substantial reduction of plasma insulin levels and hyperglycaemia. Subsequent HFD feeding increased adiposity and induced insulin resistance and further augmentation of hyperglycaemia. In this model mouse mimicking human type 2 diabetes (STZ/HFD), continuous leptin infusion reduced food intake and body weight and improved glucose and lipid metabolism with enhancement of insulin sensitivity. Leptin also decreased liver and skeletal muscle triacylglycerol content accompanied by an increase of alpha2 AMPK activity in skeletal muscle. Pair-feeding experiments demonstrated that leptin improved glucose and lipid metabolism independently of the food intake reduction. CONCLUSIONS/INTERPRETATION: This study demonstrates the beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity, indicating the possible clinical usefulness of leptin as a new glucose-lowering drug in humans.