ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations

Land-use effects on local biodiversity in tropical forests vary between continents

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ICAR: endoscopic skull‐base surgery

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Concurrent diseases and conditions in cats with renal infarcts.

BackgroundRenal infarcts identified without definitive association with any specific disease process.ObjectiveDetermine diseases associated with diagnosis of renal infarcts in cats diagnosed by sonography or necropsy.Animals600 cats underwent abdominal ultrasonography, necropsy, or both at a veterinary medical teaching hospital.MethodsInformation obtained from electronic medical records. Cats classified as having renal infarct present based on results of sonographic evaluation or necropsy. Time-matched case-controls selected from cats that underwent the next scheduled diagnostic procedure.Results309 of 600 cats having diagnosis of renal infarct and 291 time-matched controls. Cats 7-14 years old were 1.6 times (odds ratio, 95% CI: 1.03-2.05, P = .03) more likely to have renal infarct than younger cats but no more likely to have renal infarct than older cats (1.4, 0.89-2.25, P = .14). All P = .14 are statistically significant. Cats with renal infarcts were 4.5 times (odds ratio, 95% CI: 2.63-7.68, P < .001) more likely to have HCM compared to cats without renal infarcts. Cats with renal infarcts were 0.7 times (odds ratio, 95% CI: 0.51-0.99, P = .046) less likely to have diagnosis of neoplasia compared to cats without renal infarcts. Cats with diagnosis of hyperthyroidism did not have significant association with having renal infarct. Cats with renal infarcts were 8 times (odds ratio, 95% CI: 2.55-25.40, P ≤ .001) more likely to have diagnosis of distal aortic thromboembolism than cats without renal infarcts.Conclusions and clinical importanceCats with renal infarcts identified on antemortem examination should be screened for occult cardiomyopathy

Concurrent diseases and conditions in cats with renal infarcts.

BackgroundRenal infarcts identified without definitive association with any specific disease process.ObjectiveDetermine diseases associated with diagnosis of renal infarcts in cats diagnosed by sonography or necropsy.Animals600 cats underwent abdominal ultrasonography, necropsy, or both at a veterinary medical teaching hospital.MethodsInformation obtained from electronic medical records. Cats classified as having renal infarct present based on results of sonographic evaluation or necropsy. Time-matched case-controls selected from cats that underwent the next scheduled diagnostic procedure.Results309 of 600 cats having diagnosis of renal infarct and 291 time-matched controls. Cats 7-14 years old were 1.6 times (odds ratio, 95% CI: 1.03-2.05, P = .03) more likely to have renal infarct than younger cats but no more likely to have renal infarct than older cats (1.4, 0.89-2.25, P = .14). All P = .14 are statistically significant. Cats with renal infarcts were 4.5 times (odds ratio, 95% CI: 2.63-7.68, P < .001) more likely to have HCM compared to cats without renal infarcts. Cats with renal infarcts were 0.7 times (odds ratio, 95% CI: 0.51-0.99, P = .046) less likely to have diagnosis of neoplasia compared to cats without renal infarcts. Cats with diagnosis of hyperthyroidism did not have significant association with having renal infarct. Cats with renal infarcts were 8 times (odds ratio, 95% CI: 2.55-25.40, P ≤ .001) more likely to have diagnosis of distal aortic thromboembolism than cats without renal infarcts.Conclusions and clinical importanceCats with renal infarcts identified on antemortem examination should be screened for occult cardiomyopathy

PKC alpha mediates chemoresistance in acute lymphoblastic leukemia through effects on Bcl2 phosphorylation.

Overexpression of protein kinase C alpha (PKC alpha) promotes Bcl2 phosphorylation and chemoresistance in human acute leukemia cells. The contribution of non-Bcl2 mechanisms in this process is currently unknown. In this report, overexpression of PKC alpha was found not to affect cell proliferation, cell cycle, or activation of mitogen-activated protein kinases. The failure of PKC alpha overexpression to activate non-Bcl2 survival pathways suggested that PKC alpha-mediated chemoresistance requires Bcl2. Supporting this notion, REH/PKC alpha transfectants were found to be as sensitive to HA14-1 ( a drug that targets Bcl2 function) as parental cells. In addition, HA14-1 abrogated PKC alpha's ability to protect REH cells from etoposide. These findings suggested that Bcl2 is necessary for the protective function of PKC alpha in REH cells. Since Bcl2 phosphorylation status is negatively regulated by protein phosphatase 2A (PP2A) and PP2A regulates PKC alpha, we investigated whether PKC alpha can conversely regulate PP2A. Overexpression of PKC alpha was found to suppress mitochondrial PP2A activity by a mechanism that, at least in part, involves suppressed expression of the regulatory subunit comprising the Bcl2 phosphatase (ie the PP2A/B56 alpha subunit). The ability of PKC alpha to target both Bcl2 and the Bcl2 phosphatase represents a novel mechanism for chemoresistance

PKC alpha promotes chemoresistance in acute lymphoblastic leukemia derived REH cells by a Bcl2-dependent, MAPK-independent mechanism.

Protein kinase C (PKC) has important roles in regulating a number of cellular processes critical to cellular homeostasis. In general, activation PKC promotes signaling cascades that support cell survival. PKC modulates chemoresistance by diverse mechanisms including the activation of mitogen activated protein kinases (MAPK) pathways and by the phosphorylation of Bcl2. Activation of PKC in acute lymphoblastic leukemia (ALL) derived REH cells resulted in a) co-localization of PKC with Bcl2 in mitochondrial membranes, b) increased levels of Bcl2 phosphorylation, and c) increased chemoresistance of cells exposed to chemotherapeutic agents. It is not clear, however, what other mechanisms in addition to the regulation of Bcl2 may be involved in PKC-mediated chemoresistance in REH cells. In this report, we address this question by examining the effect of PKC overexpression on other survival mechanisms. Overexpression of PKC alone did not affect proliferation, cell cycle, or activation of MAPK in REH cells. These findings suggest that PKC -mediated chemoresistance in REH cells may indeed rely heavily on the regulation of Bcl2. Indeed, REH cells overexpressing PKC are similarly sensitive to HA-14, a drug that targets Bcl2 function directly. This finding suggests that the protective properties of PKC require Bcl2, at least in these cells. Interestingly, MAPK was not active in either REH cells or REH PKC transfectants. The subsequent discovery of ERK as another Bcl2 kinase raised the possibility that PKC could have promoted Bcl2 phosphorylation by the activation of ERK. The finding that PKC promotes Bcl2 phosphorylation in the absence of active ERK is significant since it demonstrates that PKC can act directly on Bcl2. Furthermore, overexpression of PKC inhibited mitochondrial Protein Phosphatase 2A (PP2A) activity by a mechanism that involves suppression of PP2A/B56 subunit expression. Since PP2A is a known Bcl2 phosphatase, one mechanism by which PKC may promote protection of REH cells from stress stimuli is via the inactivation of the Bcl2 phosphatase. The ability of PKC to directly target Bcl2 and the Bcl2 protein phosphatase represents a novel survival regulatory mechanism. This mechanism is the mirror image of PP2A-mediated stress pathways where PP2A targets both Bcl2 and the Bcl2 kinase

Discovery of three novel coccidian parasites infecting California sea lions (Zalophus californianus), with evidence of sexual replication and interspecies pathogenicity.

Enteric protozoal infection was identified in 5 stranded California sea lions (Zalophus californianus). Microscopically, the apical cytoplasm of distal jejunal enterocytes contained multiple stages of coccidian parasites, including schizonts with merozoites and spherical gametocytes, which were morphologically similar to coccidians. By histopathology, organisms appeared to be confined to the intestine and accompanied by only mild enteritis. Using electron microscopy, both sexual (microgametocytes, macrogamonts) and asexual (schizonts, merozoites) coccidian stages were identified in enterocytes within parasitophorous vacuoles, consistent with apicomplexan development in a definitive host. Serology was negative for tissue cyst-forming coccidians, and immunohistochemistry for Toxoplasma gondii was inconclusive and negative for Neospora caninum and Sarcocystis neurona. Analysis of ITS-1 gene sequences amplified from frozen or formalin-fixed paraffin-embedded intestinal sections identified DNA sequences with closest homology to Neospora sp. (80%); these novel sequences were referred to as belonging to coccidian parasites "A," "B," and "C." Subsequent molecular analyses completed on a neonatal harbor seal (Phoca vitulina) with protozoal lymphadenitis, hepatitis, myocarditis, and encephalitis showed that it was infected with a coccidian parasite bearing the "C" sequence type. Our results indicate that sea lions likely serve as definitive hosts for 3 newly described coccidian parasites, at least 1 of which is pathogenic in a marine mammal intermediate host species