In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.     

Inadvertent Propagation of Factor VII Deficiency in a Canine Mucopolysaccharidosis Type I Research Breeding Colony

Issues of cost and genetics can result in inbreeding of canine genetic disease colonies. Beagles often are used to maintain such colonies, providing stock for outcrosses. Factor VII (FVII) deficiency is a hemostatic disorder found at increased frequency in beagles and has been characterized at the DNA level. Deficiency of FVII presents obstacles in colonies founded with beagles. An initial finding of a FVII-deficient pup from a longstanding colony prompted us to evaluate FVII deficiency fully in this colony. Current and archival records and tissues were used to reconstruct the colony pedigree, assess the contribution from beagles, and test samples to document the source and frequency of the mutant FVII allele. As part of this study we developed a PCR-based diagnostic assay that was simpler than what was previously available. Pedigree analysis revealed a founder effect implicating beagles that led to high frequency (55%) of the mutant allele. In addition, affected animals were identified. The complete picture of the clinical effect within the colony remains unclear, but unusual neonatal presentations, including hemoabdomen, have occurred in pups affected with FVII deficiency. Use of a PCR-based diagnostic assay to screen all potential beagle breeding stock will prevent similar occurrences of FVII deficiency in future canine research colonies.Abbreviations: FVII, factor VII; MPS I, mucopolysaccharidosis I; PT, prothrombin timeThe importance of developing clinically relevant large animal models for human genetic diseases is becoming increasingly evident.⁴ For example, preclinical assessments of gene transfer experiments require large long-lived animal models physiologically and genetically comparable to humans. Canine models are ideal because their genome has been sequenced, they are large and long-lived, and because more than 60% of inherited diseases of dogs are known to be homologs of human diseases.⁴The maintenance of genetic diseases in research colonies results, for all practical purposes, in a founder effect by which allelic frequencies in a research colony may be skewed upward from those in the general population, due to founding of the colony by a limited number of animals. This founder effect is due to insufficient genetic outcrosses, resulting from economic constraints and considerations such as the inbreeding needed with a recessive condition. Practically speaking, colony founders may inevitably be incompletely characterized at the genetic level, potentially leading to increased prevalence of an additional genetic disease. When available, practical screening tests (clotting times, cardiac evaluation, and so on) or breed-specific genetic tests should be conducted to reduce additional genetic diseases within a research colony.The occurrence of additional genetic diseases in research colonies can limit or confound the primary research objective and affect the number of research animals used and their health and welfare. Inherited factor VII (FVII) deficiency in beagles is such a condition.⁸ Although largely an asymptomatic defect, this autosomal recessive hemostatic disorder, can lead to excessive bleeding after surgery or trauma, hematoma formation, body cavity bleeding, and persistent uterine and vaginal hemorrhage.²³ Factor VII deficiency also occurs in Alaskan malamutes,¹⁴ mixed breeds,¹³ and Alaskan klee kai dogs.¹¹ Clinical symptoms in canines can be reduced by transfusions with fresh plasma or blood, or administration of recombinant activated human FVII.^9,17 However, treatments are only a temporary solution, because the half-life of FVII protein is only 3 to 4 h and, in canines, treatment with human proteins raises concern about antibody responses to those proteins, thus potentially limiting further therapy. The FVII mutation initially described in beagles (referred to henceforth as the ‘beagle mutation’ in full recognition of its occurrence in additional breeds) is well described.¹ Furthermore the beagle mutation has been documented to cause the FVII deficiency seen in the Alaskan klee kai,¹¹ Airedale terrier, giant schnauzer, and Scottish deerhound.²² The published assay is a restriction digest to test beagles for the causative transitional missense mutation of a guanine-to-adenine located in exon 5 (leading to the G96E mutant protein).¹ However, because the assay relies on an enzyme with multiple sites in the resultant amplicon, interpretation of results can be problematic, potentially requiring direct DNA sequencing for confirmation of the genotype.Herein we present data from a long-standing canine research breeding colony for mucopolysaccharidosis type I (MPS I) which indicate that FVII deficiency should be a primary concern when developing research colonies by using beagle breeding stock. We suspected factor VII deficiency in this colony after an episode of hemoabdomen in a neonate, which was noted shortly after the colony was transferred to a different institution. Using an improved PCR-based diagnostic assay for the beagle FVII mutation, we have documented the history of this mutant allele within the colony in detail and have identified the presence of this allele in other canine colonies.     

Modeling system states in liver cells: Survival,apoptosis and their modifications in response to viral infection

Background  

The decision pro- or contra apoptosis is complex, involves a number of different inputs, and is central for the homeostasis of an individual cell as well as for the maintenance and regeneration of the complete organism.     

IFN-γ-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy

Chlamydial infection of the host cell induces Gamma interferon (IFNγ), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNγ-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen''s elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNγ-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNγ, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5−/− MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNγ-induced Atg5−/− cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6−/−) MEFs, in which chlamydial growth is enhanced, do not respond to IFNγ even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction.     

Complete Primate Skeleton from the Middle Eocene of Messel in Germany: Morphology and Paleobiology

Background

The best European locality for complete Eocene mammal skeletons is Grube Messel, near Darmstadt, Germany. Although the site was surrounded by a para-tropical rain forest in the Eocene, primates are remarkably rare there, and only eight fragmentary specimens were known until now. Messel has now yielded a full primate skeleton. The specimen has an unusual history: it was privately collected and sold in two parts, with only the lesser part previously known. The second part, which has just come to light, shows the skeleton to be the most complete primate known in the fossil record.

Methodology/Principal Findings

We describe the morphology and investigate the paleobiology of the skeleton. The specimen is described as Darwinius masillae n.gen. n.sp. belonging to the Cercamoniinae. Because the skeleton is lightly crushed and bones cannot be handled individually, imaging studies are of particular importance. Skull radiography shows a host of teeth developing within the juvenile face. Investigation of growth and proportion suggest that the individual was a weaned and independent-feeding female that died in her first year of life, and might have attained a body weight of 650–900 g had she lived to adulthood. She was an agile, nail-bearing, generalized arboreal quadruped living above the floor of the Messel rain forest.

Conclusions/Significance

Darwinius masillae represents the most complete fossil primate ever found, including both skeleton, soft body outline and contents of the digestive tract. Study of all these features allows a fairly complete reconstruction of life history, locomotion, and diet. Any future study of Eocene-Oligocene primates should benefit from information preserved in the Darwinius holotype. Of particular importance to phylogenetic studies, the absence of a toilet claw and a toothcomb demonstrates that Darwinius masillae is not simply a fossil lemur, but part of a larger group of primates, Adapoidea, representative of the early haplorhine diversification.     

Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells

Background

Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of the ErbB2 (ΔN-ErbB2).

Methodology/Principal Findings

Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic ΔN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however, normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm.

Conclusions/Significance

Our data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in autophagosome formation.     

Aspergillus alabamensis,a New Clinically Relevant Species in the Section Terrei

Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins enolase (enoA), β-tubulin (benA), and calmodulin (calM) of a large number of isolates within the section Terrei, genus Aspergillus, revealed the presence of a new cryptic species within this section, Aspergillus alabamensis. Most members of this new cryptic species were recovered as colonizing isolates from immunocompetent patient populations, had decreased in vitro susceptibilities to the antifungal drug amphotericin B, and were morphologically similar to but genetically distinct from Aspergillus terreus isolates.Invasive infections caused by Aspergillus terreus are often disseminated with increased lethality compared with infections caused by other Aspergillus species and tend to be resistant to treatment with the antifungal drug amphotericin B (6, 14, 17). Despite the clinical significance of this organism, little is known about the epidemiology, genetic diversity, and population structure of A. terreus.Historically, A. terreus has been identified in the laboratory by conventional methods such as colony morphology and microscopic characteristics. Such morphological studies have placed A. terreus as a single homogenous species within the section Terrei along with two other varieties, A. terreus var. africanus and A. terreus var. aureus (11). Recent studies have shown that morphological characteristics may not be reliable for distinguishing Aspergillus species, as inferred from the demonstration of multiple cryptic species within the section Fumigati by molecular phylogenetic methods (3-5, 13, 18).In the past, molecular methods largely based on randomly amplified polymorphic DNA-PCR-based assays have shown that A. terreus isolates can have great strain diversity (1, 8, 16). One recent genotyping study of several A. terreus clinical isolates recovered from two different medical centers using this method concluded that nosocomial acquisition of A. terreus infections was highly unlikely given the great genetic diversity observed (7). Another study demonstrated that comparative sequence analyses of the D1 and D2 regions had limited utility to study relationships within the section Terrei, while the internal transcribed spacer regions were useful since there was more nucleotide diversity in this region (16). However, the authors of this study could not resolve species within the section Terrei using these molecular approaches.In the present study, we have developed a multilocus sequence approach employing three protein-coding regions to study species diversity of the section Terrei using a large panel of isolates from both clinical and environmental origins recovered from various parts of the world. The studies outlined below demonstrate the presence of a new, clinically relevant species, Aspergillus alabamensis, and clarify the taxonomic position of the A. terreus variant A. terreus var. aureus.