[Cytologic evidence of hepatocytotropic T-cell-lymphoma in a 15-year-old male cat]. / Zytologischer Nachweis eines hepatozytotropen T-Zell-Lymphoms bei einem 15 Jahre alten Kater.

This case report describes the rare phenomenon of emperipolesis-like invasion of lymphatic blasts into the hepatocytes of a 15-year-old European Shorthair cat. The cat presented with nonspecific clinical signs (inappetence and weight loss). Cytologic examination of an ultrasound-guided fine-needle aspirate of the liver showed a subset of hepatocytes with emperipolesis-like invasion by lymphatic blasts. Few extracellularly located lymphatic blasts exhibited erythrophagia. Following the cytological diagnosis of large cell lymphoma and 2 weeks of monotherapy with prednisolone, the patient was euthanized due to his poor general condition. A post-mortem sample was obtained from the liver to confirm the suspected cytological diagnosis of hepatocytotropic lymphoma. Histopathology subsequently confirmed the cytologic findings. Immunohistochemically, the lymphatic blasts were positive for CD3 leading to a diagnosis of hepatocytotropic T-cell-lymphoma, which has rarely been described so far.

Metastasizing squamous cell carcinoma in a 50-year-old Eastern Hermann's tortoise (Testudo hermannii boettgeri).

A 50-year-old female Hermann's tortoise (Testudo hermannii boettgeri) was presented with anorexia and lethargy. Clinical examination revealed multiple, visually inconspicuous but indentable areas in the shell corresponding to osteolysis radiographically. Soft tissue nodules and osteolytic lesions were also noted in the limbs. Laboratory results revealed elevated aspartate aminotransferase activity and uric acid concentrations, hypoglycemia, and hyperphosphatemia. Klebsiella oxytoca was isolated from a biopsied scutal area, and the biopsy suggested neoplasia. After a short period of clinical improvement, the animal's condition deteriorated, and it died. Post mortem computed tomography revealed polyostotic lytic lesions of multiple bones and the shell with associated soft tissue nodules protruding into the coelom, and nodular lung lesions. Necropsy, histopathology, and immunohistochemistry secured a diagnosis of a poorly differentiated, pan-cytokeratin-positive squamous cell carcinoma with widespread soft tissue and bone metastases, osteolysis and desmoplasia.

Identification of an ADAMTS2 frameshift variant in a cat family with Ehlers-Danlos syndrome.

We investigated 4 European domestic shorthair kittens with skin lesions consistent with the dermatosparaxis type of the Ehlers-Danlos syndrome, a connective tissue disorder. The kittens were sired by the same tomcat but were born by 3 different mothers. The kittens had easily torn skin resulting in nonhealing skin wounds. Both clinically and histologically, the skin showed thin epidermis in addition to inflammatory changes. Changes in collagen fibers were visible in electron micrographs. The complete genome of an affected kitten was sequenced. A one base pair duplication leading to a frameshift in the candidate gene ADAMTS2 was identified, p.(Ser235fs*3). All 4 affected cats carried the frameshift duplication in a homozygous state. Genotypes at this variant showed perfect cosegregation with the autosomal recessive Ehlers-Danlos syndrome phenotype in the available family. The mutant allele did not occur in 48 unrelated control cats. ADAMTS2 loss-of-function variants cause autosomal recessive forms of Ehlers-Danlos syndrome in humans, mice, dogs, cattle, and sheep. The available evidence from our investigation together with the functional knowledge on ADAMTS2 in other species allows to classify the identified ADAMTS2 variant as pathogenic and most likely causative variant for the observed Ehlers-Danlos syndrome.

Quantitative approaches to study phenotypic effects of large-scale genetic perturbations.

How microbes interact with their environment and how the complex interplay of their genes enables them to survive and thrive under stress is a fundamental question in microbial system biology, which is also important from a public health perspective. Large-scale studies of gene-gene, gene-drug, and drug-drug interactions have proven to be powerful tools for elucidating gene function and functional modules in the cell. Approaches that systematically quantify phenotypes in libraries of microbial strains with genome-wide genetic perturbations are crucial for progress in this area. Here, we review recent advances in this field, and point out applications to the study of gene-drug interactions. We highlight newly developed techniques for the rapid generation of genome-wide mutant libraries and the high-throughput measurement of more complex phenotypes and other observables, such as cell morphology or thermal stability of the proteome.

Granulation tissue-type hemangioma in a 6-week-old puppy - a case report.

BACKGROUND: Hemangioma is a well-known neoplasia in veterinary and human medicine. Several subtypes have been described and are distinguished based on their histologic appearance. The classification schemes of hemangiomas in human and veterinary medicine are different, and various purpose-based schemes can be found in the literature. CASE PRESENTATION: A six-week-old puppy was presented that suffered from a neoplasia that extended to the musculature of the hind limb. After surgical excision, the mass was submitted for pathohistological examination. The mass was composed of endothelial cells forming vascular slits admixed with a fibrous stroma and spindle cells. Immunohistological examination was positive for factor VIII-related antigen and smooth muscle actin, supporting the diagnosis of hemangioma. CONCLUSION: The final diagnosis of granulation tissue-type hemangioma was given due to the histological appearance of the neoplasia. Granulation tissue-type hemangioma is a rare subtype of hemangioma. In this case an uncommonly young dog was affected.

Cyclin B3 implements timely vertebrate oocyte arrest for fertilization.

To ensure successful offspring ploidy, vertebrate oocytes must halt the cell cycle in meiosis II until sperm entry. Emi2 is essential to keep oocytes arrested until fertilization. However, how this arrest is implemented exclusively in meiosis II and not prematurely in meiosis I has until now remained enigmatic. Using mouse and frog oocytes, we show here that cyclin B3, an understudied B-type cyclin, is essential to keep Emi2 levels low in meiosis I. Direct phosphorylation of Emi2 at an evolutionarily highly conserved site by Cdk1/cyclin B3 targets Emi2 for degradation. In contrast, Cdk1/cyclin B1 is inefficient in Emi2 phosphorylation, and this provides a molecular explanation for the requirement of different B-type cyclins for oocyte maturation. Cyclin B3 degradation at exit from meiosis I enables Emi2 accumulation and thus timely arrest in meiosis II. Our findings illuminate the evolutionarily conserved mechanisms that control oocyte arrest for fertilization at the correct cell-cycle stage, which is essential for embryo viability.

Cdc48 influence on separase levels is independent of mitosis and suggests translational sensitivity of separase.

Cdc48 (p97/VCP) is a AAA-ATPase that can extract ubiquitinated proteins from their binding partners and can cooperate with the proteasome for their degradation. A fission yeast cdc48 mutant (cdc48-353) shows low levels of the cohesin protease, separase, and pronounced chromosome segregation defects in mitosis. Separase initiates chromosome segregation when its binding partner securin is ubiquitinated and degraded. The low separase levels in the cdc48-353 mutant have been attributed to a failure to extract ubiquitinated securin from separase, resulting in co-degradation of separase along with securin. If true, Cdc48 would be important in mitosis. In contrast, we show here that low separase levels in the cdc48-353 mutant are independent of mitosis. Moreover, we find no evidence of enhanced separase degradation in the mutant. Instead, we suggest that the cdc48-353 mutant uncovers specific requirements for separase translation. Our results highlight a need to better understand how this key mitotic enzyme is synthesized.

Immunohistological composition of peri-implantitis affected tissue around ceramic implants-A pilot study.

BACKGROUND: Aim of the pilot study was the histologic classification of the inflamed peri-implant soft tissue around ceramic implants (CI) in comparison with titanium implants (TI). METHODS: Peri-implant tissue were retrieved from 15 patients (aged 34 to 88 years, seven males/eight females) with severe peri-implantitis (eight CI, seven TI). The peri-implant soft tissue samples were retrieved from the sites during scheduled removal of the implant and prepared for immunohistochemical analysis. Monoclonal antibodies (targeting CD3, CD20, CD138, and CD68) were used to identify T- and B-cells, plasma cells and macrophages. Quantitative assessment was performed by one histologically trained investigator. Linear mixed regression models were used. RESULTS: A similar numerical distribution of the cell population was found in peri-implantitis around CI compared with TI. CD3 (TI, 17% to 85% versus CI, 20% to 70% of total cell number) and CD138 (TI, 1% to 73% versus CI, 12% to 69% of total cell number) were predominantly expressed. Notably, patient-individual differences of numerical cell distribution were detected. Co-localization of B- and T-lymphocytes was observed. CONCLUSIONS: Peri-implantitis around CI in comparison with TI seems to have a similar histological appearance. Differences in cellular composition of peri-implantitis lesions might also depend on the patient's specific immune status and not only on the material used.

Validation of the transferability of membrane-based fed-batch shake flask cultivations to stirred-tank reactor using three different protease producing Bacillus strains.

Most industrial fermentation processes are operated in fed-batch mode to overcome catabolite repression, undesired by-product formation and oxygen limitation. To maintain comparable process conditions during screening of optimal production strains, the implementation of a fed-batch mode at small scale is crucial. In this study, three different protease producing Bacillus species, Bacillus aeolius, B. licheniformis and B. pumilus, were cultivated using the previously described membrane-based fed-batch shake flasks. Under carbon-limited conditions, catabolite repression was avoided, so that proteases were produced in all strains. Protease yields of B. aeolius and B. licheniformis increased 1.5-fold relative to batch cultivations. To validate process scalability between shake flasks and stirred tank reactors, membrane-based fed-batch shake flask cultivations were transferred to laboratory-scale stirred tank reactors with equal feeding rates. Despite inevitable differences between the scales such as pH control, feed supply and feed start, comparable results were achieved. Oxygen transfer rates of B. licheniformis and B. pumilus measured with the respiration activity monitoring system (RAMOS) in shake flasks and in stirred tank reactor with an off-gas analyzer were almost identical in both cultivation systems. The protease activities referring to the total consumed glucose were also mostly comparable. A slight decrease from shake flask to stirred tank reactor could be observed, which is presumably due to differences in pH control. This study successfully demonstrates the transferability of membrane-based fed-batch shake flask cultivations to laboratory-scale stirred tank reactors.

Elucidation of auxotrophic deficiencies of Bacillus pumilus DSM 18097 to develop a defined minimal medium.

BACKGROUND: Culture media containing complex compounds like yeast extract or peptone show numerous disadvantages. The chemical composition of the complex compounds is prone to significant variations from batch to batch and quality control is difficult. Therefore, the use of chemically defined media receives more and more attention in commercial fermentations. This concept results in better reproducibility, it simplifies downstream processing of secreted products and enable rapid scale-up. Culturing bacteria with unknown auxotrophies in chemically defined media is challenging and often not possible without an extensive trial-and-error approach. In this study, a respiration activity monitoring system for shake flasks and its recent version for microtiter plates were used to clarify unknown auxotrophic deficiencies in the model organism Bacillus pumilus DSM 18097. RESULTS: Bacillus pumilus DSM 18097 was unable to grow in a mineral medium without the addition of complex compounds. Therefore, a rich chemically defined minimal medium was tested containing basically all vitamins, amino acids and nucleobases, which are essential ingredients of complex components. The strain was successfully cultivated in this medium. By monitoring of the respiration activity, nutrients were supplemented to and omitted from the rich chemically defined medium in a rational way, thus enabling a systematic and fast determination of the auxotrophic deficiencies. Experiments have shown that the investigated strain requires amino acids, especially cysteine or histidine and the vitamin biotin for growth. CONCLUSIONS: The introduced method allows an efficient and rapid identification of unknown auxotrophic deficiencies and can be used to develop a simple chemically defined tailor-made medium. B. pumilus DSM 18097 was chosen as a model organism to demonstrate the method. However, the method is generally suitable for a wide range of microorganisms. By combining a systematic combinatorial approach based on monitoring the respiration activity with cultivation in microtiter plates, high throughput experiments with high information content can be conducted. This approach facilitates media development, strain characterization and cultivation of fastidious microorganisms in chemically defined minimal media while simultaneously reducing the experimental effort.

Effects of combined cryopreservation and decellularization on the biomechanical, structural and biochemical properties of porcine pulmonary heart valves.

UNLABELLED: Non-fixed, decellularized allogeneic heart valve scaffolds seem to be the best choice for heart valve replacement, their availability, however, is quite limited. Cryopreservation could prolong their shelf-life, allowing for their ideal match to a recipient. In this study, porcine pulmonary valves were decellularized using detergents, either prior or after cryopreservation, and analyzed. Mechanical integrity was analyzed by uniaxial tensile testing, histoarchitecture by histological staining, and composition by DNA, collagen (hydroxyproline) and GAG (chondroitin sulfate) quantification. Residual sodium dodecyl sulfate (SDS) in the scaffold was quantified by applying a methylene blue activation assay (MBAS). Cryopreserved decellularized scaffolds (DC) and scaffolds that were decellularized after cryopreservation (CD) were compared to fresh valves (F), cryopreserved native valves (C), and decellularized only scaffolds (D). The E-modulus and tensile strength of decellularized (D) tissue showed no significant difference compared to DC and CD. The decellularization resulted in an overall reduction of DNA and GAG, with DC containing the lowest amount of GAGs. The DNA content in the valvular wall of the CD group was higher than in the D and DC groups. CD valves showed slightly more residual SDS than DC valves, which might be harmful to recipient cells. In conclusion, cryopreservation after decellularization was shown to be preferable over cryopreservation before decellularization. However, in vivo testing would be necessary to determine whether these differences are significant in biocompatibility or immunogenicity of the scaffolds. STATEMENT OF SIGNIFICANCE: Absence of adverse effects on biomechanical stability of acellular heart valve grafts by cryopreservation, neither before nor after decellularization, allows the identification of best matching patients in a less time pressure dictated process, and therefore to an optimized use of a very limited, but best-suited heart valve prosthesis.