Asynchronous nuclear division cycles in multinucleated cells

Synchronous mitosis is common in multinucleated cells. We analyzed a unique asynchronous nuclear division cycle in a multinucleated filamentous fungus, Ashbya gossypii. Nuclear pedigree analysis and observation of GFP-labeled spindle pole bodies demonstrated that neighboring nuclei in A. gossypii cells are in different cell cycle stages despite close physical proximity. Neighboring nuclei did not differ significantly in their patterns of cyclin protein localization such that both G1 and mitotic cyclins were present regardless of cell cycle stage, suggesting that the complete destruction of cyclins is not occurring in this system. Indeed, the expression of mitotic cyclin lacking NH(2)-terminal destruction box sequences did not block cell cycle progression. Cells lacking AgSic1p, a predicted cyclin-dependent kinase (CDK) inhibitor, however, showed aberrant multipolar spindles and fragmented nuclei that are indicative of flawed mitoses. We hypothesize that the continuous cytoplasm in these cells promoted the evolution of a nuclear division cycle in which CDK inhibitors primarily control CDK activity rather than oscillating mitotic cyclin proteins

Retroviral DNA—the silent winner: blood transfusion containing latent feline leukemia provirus causes infection and disease in naïve recipient cats

Additional File 7: Figure S5. Photo of a blood smear from cat R1 (group B) with lymphoblastic leukemia at the time of necropsy. Lymphoblast cells are marked with an arrow. a) The picture displays a large lymphoblast with moderate amounts of basophilic cytoplasm and a large, round nucleus with fine chromatin patterns and several large, indistinct nucleoli. There is also a medium-sized lymphocyte with moderate amounts of pale basophilic cytoplasm and a round nucleus with a coarse chromatin pattern. b) The picture shows a medium-sized to large lymphoblast with small amounts of basophilic cytoplasm and a large, round nucleus with a fine chromatin pattern and two prominent round nucleoli

Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues

ABSTRACT: BACKGROUND: Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan(R) real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats. RESULTS: RNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), beta-actin (ACTB), beta-2-microglobulin (B2M), beta-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan(R) assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~10;6-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied, followed by ACTB and ABL; B2M, HPRT, and the 18S rRNA genes were the least stable ones. CONCLUSION: The reference gene expression stability varied considerably among the feline tissues investigated. No tested gene was optimal for normalisation in all tissues. For the majority of the tissues, two to three reference genes were necessary for accurate normalisation. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analysed

Alpha-1-Acid Glycoprotein Quantification via Spatial Proximity Analyte Reagent Capture Luminescence Assay: Application as Diagnostic and Prognostic Marker in Serum and Effusions of Cats with Feline Infectious Peritonitis Undergoing GS-441524 Therapy

Until recently, the diagnosis of feline infectious peritonitis (FIP) in cats usually led to euthanasia, but recent research has revealed that antiviral drugs, including the nucleoside analog GS-441524, have the potential to effectively cure FIP. Alpha-1-acid glycoprotein (AGP) has been suggested as a diagnostic marker for FIP. However, AGP quantification methods are not easily accessible. This study aimed to establish a Spatial Proximity Analyte Reagent Capture Luminescence (SPARCLTM) assay on the VetBio-1 analyzer to determine the AGP concentrations in feline serum and effusion samples. Linearity was found in serial dilutions between 1:2000 and 1:32,000; the intra-run and inter-run precision was <5% and <15%, respectively; and AGP was stable in serum stored for at least 8 days at room temperature, at 4 °C and at −20 °C. Cats with confirmed FIP had significantly higher serum AGP concentrations (median: 2954 µg/mL (range: 200–5861 µg/mL)) than those with other inflammatory diseases (median: 1734 µg/mL (305–3449 µg/mL)) and clinically healthy cats (median 235 µg/mL (range: 78–616 µg/mL); pKW < 0.0001). The AGP concentrations were significantly higher in the effusions from cats with FIP than in those from diseased cats without FIP (pMWU < 0.0001). The AGP concentrations in the serum of cats with FIP undergoing GS-441524 treatment showed a significant drop within the first seven days of treatment and reached normal levels after ~14 days. In conclusion, the VetBio-1 SPARCLTM assay offers a precise, fast and cost-effective method to measure the AGP concentrations in serum and effusion samples of feline patients. The monitoring of the AGP concentration throughout FIP treatment provides a valuable marker to evaluate the treatment’s effectiveness and identify potential relapses at an early stage

Dominance of highly divergent feline leukemia virus A progeny variants in a cat with recurrent viremia and fatal lymphoma

ABSTRACT: BACKGROUND: In a cat that had ostensibly recovered from feline leukemia virus (FeLV) infection, we observed the reappearance of the virus and the development of fatal lymphoma 8.5 years after the initial experimental exposure to FeLV-A/Glasgow-1. The goals of the present study were to investigate this FeLV reoccurrence and molecularly characterize the progeny viruses. RESULTS: The FeLV reoccurrence was detected by the presence of FeLV antigen and RNA in the blood and saliva. The cat was feline immunodeficiency virus positive and showed CD4+ T-cell depletion, severe leukopenia, anemia and a multicentric monoclonal B-cell lymphoma. FeLV-A, but not -B or -C, was detectable. Sequencing of the envelope gene revealed three FeLV variants that were highly divergent from the virus that was originally inoculated (89-91% identity to FeLV-A/Glasgow-1). In the long terminal repeat 31 point mutations, some previously described in cats with lymphomas, were detected. The FeLV variant tissue provirus and viral RNA loads were significantly higher than the FeLV-A/Glasgow-1 loads. Moreover, the variant loads were significantly higher in lymphoma positive compared to lymphoma negative tissues. An increase in the variant provirus blood load was observed at the time of FeLV reoccurrence. CONCLUSIONS: Our results demonstrate that ostensibly recovered FeLV provirus-positive cats may act as a source of infection following FeLV reactivation. The virus variants that had largely replaced the inoculation strain had unusually heavily mutated envelopes. The mutations may have led to increased viral fitness and/or changed the mutagenic characteristics of the virus

On the dynamics of the adenylate energy system: homeorhesis vs homeostasis.

Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life

Exploring the Use of Molecular Docking to Identify Bioaccumulative Perfluorinated Alkyl Acids (PFAAs)

Methods to predict the bioaccumulation potential of per- and polyfluorinated alkyl substances (PFAS) are sorely needed, given the proliferation of these substances and lack of data on their properties and behavior. Here, we test whether molecular docking, a technique where interactions between proteins and ligands are simulated to predict both bound conformation and interaction affinity, can be used to predict PFAS binding strength and biological half-life. We show that an easy-to-implement docking program, Autodock Vina, can successfully redock perfluorooctanesulfonate (PFOS) to human serum albumin with deviations smaller than 2 Å. Furthermore, predicted binding strengths largely fall within one standard deviation of measured values for perfluorinated alkyl acids (PFAAs). Correlations with half-lives suggest both membrane partitioning and protein interactions are important, and that serum albumin is only one of a number of proteins controlling the fate of these chemicals in organisms. However, few data are available for validation of our approach as a broad screening tool, and available data are highly variable. We therefore call for collection of new data, particularly including proteins other than serum albumin and substances beyond perfluorooctanoic acid (PFOA) and PFOS. The methods we discuss in this work can serve as a framework for guiding such data collection

Adeno-associated vector-delivered CRISPR/SaCas9 system reduces Feline Leukemia Virus production in vitro

Feline leukemia virus (FeLV) is a retrovirus of cats worldwide. High viral loads are associated with progressive infection and the death of the host, due to FeLV-associated disease. In contrast, low viral loads, an effective immune response, and a better clinical outcome can be observed in cats with regressive infection. We hypothesize that by lowering viral loads in progressively infected cats, using CRISPR/SaCas9-assisted gene therapy, the cat’s immune system may be permitted to direct the infection towards a regressive outcome. In a step towards this goal, the present study evaluates different adeno-associated vectors (AAVs) for their competence in delivering a gene editing system into feline cells, followed by investigations of the CRISPR/SaCas9 targeting efficiency for different sites within the FeLV provirus. Nine natural AAV serotypes, two AAV hybrid strains, and Anc80L65, an in silico predicted AAV ancestor, were tested for their potential to infect different feline cell lines and feline primary cells. AAV-DJ revealed superior infection efficiency and was thus employed in subsequent transduction experiments. The introduction of double-strand breaks, using the CRISPR/SaCas9 system targeting 12 selected FeLV provirus sites, was confirmed by T7 endonuclease 1 (T7E1), as well as Tracking of Indels by Decomposition (TIDE) analysis. The highest percentage (up to 80%) of nonhomologous end-joining (NHEJ) was found in the highly conserved gag and pol regions. Subsequent transduction experiments, using AAV-DJ, confirmed indel formation and showed a significant reduction in FeLV p27 antigen for some targets. The targeting of the FeLV provirus was efficient when using the CRISPR/SaCas9 approach in vitro. Whether the observed extent of provirus targeting will be sufficient to provide progressively FeLV-infected cats with the means to overcome the infection needs to be further investigated in vivo