The conclusions of thousands of peer-reviewed publications rely on data obtained using fluorescence-based quantitative real-time PCR technology. However, the inadequate reporting of experimental detail, combined with the frequent use of flawed protocols is leading to the publication of papers that may not be technically appropriate. We take the view that this problem requires the delineation of a more transparent and comprehensive reporting policy from scientific journals. This editorial aims to provide practical guidance for the incorporation of absolute minimum standards encompassing the key assay parameters for accurate design, documentation and reporting of qPCR experiments (MIQE précis) and guidance on the publication of pure 'reference gene' articles.

BelgiumTHE Fas/ APO-l receptor is one of the major regulators of apoptosisl-7. We report here that Fas/ APO-l-mediated apoptosis requires the activation of a new class of cysteine proteases, including interleukin-lJl-converting enzyme (ICE)s--lo, which are homologous to the product of the Caenorhabditis elegans cell-death gene ced-3 (refs 11, 12). Triggering of Fas/ APO-l rapidly stimulated the proteolytic activity of ICE. Overexpression of ICE, achieved by electroporation and microinjection, strongly potentiated Fas/ APO-l-mediated cell death. In addition, inhibition of ICE activity by protease inhibitors, as well as by transient expression of the pox virus-derived serpin inhibitor, CrmA or an antisense ICE construct, substantially suppressed Fas/ APO-l-triggered cell death. We conclude that activation of ICE or an ICE-related protease is a critical event in Fas/ APO-l-mediated cell death.The signal transduction pathway elicited by Fas/ APO-l is almost completely unknown. Initiation of apoptosis may involve a new class of cysteine proteases, including the product of the C. elegans cell-death gene eed-3, mammalian interleukin-lfJ-converting enzyme (ICE) and the related proteases Nedd-2/Ich-l, prICE and CPP-32 (refs 11-17). Overexpression ofCED-3, ICE or Nedd-2/Ich-1 in Rat-l fibroblasts has been shown to result in apoptotic cell death 12.15. We therefore investigated whether Fas/ APO-I-mediated apoptosis involved an ICE-related proteolytic activity. In L929-APO-l cells 18 or B-lymphoblastoid SKW 6.4 cells, apoptosis triggered by the agonistic monoclonal antibody anti-APO-I was strongly inhibited by the ICE inhibitor YVAD-CHO, a tetrapeptide aldehyde (Ki = 0.76 nM)8 (Fig. la).

Background: Little is known about etiology, disease progression, treatment outcome, survival time, and factors affecting prognosis in dogs with primary hepatitis (PH).Objectives: To review retrospectively different forms of hepatitis in a referral population, by the World Small Animal Veterinary Association Standardization criteria.Animals: One-hundred and one dogs examined for histologically confirmed PH between 2002 and 2006. Dogs with nonspecific reactive hepatitis were excluded.Methods: Retrospective study. Medical records were reviewed for prevalence, signalment, clinical and clinicopathologic manifestation, outcome, survival time, and prognostic factors for shortened survival.Results: PH occurred in 0.5% of dogs in this referral population. Acute (AH) and chronic hepatitis (CH) were diagnosed in 21 and 67 dogs, respectively. Progression from AH to CH occurred in 5/12 of the repeatedly sampled dogs. CH was idiopathic in 43 (64%) dogs, and was associated with copper accumulation in 24 (36%) dogs. Median survival time was longer in dogs with AH than in dogs with CH (either idiopathic or copper associated), and dogs with lobular dissecting hepatitis had the shortest survival time. Prognostic factors predicting shortened survival were associated with decompensated liver function and cirrhosis at initial examination.Conclusions and Clinical Importance: The majority of PH in dogs is CH. Previous studies appear to have underestimated the etiologic role of copper in both AH and CH. Prognosis is reduced in dogs with hepatic cirrhosis or cirrhosis-related clinical findings. Further research into etiology and treatment effectiveness in all PH forms is needed.

IVDD is similar in humans and dogs. Both CD and NCD breeds may therefore serve as models of spontaneous IVDD for human research. However, as with all animal models, it is important to recognize interspecies differences and, indeed, the intraspecies differences between CD and NCD breeds (early vs. late onset of IVDD, respectively) to develop an optimal canine model of human IVDD.

In determining relative gene expression by quantitative measurements of mRNA levels using real-time quantitative PCR, internal standards such as reference genes are essential. Large-scale studies evaluating (candidate) reference genes for veterinary research have not been conducted as thoroughly as for human research, although they are equally important. Our goal was to design and evaluate a genome-wide panel of reference genes from different functional classes. First, primers were optimized using mRNA from canine cell lines and from 30 tissues of one dog as template and SYBR green as fluorescent probe. Second, the expression variation and stability of a gene within one specific tissue were determined. Prostate, kidney, mammary gland, left ventricle, and liver tissues from five to nine dogs of different breeds, sexes, ages, body weights, and disease status were used. Averaging relative stabilities over these tissues revealed the usefulness of individual genes as reference genes. Furthermore, according to expression variation of a reference gene within a specific tissue, usually two to four reference genes are sufficient. Taken together, ribosomal protein S19 (RPS19), ribosomal protein S5 (RPS5), b-2-microglobulin (B2M), and hypoxanthine phosphoribosyltransferase (HPRT) are advocated. However, the optimal set of reference genes depends on the tissue and should be selected and evaluated for each series of experiments.

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SummaryThe recent development of 3D-liver stem cell cultures (hepatic organoids) opens up new avenues for gene and/or stem cell therapy to treat liver disease. To test safety and efficacy, a relevant large animal model is essential but not yet established. Because of its shared pathologies and disease pathways, the dog is considered the best model for human liver disease. Here we report the establishment of a long-term canine hepatic organoid culture allowing undifferentiated expansion of progenitor cells that can be differentiated toward functional hepatocytes. We show that cultures can be initiated from fresh and frozen liver tissues using Tru-Cut or fine-needle biopsies. The use of Wnt agonists proved important for canine organoid proliferation and inhibition of differentiation. Finally, we demonstrate that successful gene supplementation in hepatic organoids of COMMD1-deficient dogs restores function and can be an effective means to cure copper storage disease.

SummaryHepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research.