Viral load, organ distribution, histopathological lesions, and cytokine mRNA expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed.

In vitro effects of thiazolides on Giardia lamblia WB clone C6 cultured axenically and in coculture with Caco2 cells

The thiazolides represent a novel class of anti-infective drugs, with the nitrothiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] (NTZ) as the parent compound. NTZ exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ), which exhibits similar activities. We have here comparatively investigated the in vitro effects of NTZ, TIZ, a number of other modified thiazolides, and metronidazole (MTZ) on Giardia lamblia trophozoites grown under axenic culture conditions and in coculture with the human cancer colon cell line Caco2. The modifications of the thiazolides included, on one hand, the replacement of the nitro group on the thiazole ring with a bromide, and, on the other hand, the differential positioning of methyl groups on the benzene ring. Of seven compounds with a bromo instead of a nitro group, only one, RM4820, showed moderate inhibition of Giardia proliferation in axenic culture, but not in coculture with Caco2 cells, with a 50% inhibitory concentration (IC50) of 18.8 microM; in comparison, NTZ and tizoxanide had IC50s of 2.4 microM, and MTZ had an IC50 of 7.8 microM. Moreover, the methylation or carboxylation of the benzene ring at position 3 resulted in a significant decrease of activity, and methylation at position 5 completely abrogated the antiparasitic effect of the nitrothiazole compound. Trophozoites treated with NTZ showed distinct lesions on the ventral disk as soon as 2 to 3 h after treatment, whereas treatment with metronidazole resulted in severe damage to the dorsal surface membrane at later time points.

Differential expression of TGFbeta-stimulated clone 22 in normal prostate and prostate cancer

The transforming growth factor-beta (TGFbeta) superfamily and its downstream effector genes are key regulators of epithelial homeostasis. Altered expression of these genes may be associated with malignant transformation of the prostate gland. The cDNA array analysis of differential expression of the TGFbeta superfamily and functionally related genes between patient-matched noncancerous prostate (NP) and prostate cancer (PC) bulk tissue specimens highlighted two genes, namely TGFbeta-stimulated clone-22 (TSC-22) and Id4. Verification of their mRNA expression by real-time PCR in patient-matched NP and PC bulk tissue, in laser-captured pure epithelial and cancer cells and in NP and PC cell lines confirmed TSC-22 underexpression, but not Id4 overexpression, in PC and in human PC cell lines. Immunohistochemical analysis showed that TSC-22 protein expression in NP is restricted to the basal cells and colocalizes with the basal cell marker cytokeratin 5. In contrast, all matched PC samples lack TSC-22 immunoreactivity. Likewise, PC cell lines do not show detectable TSC-22 protein expression as shown by immunoblotting. TSC-22 should be considered as a novel basal cell marker, potentially useful for studying lineage determination within the epithelial compartment of the prostate. Conversely, lack of TSC-22 seems to be a hallmark of malignant transformation of the prostate epithelium. Accordingly, TSC-22 immunohistochemistry may prove to be a diagnostic tool for discriminating benign lesions from malignant ones of the prostate. The suggested tumour suppressor function of TSC-22 warrants further investigation on its role in prostate carcinogenesis and on the TSC-22 pathway as a candidate therapeutic target in PC.

Characterization of a Giardia lamblia WB C6 clone resistant to the isoflavone formononetin

Giardia lamblia is a common intestinal-dwelling protozoan and causes diarrhoea in humans and animals worldwide. For several years, a small number of drugs such as the 5-nitroimidazole metronidazole (MET) or the thiazolide nitazoxanide (NTZ) have been used for chemotherapy against giardiasis. However, various pre-clinical and clinical investigations revealed that antigiardial chemotherapy may be complicated by emergence of giardial resistance to these drugs. The present study addressed the question if isoflavones with antigiardial activity, such as daidzein (DAI) or formononetin (FOR), may serve as alternative compounds for treatment of giardiasis. For this purpose, the potential of G. lamblia clone WB C6 to form resistance to FOR and related isoflavones was tested in vitro. In the line of these experiments, a clone (C3) resistant to isoflavones, but sensitive to MET and NTZ, was generated. Affinity chromatography on DAI-agarose using cell-free extracts of G. lamblia trophozoites resulted in the isolation of a polypeptide of approximately 40 kDa, which was identified by mass spectrometry as a nucleoside hydrolase (NH) homologue (EAA37551.1). In a nucleoside hydrolase assay, recombinant NH hydrolysed all nucleosides with a preference for purine nucleosides and was inhibited by isoflavones. Using quantitative RT-PCR, the expression of genes that are potentially involved in resistance formation was analysed, namely NH and genes encoding variant surface proteins (VSPs, TSA417). The transcript level of the potential target NH was found to be significantly reduced in C3. Moreover, drastic changes were observed in VSP gene expression. This may indicate that resistance formation in Giardia against isoflavones is linked to, and possibly mediated by, altered gene expression. Taken together, our results suggest FOR or related isoflavones as an alternative antigiardial agent to overcome potential problems of resistance to drugs like MET or NTZ. However, the capacity of Giardia to develop resistance to isoflavones can potentially interfere with this alternative treatment of the disease.

Telomere length in paroxysmal nocturnal hemoglobinuria correlates with clone size

OBJECTIVE: To study if telomere length can be used as a surrogate marker for the mitotic history in normal and affected hematopoietic cells from patients with paroxysmal nocturnal hemoglobinuria (PNH). METHODS: The telomere length was measured by automated multicolor flow fluorescence in situ hybridization in glycosyl-phosphatidyl-inositol anchored protein (GPI)-negative and GPI-positive peripheral blood leukocytes. Eleven patients were studied, two with predominantly hemolytic PNH and nine with PNH associated with marrow failure. RESULTS: Telomere length in GPI-negative cells was significantly shorter than in GPI-positive cells of the same patient (p < 0.01, n = 11). The difference in telomere length (telomere length in GPI-positive minus telomere length in GPI-negative cells) correlated with the percentage of GPI-negative white blood cells. CONCLUSION: Our results support the hypothesis that telomere length is correlated to the replicative history of GPI-positive and GPI-negative cells and warrant further studies of telomere length in relation to disease progression in PNH.

Streptococcus bovis clone causing two episodes of endocarditis 8 years apart

A patient had endocarditis caused by Streptococcus bovis twice 8 years apart. According to pulsed-field gel electrophoresis (PFGE) the two isolates were identical. Seven unrelated blood isolates of S. bovis yielded unique PFGE patterns. Considering this heterogeneous population structure our findings demonstrate the long-term persistence of an S. bovis clone in a patient with recurrent endocarditis.

Scaleable Code Clone Detection

Code clone detection helps connect developers across projects, if we do it on a large scale. The cornerstones that allow clone detection to work on a large scale are: (1) bad hashing (2) lightweight parsing using regular expressions and (3) MapReduce pipelines. Bad hashing means to determine whether or not two artifacts are similar by checking whether their hashes are identical. We show a bad hashing scheme that works well on source code. Lightweight parsing using regular expressions is our technique of obtaining entire parse trees from regular expressions, robustly and efficiently. We detail the algorithm and implementation of one such regular expression engine. MapReduce pipelines are a way of expressing a computation such that it can automatically and simply be parallelized. We detail the design and implementation of one such MapReduce pipeline that is efficient and debuggable. We show a clone detector that combines these cornerstones to detect code clones across all projects, across all versions of each project.

Third-Generation-Cephalosporin-Resistant Klebsiella pneumoniae Isolates from Humans and Companion Animals in Switzerland: Spread of a DHA-Producing Sequence Type 11 Clone in a Veterinary Setting.

Characterization of third-generation-cephalosporin-resistant Klebsiella pneumoniae isolates originating mainly from one human hospital (n = 22) and one companion animal hospital (n = 25) in Bern (Switzerland) revealed the absence of epidemiological links between human and animal isolates. Human infections were not associated with the spread of any specific clone, while the majority of animal infections were due to K. pneumoniae sequence type 11 isolates producing plasmidic DHA AmpC. This clonal dissemination within the veterinary hospital emphasizes the need for effective infection control practices.

Molecular cloning, expression analysis and assignment of the porcine tumor necrosis factor superfamily member 10 gene (TNFSF10) to SSC13q34-->q36 by fluorescence in situ hybridization and radiation hybrid mapping

We have cloned the complete coding region of the porcine TNFSF10 gene. The porcine TNFSF10 cDNA has an ORF of 870 nucleotides and shares 85% identity with human TNFSF10, and 75% and 72% identity with rat and mouse Tnfsf10 coding sequences, respectively. The deduced porcine TNFSF10 protein consists of 289 amino acids with the calculated molecular mass of 33.5 kDa and a predicted pI of 8.15. The amino acid sequence similarities correspond to 86, 72 and 70% when compared with human, rat and mouse sequences, respectively. Northern blot analysis detected TNFSF10-specific transcripts (approximately 1.7 kb) in various organs of a 10-week-old pig, suggesting ubiquitous expression. Real-time RT-PCR studies of various organs from fetal (days 73 and 98) and postnatal stages (two weeks, eight months) demonstrated developmental and tissue-specific regulation of TNFSF10 mRNA abundance. The chromosomal location of the porcine TNFSF10 gene was determined by FISH of a specific BAC clone to metaphase chromosomes. This TNFSF10 BAC clone has been assigned to SSC13q34-->q36. Additionally, the localization of the TNFSF10 gene was verified by RH mapping on the porcine IMpRH panel.

Double Copies of bla(KPC-3)::Tn4401a on an IncX3 Plasmid in Klebsiella pneumoniae Successful Clone ST512 from Italy.

A carbapenem-resistant sequence type 512 (ST512) Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing K. pneumoniae strain showing a novel variant plasmid content was isolated in Palermo, Italy, in 2014. ST512 is a worldwide successful clone associated with the spread of bla(KPC) genes located on the IncFIIk pKpQIL plasmid. In our ST512 strain, the bla(KPC-3) gene was unusually located on an IncX3 plasmid, whose complete sequence was determined. Two copies of bla(KPC-3)::Tn4401a caused by intramolecular transposition events were detected in the plasmid.

How to be in a good shape? The influence of clone morphology on cell competition

Cell competition is a conserved mechanism where slow proliferating cells (so called losers) are eliminated by faster proliferating neighbors (so called winners) through apoptosis.(1) It is an important process which prevents developmental malformations and maintains tissue fitness in aging adults.(2) Recently, we have shown that the probability of elimination of loser cells correlates with the surface of contact between losers and winners in Myc-induced competition.(3) Moreover, we have characterized an active mechanism that increases the surface of contact between losers and winners, hence accelerating the elimination of loser cells. This is the first indication that cell shape and mechanics can influence cell competition. Here, we will discuss the consequence of the relationship between shape and competition, as well as the relevance of this model for other modes of competition.