Abstract. Calf diarrhea is a major economic burden to the bovine industry. Since multiple infectious agents can be involved in calf diarrhea, and the detection of each of the causative agents by traditional methods is laborious and expensive, a panel of 2 multiplex real-time polymerase chain reaction (PCR) assays was developed for rapid and simultaneous detection of the 5 major bovine enteric pathogens (i.e., Bovine coronavirus [BCoV; formally known as Betacoronavirus 1], group A Bovine rotavirus [BRV], Salmonella spp., Escherichia coli K99 + , and Cryptosporidium parvum). The estimated detection limit (i.e., analytic sensitivity) of the panel was 0.1 TCID 50 (50% tissue culture infective dose) for BCoV and group A BRV; 5 and 0.5 colonyforming units for E. coli K99 + and Salmonella, respectively; and 50 oocysts for Cryptosporidium per reaction. In testing 243 fecal samples obtained from submissions to the Iowa State University Veterinary Diagnostic Laboratory or from experimental animals with known infection status, the newly developed multiplex realtime PCR panel simultaneously detected all 5 pathogens directly from fecal samples and was more rapid and sensitive than the traditional diagnostic tests. The PCR panel showed 89%-97% agreement with those conventional diagnostic tests, demonstrating diagnostic sensitivity equal to or better than that of the conventional tests. In conclusion, the multiplex real-time PCR panel can be a tool for a timely and accurate diagnosis of calf diarrhea associated with BCoV, group A BRV, E. coli K99 + , Salmonella, and/or Cryptosporidium.

The ancient wisdom found in iron gall ink guides this work to a simple but advanced solution to the molecular engineering of fluidic interfaces. The Fe(II)–tannin coordination complex, a precursor of the iron gall ink, transforms into interface‐active Fe(III)–tannin species, by oxygen molecules, which form a self‐assembled layer at the fluidic interface spontaneously but still controllably. Kinetic studies show that the oxidation rate is directed by the counteranion of Fe(II) precursor salts, and FeCl2 is found to be more effective than FeSO4—an ingredient of iron gall ink—in the interfacial‐film fabrication. The optimized protocol leads to the formation of micrometer‐thick, free‐standing films at the air–water interface by continuously generating Fe(III)–tannic acid complexes in situ. The durable films formed are transferable, self‐healable, pliable, and postfunctionalizable, and are hardened further by transfer to the basic buffer. This O2‐instructed film formation can be applied to other fluidic interfaces that have high O2 level, demonstrated by emulsion stabilization and concurrent capsule formation at the oil–water interface with no aid of surfactants. The system, inspired by the iron gall ink, provides new vistas on interface engineering and related materials science.

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Purpose Histone deacetylase inhibitors (HDACi) have recently emerged as efficacious therapies that target epigenetic mechanisms in hematologic malignancies. One such hematologic malignancy, B-cell acute lymphoblastic leukemia (B-ALL), may be highly dependent on epigenetic regulation for leukemia development and maintenance, and thus sensitive to small molecule inhibitors that target epigenetic mechanisms. Experimental Design A panel of B-ALL cell lines was tested for sensitivity to HDACi with varying isoform sensitivity. Isoform specific shRNAs were used as further validation of HDACs as relevant therapeutic targets in B-ALL. Mouse xenografts of B cell malignancy derived cell lines and a pediatric B-ALL were used to demonstrate pharmacological efficacy. Results Non-selective HDAC inhibitors were cytotoxic to a panel of B-ALL cell lines as well as to xenografted human leukemia patient samples. Assessment of isoform specific HDACi indicated that targeting HDAC1-3 with class I HDAC specific inhibitors was sufficient to inhibit growth of B-ALL cell lines. Furthermore, shRNA mediated knockdown of HDAC1 or HDAC2 resulted in growth inhibition in these cells. We then assessed a compound that specifically inhibits only HDAC1 and HDAC2. This compound suppressed growth and induced apoptosis in B-ALL cell lines in vitro and in vivo while it was far less effective against other B-cell derived malignancies. Conclusions Here we show that HDAC inhibitors are a potential therapeutic option for B-ALL, and that a more specific inhibitor of HDAC1 and HDAC2 could be therapeutically useful for patients with B-ALL.

An enzyme-instructed method is developed for material-independent, cytocompatible coating of phenolic amines, inspired by melanogenesis found in nature. Tyrosinase-based film formation proceeds smoothly in an aqueous solution at neutral pH, and can use various phenolic amines including catecholamines, such as tyrosine, tyramine, dopamine, norepinephrine, and DOPA, as a coating precursor. Compared with polydopamine coating, the method is fast and efficient, and forms uniform films. Its high cytocompatibility is advantageously applied to cell-surface engineering, where chemically labile Jurkat cells are coated individually without any noticeable decrease in viability. Considering the huge potential of polyphenolic-based coatings, the strategy developed herein will provide an advanced tool for manipulating biological entities, including living cells, in biomedical and medicinal applications.

To study the oncogenic role of the NRAS oncogene (NRAS G12V ) in the context of acute myeloid leukemia (AML), we used a Vav promoter-tetracycline transactivator (Vav-tTA)-driven repressible TRE-NRAS G12V transgene system in Mll-AF9 knock-in mice developing AML. Conditional repression of NRAS G12V expression greatly reduced peripheral white blood cell (WBC) counts in leukemia recipient mice and induced apoptosis in the transplanted AML cells correlated with reduced Ras/Erk signaling. After marked decrease of AML blast cells, myeloproliferative disease (MPD)-like AML relapsed characterized by cells that did not express NRAS G12V . In comparison with primary AML, the MPD-like AML showed significantly reduced aggressiveness, reduced myelosuppression, and a more differentiated phenotype. We conclude that, in AML induced by an Mll-AF9 transgene, NRAS G12V expression contributes to acute leukemia maintenance by suppressing apoptosis and reducing differentiation of leukemia cells. Moreover, NRAS G12V oncogene has a cell nonautonomous role in suppressing erythropoiesis that results in the MPD-like AML show significantly reduced ability to induce anemia. Our results imply that targeting NRAS or RAS oncogene-activated pathways is a good therapeutic strategy for AML and attenuating aggressiveness of relapsed AML. IntroductionKelly and Gilliand have proposed that acute myeloid leukemia (AML) is induced by the cooperation of 2 general classes of mutations. 1 Class I mutations confer cell survival and proliferation advantages and generally result from mutations in genes encoding cell-signaling molecules like NRAS and FLT3. Class II mutations impair differentiation and subsequent apoptosis and, in general, result from mutations in genes encoding transcription factors or chromatin-modifying proteins such as AML1 or mixed lineage leukemia (MLL). Most studies to develop new anticancer drugs have focused on finding efficient inhibitors against the mutant products of class I oncogenes such as ABL, c-KIT, and FLT3. [2][3][4][5][6][7][8] Although previous studies showed that FLT3 inhibition effectively suppresses the cell growth of leukemia induced by the cooperation of an FLT3-activating mutation and an MLL translocation, 9,10 the appropriateness of class I oncogene inhibitors for AML therapy has not been well studied, particularly in leukemia induced in cooperation with a class II oncogene. Conditionally expressed transgenes in mice provide an ideal setting in which to address this issue. Studies have shown that continued expression of oncogenes such as c-Myc or Bcr/Abl is required for maintaining leukemia in transgenic mouse models. [11][12][13] Cancer cells in these models are said to have "oncogene addiction," because they die, differentiate, or become quiescent upon shutting off oncogene expression. 13 To study the functions of a class I mutation in AML, we investigated the role of an NRAS-activating mutation, NRAS G12V , in murine AML induced in combination with the Mll-AF9 oncogene.RAS mutants, which abnormally stimulate RAF/...

p16INK4A and p57 KIP2 are inhibitors of cyclin-dependent kinases and their inactivation by methylation has been reported as a major tumorigenic mechanism in tumors. To examine whether methylation of p16INK4A and p57 KIP2is involved in the development and progression of gastric MALT lymphomas, 24 gastric low-grade lymphomas of MALT, 11 diffuse large B-cell lymphomas, and 10 each case of gastric lymphoid follicles with and without Helicobacter pylori infection were studied. H. pylori infection was positive in 85.7% of the gastric lymphomas. In the gastric lymphoid follicles positive for H. pylori, methylation of p16 INK4A was detected in 10% of cases, while methylation of p57 KIP2 was not detected. In low-grade MALT lymphomas, p16 INK4A and p57 KIP2 were methylated in 41.7 and 29.2% of the cases, respectively. In diffuse large B-cell lymphomas, methylation of p16 INK4A and p57 KIP2was found in 72.7 and 36.4% of the cases, respectively. All but one case with p16 INK4A and p57 KIP2 methylation was H. pylori positive and most of them were stage I. Our results indicate that methylation of p16 INK4A followed by p57 KIP2 methylation involves during the tumorigenesis of gastric MALT lymphomas associated with H. pylori infection. As methylation of these two genes was more frequent in the higher grade (Po0.05), it may contribute to the malignant progression of gastric MALT lymphomas.

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