Presence of Leishmania infantum in red foxes (Vulpes vulpes) in southern Italy

Skin, lymph node (popliteal), and bone marrow samples were collected from 50 red foxes (Vulpes vulpes) from May 2004 to May 2005 in southern Italy. Samples were tested for Leishmania infantum by polymerase chain reaction (PCR). The parasite was detected by PCR from 20 of 50 (40%) fox carcasses. All 20 positive cases were PCR-positive from lymph node and bone marrow samples, whereas 17 of 20 positive cases were PCR-positive from skin samples. Infection status was not related to age or sex. This is the first report of leishmaniasis in red foxes in Italy based on PCR results, and these results reinforce the assumption that this wild canid can serve as a reservoir for Leishmania.     

Anti-inflammatory and antioxidant activity of coumarins designed as potential fluorescent zinc sensors

A series of coumarin analogs, designed and synthesised as potential fluorescent zinc probes were evaluated for their biological activity as anti-inflammatory and antioxidant agents. The effect of the synthesised compounds on inflammation, using the carrageenin-induced rat paw oedema model, was studied. In general, the compounds were found to be potent anti-inflammatory agents (26.5-64%). Compound 5 was found to interact significantly with 1,1-diphenyl-2-picryl-hydrazyl stable free radical (DPPH) whereas the remainder were inactive in this assay. The compounds inhibit in general the soybean lipoxygenase and scavenge superoxide anion radicals. The anti-inflammatory activity seems to be connected with their reducing activity. Their RM values were determined as an expression of their lipophilicity. Theoretical calculations of their lipophilicity as clog P were performed indicating that only a poor relationship exists between their lipophilicity and anti-inflammatory activity.     

Nontransgenic Genome Modification in Plant Cells

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods.Methods for genome editing in plant cells have fallen behind the remarkable progress made in whole-genome sequencing projects. The availability of reliable and efficient methods for genome editing would foster gene discovery and functional gene analyses in model plants and the introduction of novel traits in agriculturally important species (Puchta, 2002; Hanin and Paszkowski, 2003; Reiss, 2003; Porteus, 2009). Genome editing in various species is typically achieved by integrating foreign DNA molecules into the target genome by homologous recombination (HR). Genome editing by HR is routine in yeast (Saccharomyces cerevisiae) cells (Scherer and Davis, 1979) and has been adapted for other species, including Drosophila, human cell lines, various fungal species, and mouse embryonic stem cells (Baribault and Kemler, 1989; Venken and Bellen, 2005; Porteus, 2007; Hall et al., 2009; Laible and Alonso-González, 2009; Tenzen et al., 2009). In plants, however, foreign DNA molecules, which are typically delivered by direct gene-transfer methods (e.g. Agrobacterium and microbombardment of plasmid DNA), often integrate into the target cell genome via nonhomologous end joining (NHEJ) and not HR (Ray and Langer, 2002; Britt and May, 2003).Various methods have been developed to indentify and select for rare site-specific foreign DNA integration events or to enhance the rate of HR-mediated DNA integration in plant cells. Novel T-DNA molecules designed to support strong positive- and negative-selection schemes (e.g. Thykjaer et al., 1997; Terada et al., 2002), altering the plant DNA-repair machinery by expressing yeast chromatin remodeling protein (Shaked et al., 2005), and PCR screening of large numbers of transgenic plants (Kempin et al., 1997; Hanin et al., 2001) are just a few of the experimental approaches used to achieve HR-mediated gene targeting in plant species. While successful, these approaches, and others, have resulted in only a limited number of reports describing the successful implementation of HR-mediated gene targeting of native and transgenic sequences in plant cells (for review, see Puchta, 2002; Hanin and Paszkowski, 2003; Reiss, 2003; Porteus, 2009; Weinthal et al., 2010).HR-mediated gene targeting can potentially be enhanced by the induction of genomic double-strand breaks (DSBs). In their pioneering studies, Puchta et al. (1993, 1996) showed that DSB induction by the naturally occurring rare-cutting restriction enzyme I-SceI leads to enhanced HR-mediated DNA repair in plants. Expression of I-SceI and another rare-cutting restriction enzyme (I-CeuI) also led to efficient NHEJ-mediated site-specific mutagenesis and integration of foreign DNA molecules in plants (Salomon and Puchta, 1998; Chilton and Que, 2003; Tzfira et al., 2003). Naturally occurring rare-cutting restriction enzymes thus hold great promise as a tool for genome editing in plant cells (Carroll, 2004; Pâques and Duchateau, 2007). However, their wide application is hindered by the tedious and next to impossible reengineering of such enzymes for novel DNA-target specificities (Pâques and Duchateau, 2007).A viable alternative to the use of rare-cutting restriction enzymes is the zinc finger nucleases (ZFNs), which have been used for genome editing in a wide range of eukaryotic species, including plants (e.g. Bibikova et al., 2001; Porteus and Baltimore, 2003; Lloyd et al., 2005; Urnov et al., 2005; Wright et al., 2005; Beumer et al., 2006; Moehle et al., 2007; Santiago et al., 2008; Shukla et al., 2009; Tovkach et al., 2009; Townsend et al., 2009; Osakabe et al., 2010; Petolino et al., 2010; Zhang et al., 2010). Here too, ZFNs have been used to enhance DNA integration via HR (e.g. Shukla et al., 2009; Townsend et al., 2009) and as an efficient tool for the induction of site-specific mutagenesis (e.g. Lloyd et al., 2005; Zhang et al., 2010) in plant species. The latter is more efficient and simpler to implement in plants as it does not require codelivery of both ZFN-expressing and donor DNA molecules and it relies on NHEJ—the dominant DNA-repair machinery in most plant species (Ray and Langer, 2002; Britt and May, 2003).ZFNs are artificial restriction enzymes composed of a fusion between an artificial Cys₂His₂ zinc-finger protein DNA-binding domain and the cleavage domain of the FokI endonuclease. The DNA-binding domain of ZFNs can be engineered to recognize a variety of DNA sequences (for review, see Durai et al., 2005; Porteus and Carroll, 2005; Carroll et al., 2006). The FokI endonuclease domain functions as a dimer, and digestion of the target DNA requires proper alignment of two ZFN monomers at the target site (Durai et al., 2005; Porteus and Carroll, 2005; Carroll et al., 2006). Efficient and coordinated expression of both monomers is thus required for the production of DSBs in living cells. Transient ZFN expression, by direct gene delivery, is the method of choice for targeted mutagenesis in human and animal cells (e.g. Urnov et al., 2005; Beumer et al., 2006; Meng et al., 2008). Among the different methods used for high and efficient transient ZFN delivery in animal and human cell lines are plasmid injection (Morton et al., 2006; Foley et al., 2009), direct plasmid transfer (Urnov et al., 2005), the use of integrase-defective lentiviral vectors (Lombardo et al., 2007), and mRNA injection (Takasu et al., 2010).In plant species, however, efficient and strong gene expression is often achieved by stable gene transformation. Both transient and stable ZFN expression have been used in gene-targeting experiments in plants (Lloyd et al., 2005; Wright et al., 2005; Maeder et al., 2008; Cai et al., 2009; de Pater et al., 2009; Shukla et al., 2009; Tovkach et al., 2009; Townsend et al., 2009; Osakabe et al., 2010; Petolino et al., 2010; Zhang et al., 2010). In all cases, direct gene-transformation methods, using polyethylene glycol, silicon carbide whiskers, or Agrobacterium, were deployed. Thus, while mutant plants and tissues could be recovered, potentially without any detectable traces of foreign DNA, such plants were generated using a transgenic approach and are therefore still likely to be classified as transgenic. Furthermore, the recovery of mutants in many cases is also dependent on the ability to regenerate plants from protoplasts, a procedure that has only been successfully applied in a limited number of plant species. Therefore, while ZFN technology is a powerful tool for site-specific mutagenesis, its wider implementation for plant improvement may be somewhat limited, both by its restriction to certain plant species and by legislative restrictions imposed on transgenic plants.Here we describe an alternative to direct gene transfer for ZFN delivery and for the production of mutated plants. Our approach is based on the use of a novel Tobacco rattle virus (TRV)-based expression system, which is capable of systemically infecting its host and spreading into a variety of tissues and cells of intact plants, including developing buds and regenerating tissues. We traced the indirect ZFN delivery in infected plants by activation of a mutated reporter gene and we demonstrate that this approach can be used to recover mutated plants.     

Microbial Community Structure and Dynamics of Dark Fire-Cured Tobacco Fermentation

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.     

Glocal Clinical Registries: Pacemaker Registry Design and Implementation for Global and Local Integration – Methodology and Case Study

Background

The ability to apply standard and interoperable solutions for implementing and managing medical registries as well as aggregate, reproduce, and access data sets from legacy formats and platforms to advanced standard formats and operating systems are crucial for both clinical healthcare and biomedical research settings.

Purpose

Our study describes a reproducible, highly scalable, standard framework for a device registry implementation addressing both local data quality components and global linking problems.

Methods and Results

We developed a device registry framework involving the following steps: (1) Data standards definition and representation of the research workflow, (2) Development of electronic case report forms using REDCap (Research Electronic Data Capture), (3) Data collection according to the clinical research workflow and, (4) Data augmentation by enriching the registry database with local electronic health records, governmental database and linked open data collections, (5) Data quality control and (6) Data dissemination through the registry Web site. Our registry adopted all applicable standardized data elements proposed by American College Cardiology / American Heart Association Clinical Data Standards, as well as variables derived from cardiac devices randomized trials and Clinical Data Interchange Standards Consortium. Local interoperability was performed between REDCap and data derived from Electronic Health Record system. The original data set was also augmented by incorporating the reimbursed values paid by the Brazilian government during a hospitalization for pacemaker implantation. By linking our registry to the open data collection repository Linked Clinical Trials (LinkedCT) we found 130 clinical trials which are potentially correlated with our pacemaker registry.

Conclusion

This study demonstrates how standard and reproducible solutions can be applied in the implementation of medical registries to constitute a re-usable framework. Such approach has the potential to facilitate data integration between healthcare and research settings, also being a useful framework to be used in other biomedical registries.     

Stereoselective reactions of (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene derivatives form 27-nor-3,20,23,26-tetrahydroxy-cholesten-22-ones and related bromo ketones

The previously reported analog of pregnenolone having a 3,4-dihydro-2H-pyran attached via a Cz.sbnd;C bond to the C-20 position (1), stereoselectively reacts with m-chloroperoxybenzoic acid in methanol at -5 degrees C. Acid-catalyzed hydrolysis of the isolated intermediates gives good yields of mostly a new 27-norcholesterol analog: (20R,23R)-3,20,23,26-tetrahydroxy-27-norcholest-5-en-22-one-3-acetate (2a, and a smaller amount of its 23S enantiomer 2b). Three different conditions of epoxidation and methanolysis followed by acid-catalyzed hydrolysis typically produce approximately 2:1 ratios of the 23R:23S diastereoisomers with a C-23 hydroxy group at the new asymmetric center. Bromine also reacts stereoselectively with (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene (4) giving mostly (20R,23R)-23-bromo-3,20,26-trihydroxy-27-norcholest-5-en-22-one (7a). Thus both major steroidal products 2a and 7a have the same C-23R configuration. Assignment of molecular structures and the absolute configurations to 1 and 2a were based on elemental analysis, mass spectra, nuclear magnetic resonance, FTIR infrared spectroscopic analysis and X-ray crystallography. Mechanisms are discussed for stereochemical selectivity during epoxidation and bromination of the 3,4-dihydro-2H-pyranyl ring in 1 and 4.     

Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta ) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent

Hepatic stellate cells are the primary cell type responsible for matrix deposition in liver fibrosis, undergoing a process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic agent transforming growth factor-beta (TGF-beta). We have studied activation of the TGF-beta downstream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for 1, 4, and 7 days, with quiescent, intermediate, and fully transdifferentiated phenotypes, respectively. Total levels of Smad4, common to multiple TGF-beta superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-beta or the nuclear export inhibitor leptomycin B. TGF-beta mediates activation of Smad2 primarily in early cultured cells and that of Smad3 primarily in transdifferentiated cells. The linker protein SARA, which is required for Smad2 signaling, disappears with transdifferentiation. Additionally, day 7 cells demonstrate constitutive phosphorylation and nuclear localization of Smad 2, which is not affected by pretreatment with TGF-beta-neutralizing antibodies, a type I TGF-beta receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-beta-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.