Three-dimensional in vivo imaging of green fluorescent protein-expressing T cells in mice with noncontact fluorescence molecular tomography

Given that optical tomography is capable of quantitatively imaging the distribution of several important chromophores and fluorophores in vivo, there has been a great deal of interest in developing optical imaging systems with increased numbers of measurements under optimal experimental conditions. In this article, we present a novel system that enables three-dimensional imaging of fluorescent probes in whole animals using a noncontact setup, in parallel with a three-dimensional surface reconstruction algorithm. This approach is directed toward the in vivo imaging of fluorophore or fluorescent protein concentration in small animals. The system consists of a rotating sample holder and a lens-coupled charge-coupled device camera in combination with a fiber-coupled laser scanning device. By measuring multiple projections, large data sets can be obtained, thus improving the accuracy of the inversion models used for quantitative three-dimensional reconstruction of fluorochrome distribution, as well as facilitating a higher spatial resolution. In this study, the system was applied to determining the distribution of green fluorescent protein (GFP)-expressing T lymphocytes in a transgenic mouse model, thus demonstrating the potential of the system for studying immune system function. The technique was used to image and reconstruct fluorescence originating from 32 x 10(6) T cells in the thymus and 3 x 10(5) T cells in the spleen.     

Antifungal chemical compounds identified using a C. elegans pathogenicity assay

There is an urgent need for the development of new antifungal agents. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery. We show that Candida albicans, as well as other Candida species, are ingested by Caenorhabditis elegans and establish a persistent lethal infection in the C. elegans intestinal track. Importantly, key components of Candida pathogenesis in mammals, such as filament formation, are also involved in nematode killing. We devised a Candida-mediated C. elegans assay that allows high-throughput in vivo screening of chemical libraries for antifungal activities, while synchronously screening against toxic compounds. The assay is performed in liquid media using standard 96-well plate technology and allows the study of C. albicans in non-planktonic form. A screen of 1,266 compounds with known pharmaceutical activities identified 15 (approximately 1.2%) that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Two compounds identified in the screen, caffeic acid phenethyl ester, a major active component of honeybee propolis, and the fluoroquinolone agent enoxacin exhibited antifungal activity in a murine model of candidiasis. The whole-animal C. elegans assay may help to study the molecular basis of C. albicans pathogenesis and identify antifungal compounds that most likely would not be identified by in vitro screens that target fungal growth. Compounds identified in the screen that affect the virulence of Candida in vivo can potentially be used as "probe compounds" and may have antifungal activity against other fungi.     

Enzymatic properties of human kallikrein-related peptidase 12 (KLK12)

Human kallikrein-related peptidase 12 (KLK12) is a new member of the human tissue kallikrein family. Preliminary studies suggest that KLK12 is differentially expressed in breast cancer and may have potential use as a cancer biomarker. It has been predicted that KLK12 is a secreted serine protease. However, the enzymatic properties of this protein have not been reported so far. Here, we report the production of recombinant KLK12 and analyses of its enzymatic characteristics, including zymogen activation, substrate specificity, and regulation of its activity. KLK12 is secreted as an inactive pro-enzyme, which is able to autoactivate to gain enzymatic activity. Through screening of a panel of fluorogenic and chromogenic peptide substrates, we establish that active KLK12 possesses trypsin-like activity, cleaving peptide bonds after both arginine and lysine. Active KLK12 quickly loses its activity due to autodegradation, and its activity can also be rapidly inhibited by zinc ions and by alpha2-antiplasmin through covalent complex formation. Furthermore, we demonstrate that KLK12 is able to activate KLK11 zymogen in vitro. Our results indicate that KLK12 may participate in enzymatic cascades involving other kallikreins.     

Human tissue kallikrein 5 is a member of a proteolytic cascade pathway involved in seminal clot liquefaction and potentially in prostate cancer progression

Human tissue kallikreins (hKs) are a family of fifteen serine proteases. Several lines of evidence suggest that hKs participate in proteolytic cascade pathways. Human kallikrein 5 (hK5) has trypsin-like activity, is able to self-activate, and is co-expressed in various tissues with other hKs. In this study, we examined the ability of hK5 to activate other hKs. By using synthetic heptapeptides that encompass the activation site of each kallikrein and recombinant pro-hKs, we demonstrated that hK5 is able to activate pro-hK2 and pro-hK3. We then showed that, following their activation, hK5 can internally cleave and deactivate hK2 and hK3. Given the predominant expression of hK2 and hK3 in the prostate, we examined the pathophysiological role of hK5 in this tissue. We studied the regulation of hK5 activity by cations (Zn2+, Ca2+, Mg2+, Na2+, and K+) and citrate and showed that Zn can efficiently inhibit hK5 activity at levels well below its normal concentration in the prostate. We also show that hK5 can degrade semenogelins I and II, the major components of the seminal clot. Semenogelins can reverse the inhibition of hK5 by Zn2+, providing a novel regulatory mechanism of its serine protease activity. hK5 is also able to internally cleave insulin-like growth factor-binding proteins 1, 2, 3, 4, and 5, but not 6, suggesting that it might be involved in prostate cancer progression through growth factor regulation. Our results uncover a kallikrein proteolytic cascade pathway in the prostate that participates in seminal clot liquefaction and probably in prostate cancer progression.     

The Control Region of Maternally and Paternally Inherited Mitochondrial Genomes of Three Species of the Sea Mussel Genus Mytilus

Species of the mussel genus Mytilus possess maternally and paternally transmitted mitochondrial genomes. In the interbreeding taxa Mytilus edulis and M. galloprovincialis, several genomes of both types have been fully sequenced. The genome consists of the coding part (which, in addition to protein and RNA genes, contains several small noncoding sequences) and the main control region (CR), which in turn consists of three distinct parts: the first variable (VD1), the conserved (CD), and the second variable (VD2) domain. The maternal and paternal genomes are very similar in gene content and organization, even though they differ by >20% in primary sequence. They differ even more at VD1 and VD2, yet they are remarkably similar at CD. The complete sequence of a genome from the closely related species M. trossulus was previously reported and found to consist of a maternal-like coding part and a paternal-like and a maternal-like CR. From this and from the fact that it was extracted from a male individual, it was inferred that this is a genome that switched from maternal to paternal transmission. Here we provide clear evidence that this genome is the maternal genome of M. trossulus. We have found that in this genome the tRNA^Gln in the coding region is apparently defective and that an intact copy of this tRNA occurs in the CR, that one of the two conserved domains is missing essential motifs, and that one of the two first variable domains has a high rate of divergence. These features may explain the large size and mosaic structure of the CR of the maternal genome of M. trossulus. We have also obtained CR sequences of the maternal and paternal genomes of M. californianus, a more distantly related species. We compare the control regions from all three species, focusing on the divergence among genomes of different species origin and among genomes of different transmission routes.     

Candida albicans Hyphal Formation and Virulence Assessed Using a Caenorhabditis elegans Infection Model

Candida albicans colonizes the human gastrointestinal tract and can cause life-threatening systemic infection in susceptible hosts. We study here C. albicans virulence determinants using the nematode Caenorhabditis elegans in a pathogenesis system that models candidiasis. The yeast form of C. albicans is ingested into the C. elegans digestive tract. In liquid media, the yeast cells then undergo morphological change to form hyphae, which results in aggressive tissue destruction and death of the nematode. Several lines of evidence demonstrate that hyphal formation is critical for C. albicans pathogenesis in C. elegans. First, two yeast species unable to form hyphae (Debaryomyces hansenii and Candida lusitaniae) were less virulent than C. albicans in the C. elegans assay. Second, three C. albicans mutant strains compromised in their ability to form hyphae (efg1Δ/efg1Δ, flo8Δ/flo8Δ, and cph1Δ/cph1Δ efg1Δ/efg1Δ) were dramatically attenuated for virulence. Third, the conditional tet-NRG1 strain, which enables the external manipulation of morphogenesis in vivo, was more virulent toward C. elegans when the assay was conducted under conditions that permit hyphal growth. Finally, we demonstrate the utility of the C. elegans assay in a screen for C. albicans virulence determinants, which identified several genes important for both hyphal formation in vivo and the killing of C. elegans, including the recently described CAS5 and ADA2 genes. These studies in a C. elegans-C. albicans infection model provide insights into the virulence mechanisms of an important human pathogen.Candida albicans is the most common human fungal pathogen; however, our knowledge of its virulence mechanisms is incomplete, and our best antifungal agents are often ineffective in treating severe candidiasis (3). Infections with Candida species account for 70 to 90% of all invasive mycoses (32) and can be associated with devastating consequences, particularly in intensive care units where mortality rates reach 40% (24, 34). The drug resistance of pathogenic fungi exacerbates this problem and often limits therapeutic options (35). The identification of virulence pathways that can be targeted with novel antifungal therapies is urgently needed (31, 38, 46).One approach to understand the genetic mechanisms of virulence is to use invertebrates, such as the nematode Caenorhabditis elegans, as model hosts (43). Studies of C. elegans infection with Pseudomonas aeruginosa and Cryptococcus neoformans, for example, have led to the identification of evolutionarily conserved mechanisms of host immunity and microbial virulence (1, 21, 50). However, efforts to design an accurate nonmammalian model of C. albicans pathogenesis have been stymied, in part because it has been difficult to capture the role of Candida dimorphism in these systems.Morphogenesis in C. albicans is intricately related to pathogenesis and thus has been intensively studied. C. albicans hyphae are important for tissue destruction and host invasion (3). As such, C. albicans mutants and non-albicans Candida species that are unable to form true hyphae are attenuated for virulence (3, 37). However, C. albicans yeast cells also have virulence attributes (4, 33) that are likely involved in dissemination of the fungus through the bloodstream, and the establishment of infection at distant sites. To date, genetic screens to identify the determinants of Candida morphology have been conducted in vitro. Determining the role of these genes in virulence has traditionally involved separate and often laborious studies in mammals. Therefore, an expedient system to study morphogenesis of C. albicans in vivo and accurately model pathogenesis would offer many important advantages.Here, we study C. albicans pathogenesis using the invertebrate host C. elegans. C. albicans yeast cells are ingested into the gastrointestinal tract. In liquid media, the yeast cells form hyphae, which results in an aggressive infection that ultimately kills the nematode. Fungal hyphae destroy worm tissues and pierce the collagenous cuticle of the animal, a phenotype that is easily visible using a dissecting microscope. By studying mutants and genetically engineered C. albicans strains, we show that hyphal formation is required for full virulence in this system. Finally, we illustrate the utility of the C. elegans-C. albicans infection assay in a screen for genes involved in Candida morphogenesis and virulence.     

Building arks for tRNA: Structure and function of the Arc1p family of non-catalytic tRNA-binding proteins

Following the intricate architecture of the eukaryotic cell, protein synthesis involves formation of many macromolecular assemblies, some of which are composed by tRNA-aminoacylation enzymes. Protein-protein and protein-tRNA interactions in these complexes can be facilitated by non-catalytic tRNA-binding proteins. This review focuses on the dissection of the molecular, structural and functional properties of a particular family of such proteins: yeast Arc1p and its homologues in prokaryotes and higher eukaryotes. They represent paradigms of the strategies employed for the organization of sophisticated and dynamic nanostructures supporting spatio-temporal cellular organization.     

The Quest for Renal Disease Proteomic Signatures: Where Should We Look?

Renal diseases are prevalent and important. However, despite significant strides in medicine, clinical nephrology still relies on nonspecific and inadequate markers such as serum creatinine and total urine protein for monitoring and diagnosis of renal disease. In case of glomerular renal diseases, biopsy is often necessary to establish the diagnosis. With new developments in proteomics technology, numerous studies have emerged, searching for better markers of kidney disease diagnosis and/or prognosis. Blood, urine, and renal biopsy tissue have been explored as potential sources of biomarkers. Some interesting individual or multiparametric biomarkers have been found; however, none have yet been validated or entered clinical practice. This review focuses on some studies of biomarkers of glomerular renal diseases, as well as addresses the question of which sample type(s) might be most promising in preliminary discovery phases of candidate proteins.     

Inhibitors of foot and mouth disease virus targeting a novel pocket of the RNA-dependent RNA polymerase

Background

Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections.

Methodology/Principal Findings

Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC₅₀s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC₅₀ = 12 µM and EC₉₀ = 20 µM).

Significance

We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD.