Fluorescence spectroscopy of H‐ras transfected murine fibroblasts: A comparison with Monte Carlo simulations

Autofluorescence properties of tissues have been widely used to diagnose various types of malignancies. In this study, we measured the autofluorescence properties of H‐ras transfected murine fibroblasts and the counterpart control cells. The pair of cells is genetically identical except for the transfected H‐ras gene. We applied Monte Carlo simulations to evaluate the relative contributions of Rayleigh and Mie scattering effects towards fluorescence in an in vitro model system of normal and H‐ras transfected fibroblasts. The experimental results showed that fluorescence emission intensity was higher for normal cells than the malignant counterpart cells by about 30%. In normal cells, linearity in emission intensity was observed for cell densities of up to 1.0 × 10⁶ cells/ml whereas for transformed cells it was up to 1.4 × 10⁶ cells/ml. Nuclear volume changes give good account for the differences in the intrinsic fluorescence between normal and malignant cells. The Monte Carlo (MC) code, newly developed for this study, explains both predominant experimental features: the large fluorescence intensity differences between the transfected and the corresponding control cells as well as the phenomena of the red shift in the excitation spectra as a function of cell density. The contribution of Rayleigh scattering was found to be predominant compared to Mie scattering. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 132–140, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mre 11 p nuclease activity is dispensable for telomeric rapid deletion

Telomeric rapid deletion (TRD) is an intrachromatid recombination process that truncates over-elongated telomeres to the genetically determined average telomere length. We have proposed that TRD is initiated by invasion of the 3' G-rich overhang into centromere-proximal telomere sequence, forming an intermediate that leads to excision of the distal telomere tract. TRD efficiency is dependent on Mre 11p and Rad50p, two members of the widely conserved Mre 11p/Rad50p/Xrs2p (MRX) complex. To investigate the role of Mre 11p in TRD, we conducted a structure/function analysis by testing the TRD rate and precision of mutations within known functional domains. We analyzed 12 alleles that disrupt different Mre 11p activities. Surprisingly, mutations in essential residues of the nuclease domain do not inhibit TRD, effectively ruling out nuclease activity as the source of the Mre 11p requirement. Interestingly, loss of Exo1p alone or loss of Exo1p in an Mre 11 nuclease deficient background does not eliminate TRD, suggesting the presence of an additional nuclease. Second, deletion of DNA binding sites A (residues 410--420) and B (residues 644--692) actually enhances the TRD rate. Even deletion of both DNA binding domains does not abrogate TRD, although its kinetics and precision are variable. This suggests altered DNA binding or a conformational defect in the MRX complex may affect the rate of TRD product formation and indicates that the DNA binding sites formally act as repressors of TRD. Remarkably, the H213Y allele (nuclease motif IV) confers an extraordinarily rapid kinetics, with the vast majority of elongated telomeres deleted imprecisely in a single round of subculturing. In striking contrast, the P162S allele that confers dissolution of the complex also exhibits the null phenotype. These data suggest that Mre 11p can act as a positive and negative regulator of TRD in context of the MRX complex that is essential for TRD.     

The paradoxical relationship between NHEJ and telomeric fusion

Recent results shed new light on the origin of fusion products observed in the destabilized chromosomes of cancer and related diseases. These findings define an unusual relationship between nonhomologous end joining (NHEJ) and telomere "capping," with identical proteins playing opposing roles.     

Proteome Analysis of the Surface of Trichomonas vaginalis Reveals Novel Proteins and Strain-dependent Differential Expression

The identification of surface proteins on the plasma membrane of pathogens is of fundamental importance in understanding host-pathogen interactions. Surface proteins of the extracellular parasite Trichomonas are implicated in the initial adherence to mucosal tissue and are likely to play a critical role in the long term survival of this pathogen in the urogenital tract. In this study, we used cell surface biotinylation and multidimensional protein identification technology to identify the surface proteome of six strains of Trichomonas vaginalis with differing adherence capacities to vaginal epithelial cells. A combined total of 411 proteins were identified, and of these, 11 were found to be more abundant in adherent strains relative to less adherent parasites. The mRNA levels of five differentially expressed proteins selected for quantitative RT-PCR analysis mirrored their observed protein levels, confirming their up-regulation in highly adherent strains. As proof of principle and to investigate a possible role in pathogenesis for differentially expressed proteins, gain of function experiments were performed using two novel proteins that were among the most highly expressed surface proteins in adherent strains. Overexpression of either of these proteins, TVAG_244130 or TVAG_166850, in a relatively non-adherent strain increased attachment of transfected parasites to vaginal epithelial cells ∼2.2-fold. These data support a role in adhesion for these abundant surface proteins. Our analyses demonstrate that comprehensive profiling of the cell surface proteome of different parasite strains is an effective approach to identify potential new adhesion factors as well as other surface molecules that may participate in establishing and maintaining infection by this extracellular pathogen.The flagellated protozoan parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide with an estimated 174 million new cases annually (1). Although asymptomatic infection by T. vaginalis is common, multiple symptoms and pathologies can arise in both men and women, including vaginitis, urethritis, prostatitis, low birth weight infants and preterm delivery, premature rupture of membranes, and infertility (2–5). T. vaginalis has also emerged as an important cofactor in amplifying human immunodeficiency virus spread (6) as individuals infected with T. vaginalis have a significantly increased incidence of human immunodeficiency virus transmission (7, 8). T. vaginalis infection likewise increases the risk of cervical and aggressive prostate cancers (9–11).Despite the serious consequences that can arise from trichomoniasis, the underlying biochemical processes that lead to T. vaginalis pathogenesis are not well defined. Because T. vaginalis is an obligate extracellular pathogen, adherence to epithelial cells is critical for parasite survival within the human host (12). Several in vitro studies indicate that adhesion of the parasite to target mucosal epithelial cells is essential for the maintenance of infection and for cytopathogenicity (13, 14). T. vaginalis adherence to host cells is mediated, in part, by a lipophosphoglycan (LPG)¹ that coats the surface of the parasite, and altering the sugar content of this LPG reduces both adherence and cytotoxicity (15). Moreover, the mammalian protein galectin-1 binds to T. vaginalis in a carbohydrate-dependent manner via a direct interaction with parasite LPG (16). Knockdown of galectin-1 in mammalian cells, however, reduces parasite binding only by ∼17% (16). Although galectin-1-mediated interactions between T. vaginalis LPG and host cell glycoconjugates may be central in establishing infection, it is clear that parasite adhesion factors in addition to LPG are likely to be involved in host-parasite interaction. Surface proteins are likely to play important roles in the initial adherence to mucosal tissue as well as the long term survival of the pathogen on mucosal surfaces.The outcome of infection with T. vaginalis is highly variable. Possible explanations for this phenomenon include host immunity, host nutritional status, and the vaginal microbiota. Additionally, genetic differences between T. vaginalis isolates leading to differences in adherence and cytotoxicity capacities are likely to result in differences in disease progression. Recently, geographically diverse T. vaginalis strains that are significantly more cytotoxic to host cells than laboratory-adapted strains have become available (17, 18), paving the way toward comparative studies aimed at identifying proteins that correlate with virulent phenotypes.Despite the importance of T. vaginalis surface proteins as a critical interface for pathogen-host interactions, there has been no systematic investigation of the surface proteins of this parasite. The T. vaginalis genome is large and encodes a massive proteome with a considerable and diverse repertoire of candidate surface proteins (19). For example, sequence analysis programs that predict transmembrane protein topology identified over 5100 T. vaginalis proteins with one or more transmembrane domains (20). Furthermore, over 300 annotated proteins with predicted transmembrane domains also contain protein motifs common to surface proteins from other pathogens known to contribute to mucosal colonization and other pathogenic processes (20). The vast number and diversity of possible surface proteins necessitates a multitiered approach using complementary genomics and proteomics analyses to identify candidates for focused functional studies.Biotinylation of proteins at the cell surface with an impermeable reagent followed by specific purification of these proteins using streptavidin has successfully been used for the enrichment and identification of surface proteins (21–24). The high avidity binding of biotin to streptavidin greatly enhances membrane protein purification, a challenging feat because of the low abundance of membrane proteins in total cellular extracts. Here, we used this approach to profile the surface plasma membrane proteome of T. vaginalis and to identify proteins that are differentially expressed in adherent relative to less adherent strains of the parasite. To the best of our knowledge, this is the first study to systematically identify and characterize proteins at the surface of Trichomonas parasites. Defining the parasite cell surface proteome is a critical step toward understanding the relative abundance of surface proteins in strains with varying virulence properties. This information will be critical for defining the role surface proteins play in mediating contact between the parasite and host cells as well as the resulting intracellular and extracellular signals that contribute to establishing and maintaining infection. Additionally, conserved surface molecules unique to T. vaginalis that might serve as specific vaccine candidates can be revealed using this approach. The prevalence of trichomoniasis among women of reproductive age (25) and its correlation with AIDS transmission and cervical and prostate cancers (6, 8–11) provide strong arguments for the need to develop vaccines against this human pathogen.     

Microbial Source Tracking in Adjacent Karst Springs

Modern man-made environments, including urban, agricultural, and industrial environments, have complex ecological interactions among themselves and with the natural surroundings. Microbial source tracking (MST) offers advanced tools to resolve the host source of fecal contamination beyond indicator monitoring. This study was intended to assess karst spring susceptibilities to different fecal sources using MST quantitative PCR (qPCR) assays targeting human, bovine, and swine markers. It involved a dual-time monitoring frame: (i) monthly throughout the calendar year and (ii) daily during a rainfall event. Data integration was taken from both monthly and daily MST profile monitoring and improved identification of spring susceptibility to host fecal contamination; three springs located in close geographic proximity revealed different MST profiles. The Giach spring showed moderate fluctuations of MST marker quantities amid wet and dry samplings, while the Zuf spring had the highest rise of the GenBac3 marker during the wet event, which was mirrored in other markers as well. The revelation of human fecal contamination during the dry season not connected to incidents of raining leachates suggests a continuous and direct exposure to septic systems. Pigpens were identified in the watersheds of Zuf, Shefa, and Giach springs and on the border of the Gaaton spring watershed. Their impact was correlated with partial detection of the Pig-2-Bac marker in Gaaton spring, which was lower than detection levels in all three of the other springs. Ruminant and swine markers were detected intermittently, and their contamination potential during the wet samplings was exposed. These results emphasized the importance of sampling design to utilize the MST approach to delineate subtleties of fecal contamination in the environment.     

The Relationship of Sugar to Population-Level Diabetes Prevalence: An Econometric Analysis of Repeated Cross-Sectional Data

While experimental and observational studies suggest that sugar intake is associated with the development of type 2 diabetes, independent of its role in obesity, it is unclear whether alterations in sugar intake can account for differences in diabetes prevalence among overall populations. Using econometric models of repeated cross-sectional data on diabetes and nutritional components of food from 175 countries, we found that every 150 kcal/person/day increase in sugar availability (about one can of soda/day) was associated with increased diabetes prevalence by 1.1% (p <0.001) after testing for potential selection biases and controlling for other food types (including fibers, meats, fruits, oils, cereals), total calories, overweight and obesity, period-effects, and several socioeconomic variables such as aging, urbanization and income. No other food types yielded significant individual associations with diabetes prevalence after controlling for obesity and other confounders. The impact of sugar on diabetes was independent of sedentary behavior and alcohol use, and the effect was modified but not confounded by obesity or overweight. Duration and degree of sugar exposure correlated significantly with diabetes prevalence in a dose-dependent manner, while declines in sugar exposure correlated with significant subsequent declines in diabetes rates independently of other socioeconomic, dietary and obesity prevalence changes. Differences in sugar availability statistically explain variations in diabetes prevalence rates at a population level that are not explained by physical activity, overweight or obesity.     

Excessive attractor instability accounts for semantic priming in schizophrenia

One of the most pervasive findings in studies of schizophrenics with thought disorders is their peculiar pattern of semantic priming, which presumably reflects abnormal associative processes in the semantic system of these patients. Semantic priming is manifested by faster and more accurate recognition of a word-target when preceded by a semantically related prime, relative to an unrelated prime condition. Compared to control, semantic priming in schizophrenics is characterized by reduced priming effects at long prime-target Stimulus Onset Asynchrony (SOA) and, sometimes, augmented priming at short SOA. In addition, unlike controls, schizophrenics consistently show indirect (mediated) priming (such as from the prime 'wedding' to the target 'finger', mediated by 'ring'). In a previous study, we developed a novel attractor neural network model with synaptic adaptation mechanisms that could account for semantic priming patterns in healthy individuals. Here, we examine the consequences of introducing attractor instability to this network, which is hypothesized to arise from dysfunctional synaptic transmission known to occur in schizophrenia. In two simulated experiments, we demonstrate how such instability speeds up the network's dynamics and, consequently, produces the full spectrum of priming effects previously reported in patients. The model also explains the inconsistency of augmented priming results at short SOAs using directly related pairs relative to the consistency of indirect priming. Further, we discuss how the same mechanism could account for other symptoms of the disease, such as derailment ('loose associations') or the commonly seen difficulty of patients in utilizing context. Finally, we show how the model can statistically implement the overly-broad wave of spreading activation previously presumed to characterize thought-disorders in schizophrenia.