Comparison of different NAT assays for the detection of microorganisms belonging to the class Mollicutes

Abstract Background Mollicutes detection can be cumbersome due to their slow growth in vitro. For this reason, the use of DNA based on generic molecular tests represents an alternative for rapid, sensitive and specific detection of these microorganism. For this reason, six previously described nucleic acid testing assays were compared to evaluate their ability to detect microorganisms belonging to the class Mollicutes. Methods A panel of 61 mollicutes, including representatives from the Mycoplasma, Acholeplasma, Mesoplasma, Spiroplasma and Ureaplasma genus, were selected to evaluate the sensitivity and specificity of these assays. A total of 21 non-mollicutes, including closely related non-mollicutes species, were used to evaluate specificity. Limits of detection were calculated to determine the analytical sensitivity of the assays. The two best performing assays were subsequently adapted into real-time PCR format, followed by melting curve analysis. Results Both assays performed satisfactorily, with a 100% specificity described for both assays. The detection limits were found to be between 10−4 and 10−5 dilutions, equivalent to 15 to 150 genome copies approximately. Based on our work, both van Kuppeveld and Botes real-time PCR assays were found to be the best performing tests in terms of sensitivity and specificity. Furthermore, Botes real-time PCR assay could detect phytoplasmas as well. Conclusions These assays can be very useful for the rapid, specific and sensitive screening cell line contaminants, clinical samples as well as detecting non-culturable, unknown species of mollicutes or mollicutes whose growth is slow or difficult

Beautiful Mirrors at the LHC

We explore the "Beautiful Mirrors" model, which aims to explain the measured value of $A^b_{FB}$, discrepant at the $2.9\sigma$ level. This scenario introduces vector-like quarks which mix with the bottom, subtly affecting its coupling to the $Z$. The spectrum of the new particles consists of two bottom-like quarks and a charge -4/3 quark, all of which have electroweak interactions with the third generation. We explore the phenomenology and discovery reach for these new particles at the LHC, exploring single mirror quark production modes whose rates are proportional to the same mixing parameters which resolve the $A_{FB}^b$ anomaly. We find that for mirror quark masses $\lesssim 500 GeV, a 14 TeV LHC with 300 {\rm fb}^{-1}$ is required to reasonably establish the scenario and extract the relevant mixing parameters.Comment: version to be published in JHE

Gross-Neveu Models, Nonlinear Dirac Equations, Surfaces and Strings

Recent studies of the thermodynamic phase diagrams of the Gross-Neveu model (GN2), and its chiral cousin, the NJL2 model, have shown that there are phases with inhomogeneous crystalline condensates. These (static) condensates can be found analytically because the relevant Hartree-Fock and gap equations can be reduced to the nonlinear Schr\"odinger equation, whose deformations are governed by the mKdV and AKNS integrable hierarchies, respectively. Recently, Thies et al have shown that time-dependent Hartree-Fock solutions describing baryon scattering in the massless GN2 model satisfy the Sinh-Gordon equation, and can be mapped directly to classical string solutions in AdS3. Here we propose a geometric perspective for this result, based on the generalized Weierstrass spinor representation for the embedding of 2d surfaces into 3d spaces, which explains why these well-known integrable systems underlie these various Gross-Neveu gap equations, and why there should be a connection to classical string theory solutions. This geometric viewpoint may be useful for higher dimensional models, where the relevant integrable hierarchies include the Davey-Stewartson and Novikov-Veselov systems.Comment: 27 pages, 1 figur

Thermal Tolerance of the Coffee Berry Borer Hypothenemus hampei: Predictions of Climate Change Impact on a Tropical Insect Pest

Coffee is predicted to be severely affected by climate change. We determined the thermal tolerance of the coffee berry borer , Hypothenemus hampei, the most devastating pest of coffee worldwide, and make inferences on the possible effects of climate change using climatic data from Colombia, Kenya, Tanzania, and Ethiopia. For this, the effect of eight temperature regimes (15, 20, 23, 25, 27, 30, 33 and 35°C) on the bionomics of H. hampei was studied. Successful egg to adult development occurred between 20–30°C. Using linear regression and a modified Logan model, the lower and upper thresholds for development were estimated at 14.9 and 32°C, respectively. In Kenya and Colombia, the number of pest generations per year was considerably and positively correlated with the warming tolerance. Analysing 32 years of climatic data from Jimma (Ethiopia) revealed that before 1984 it was too cold for H. hampei to complete even one generation per year, but thereafter, because of rising temperatures in the area, 1–2 generations per year/coffee season could be completed. Calculated data on warming tolerance and thermal safety margins of H. hampei for the three East African locations showed considerably high variability compared to the Colombian site. The model indicates that for every 1°C rise in thermal optimum (Topt.), the maximum intrinsic rate of increase (rmax) will increase by an average of 8.5%. The effects of climate change on the further range of H. hampei distribution and possible adaption strategies are discussed. Abstracts in Spanish and French are provided as supplementary material Abstract S1 and Abstract S2

Two Pathways Recruit Telomerase to Saccharomyces cerevisiae Telomeres

The catalytic subunit of yeast telomerase, Est2p, is a telomere associated throughout most of the cell cycle, while the Est1p subunit binds only in late S/G2 phase, the time of telomerase action. Est2p binding in G1/early S phase requires a specific interaction between telomerase RNA (TLC1) and Ku80p. Here, we show that in four telomerase-deficient strains (cdc13-2, est1Ä, tlc1-SD, and tlc1-BD), Est2p telomere binding was normal in G1/early S phase but reduced to about 40–50% of wild type levels in late S/G2 phase. Est1p telomere association was low in all four strains. Wild type levels of Est2p telomere binding in late S/G2 phase was Est1p-dependent and required that Est1p be both telomere-bound and associated with a stem-bulge region in TLC1 RNA. In three telomerase-deficient strains in which Est1p is not Est2p-associated (tlc1-SD, tlc1-BD, and est2Ä), Est1p was present at normal levels but its telomere binding was very low. When the G1/early S phase and the late S/G2 phase telomerase recruitment pathways were both disrupted, neither Est2p nor Est1p was telomere-associated. We conclude that reduced levels of Est2p and low Est1p telomere binding in late S/G2 phase correlated with an est phenotype, while a WT level of Est2p binding in G1 was not sufficient to maintain telomeres. In addition, even though Cdc13p and Est1p interact by two hybrid, biochemical and genetic criteria, this interaction did not occur unless Est1p was Est2p-associated, suggesting that Est1p comes to the telomere only as part of the holoenzyme. Finally, the G1 and late S/G2 phase pathways for telomerase recruitment are distinct and are likely the only ones that bring telomerase to telomeres in wild-type cells

Introgression and pyramiding into common bean market class fabada of genes conferring resistance to anthracnose and potyvirus

Anthracnose and bean common mosaic (BCM) are considered major diseases in common bean crop causing severe yield losses worldwide. This work describes the introgression and pyramiding of genes conferring genetic resistance to BCM and anthracnose local races into line A25, a bean genotype classified as market class fabada. Resistant plants were selected using resistance tests or combining resistance tests and marker-assisted selection. Lines A252, A321, A493, Sanilac BC6-Are, and BRB130 were used as resistance sources. Resistance genes to anthracnose (Co-2 ( C ), Co-2 ( A252 ) and Co-3/9) and/or BCM (I and bc-3) were introgressed in line A25 through six parallel backcrossing programs, and six breeding lines showing a fabada seed phenotype were obtained after six backcross generations: line A1258 from A252; A1231 from A321; A1220 from A493; A1183 and A1878 from Sanilac BC6-Are; and line A2418 from BRB130. Pyramiding of different genes were developed using the pedigree method from a single cross between lines obtained in the introgression step: line A1699 (derived from cross A1258 × A1220), A2438 (A1220 × A1183), A2806 (A1878 × A2418), and A3308 (A1699 × A2806). A characterization based on eight morpho-agronomic traits revealed a limited differentiation among the obtained breeding lines and the recurrent line A25. However, using a set of seven molecular markers linked to the loci used in the breeding programs it was possible to differentiate the 11 fabada lines. Considering the genetic control of the resistance in resistant donor lines, the observed segregations in the last backcrossing generation, the reaction against the pathogens, and the expression of the molecular markers it was also possible to infer the genotype conferring resistance in the ten fabada breeding lines obtained. As a result of these breeding programs, genetic resistance to three anthracnose races controlled by genes included in clusters Co-2 and Co-3/9, and genetic resistance to BCM controlled by genotype I + bc-3 was combined in the fabada line A3308

K-Ras Mediated Murine Epidermal Tumorigenesis Is Dependent upon and Associated with Elevated Rac1 Activity

A common goal for potential cancer therapies is the identification of differences in protein expression or activity that would allow for the selective targeting of tumor vs. normal cells. The Ras proto-oncogene family (K-Ras, H-Ras and N-Ras) are amongst the most frequently mutated genes in human cancers. As a result, there has been substantial effort dedicated to determining which pathways are activated by Ras signaling and, more importantly, which of these contribute to cancer. Although the most widely studied Ras-regulated signaling pathway is the Raf/mitogen-activated protein kinase cascade, previous research in model systems has revealed that the Rac1 GTP-binding protein is also required for Ras-induced biological responses. However, what have been lacking are rigorous in vivo Rac1 target validation data and a clear demonstration that in Ras-driven hyperplastic lesions, Rac1 activity is increased. Using a combination of genetically-modified mouse models that allow for the tissue-selective activation or deletion of signaling molecules and an activation-state sensitive Rac1 antibody that detects GTP-bound Rac1, we found that Rac1 contributes to K-Ras induced epidermal papilloma initiation and growth and that Rac1 activity is elevated by oncogenic K-Ras in vivo. Previously, it was not practical to assess Rac1 activation status in the most commonly used format for clinical tumor specimens, formalin-fixed paraffin embedded (FFPE) tissues samples. However, this study clearly demonstrates that Rac1 is essential for K-Ras driven epithelial cell hyperproliferation and that Rac1 activity is elevated in tissues expressing mutant oncogenic K-Ras, while also characterizing the activation-state specific Rac1-GTP antibody as a probe to examine Rac1 activation status in FFPE samples. Our findings will facilitate further research on the status of Rac1 activity in human tumors and will help to define the tumor types of the patient population that could potentially benefit from therapies targeting Rac activation or downstream effector signaling pathways

Immunization with Pre-Erythrocytic Antigen CelTOS from Plasmodium falciparum Elicits Cross-Species Protection against Heterologous Challenge with Plasmodium berghei

BACKGROUND: The Plasmodium protein Cell-traversal protein for ookinetes and sporozoites (CelTOS) plays an important role in cell traversal of host cells in both, mosquito and vertebrates, and is required for successful malaria infections. CelTOS is highly conserved among the Plasmodium species, suggesting an important functional role across all species. Therefore, targeting the immune response to this highly conserved protein and thus potentially interfering with its biological function may result in protection against infection even by heterologous species of Plasmodium. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, we developed a recombinant codon-harmonized P. falciparum CelTOS protein that can be produced to high yields in the E. coli expression system. Inbred Balb/c and outbred CD-1 mice were immunized with various doses of the recombinant protein adjuvanted with Montanide ISA 720 and characterized using in vitro and in vivo analyses. CONCLUSIONS/SIGNIFICANCE: Immunization with PfCelTOS resulted in potent humoral and cellular immune responses and most importantly induced sterile protection against a heterologous challenge with P. berghei sporozoites in a proportion of both inbred and outbred mice. The biological activity of CelTOS-specific antibodies against the malaria parasite is likely linked to the impairment of sporozoite motility and hepatocyte infectivity. The results underscore the potential of this antigen as a pre-erythrocytic vaccine candidate and demonstrate for the first time a malaria vaccine that is cross-protective between species

Vaccinia-Related Kinase 1 Is Required for the Maintenance of Undifferentiated Spermatogonia in Mouse Male Germ Cells

Vaccinia-related kinase 1 (VRK1) is a crucial protein kinase for mitotic regulation. VRK1 is known to play a role in germ cell development, and its deficiency results in sterility. Here we describe that VRK1 is essential for the maintenance of spermatogonial stem cells. To determine whether VRK1 plays a role in these cells, we assessed the population size of undifferentiated spermatogonia. Flow cytometry analyses showed that the number of undifferentiated spermatogonia was markedly reduced in VRK1-deficient testes. VRK1 was highly expressed in spermatogonial populations, and approximately 66% of undifferentiated spermatogonia that were sorted as an Ep-CAM+/c-kit−/alpha-6-integrin+ population showed a positive signal for VRK1. Undifferentiated stem cells expressing Plzf and Oct4 but not c-kit also expressed VRK1, suggesting that VRK1 is an intrinsic factor for the maintenance of spermatogonial stem cells. Microarray analyses of the global testicular transcriptome and quantitative RT-PCR of VRK1-deficient testes revealed significantly reduced expression levels of undifferentiated spermatogonial marker genes in early postnatal mice. Together, these results suggest that VRK1 is required for the proliferation and differentiation of undifferentiated spermatogonia, which are essential for spermatogenic cell maintenance