Development of Shuttle Vectors for Transformation of Diverse Rickettsia Species

Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 – 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae

Towards pain-free diagnosis of skin diseases through multiplexed microneedles: biomarker extraction and detection using a highly sensitive blotting method

Immunodiagnostic microneedles provide a novel way to extract protein biomarkers from the skin in a minimally invasive manner for analysis in vitro. The technology could overcome challenges in biomarker analysis specifically in solid tissue, which currently often involves invasive biopsies. This study describes the development of a multiplex immunodiagnostic device incorporating mechanisms to detect multiple antigens simultaneously, as well as internal assay controls for result validation. A novel detection method is also proposed. It enables signal detection specifically at microneedle tips and therefore may aid the construction of depth profiles of skin biomarkers. The detection method can be coupled with computerised densitometry for signal quantitation. The antigen specificity, sensitivity and functional stability of the device were assessed against a number of model biomarkers. Detection and analysis of endogenous antigens (interleukins 1α and 6) from the skin using the device was demonstrated. The results were verified using conventional enzyme-linked immunosorbent assays. The detection limit of the microneedle device, at ≤10 pg/mL, was at least comparable to conventional plate-based solid-phase enzyme immunoassays

Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes

<p>Abstract</p> <p>Background</p> <p>Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered.</p> <p>Results</p> <p>Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching.</p> <p>Conclusions</p> <p>We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (α-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this technique promises to further increase the general usability of photoconvertible PAFPs to track the dynamic movement of cells and proteins over time.</p

Trophic Shifts of a Generalist Consumer in Response to Resource Pulses

Trophic shifts of generalist consumers can have broad food-web and biodiversity consequences through altered trophic flows and vertical diversity. Previous studies have used trophic shifts as indicators of food-web responses to perturbations, such as species invasion, and spatial or temporal subsidies. Resource pulses, as a form of temporal subsidies, have been found to be quite common among various ecosystems, affecting organisms at multiple trophic levels. Although diet switching of generalist consumers in response to resource pulses is well documented, few studies have examined if the switch involves trophic shifts, and if so, the directions and magnitudes of the shifts. In this study, we used stable carbon and nitrogen isotopes with a Bayesian multi-source mixing model to estimate proportional contributions of three trophic groups (i.e. producer, consumer, and fungus-detritivore) to the diets of the White-footed mouse (Peromyscus leucopus) receiving an artificial seed pulse or a naturally-occurring cicadas pulse. Our results demonstrated that resource pulses can drive trophic shifts in the mice. Specifically, the producer contribution to the mouse diets was increased by 32% with the seed pulse at both sites examined. The consumer contribution to the mouse diets was also increased by 29% with the cicadas pulse in one of the two grids examined. However, the pattern was reversed in the second grid, with a 13% decrease in the consumer contribution with the cicadas pulse. These findings suggest that generalist consumers may play different functional roles in food webs under perturbations of resource pulses. This study provides one of the few highly quantitative descriptions on dietary and trophic shifts of a key consumer in forest food webs, which may help future studies to form specific predictions on changes in trophic interactions following resource pulses

Docking of LDCVs Is Modulated by Lower Intracellular [Ca2+] than Priming

Many regulatory steps precede final membrane fusion in neuroendocrine cells. Some parts of this preparatory cascade, including fusion and priming, are dependent on the intracellular Ca2+ concentration ([Ca2+]i). However, the functional implications of [Ca2+]i in the regulation of docking remain elusive and controversial due to an inability to determine the modulatory effect of [Ca2+]i. Using a combination of TIRF-microscopy and electrophysiology we followed the movement of large dense core vesicles (LDCVs) close to the plasma membrane, simultaneously measuring membrane capacitance and [Ca2+]i. We found that a free [Ca2+]i of 700 nM maximized the immediately releasable pool and minimized the lateral mobility of vesicles, which is consistent with a maximal increase of the pool size of primed LDCVs. The parameters that reflect docking, i.e. axial mobility and the fraction of LDCVs residing at the plasma membrane for less than 5 seconds, were strongly decreased at a free [Ca2+]i of 500 nM. These results provide the first evidence that docking and priming occur at different free intracellular Ca2+ concentrations, with docking efficiency being the most robust at 500 nM

Single-Cell Profiling Reveals the Origin of Phenotypic Variability in Adipogenesis

Phenotypic heterogeneity in a clonal cell population is a well-observed but poorly understood phenomenon. Here, a single-cell approach is employed to investigate non-mutative causes of phenotypic heterogeneity during the differentiation of 3T3-L1 cells into fat cells. Using coherent anti-Stokes Raman scattering microscopy and flow cytometry, adipogenic gene expression, insulin signaling, and glucose import are visualized simultaneously with lipid droplet accumulation in single cells. Expression of adipogenic genes PPARγ, C/EBPα, aP2, LP2 suggests a commitment to fat cell differentiation in all cells. However, the lack of lipid droplet in many differentiating cells suggests adipogenic gene expression is insufficient for lipid droplet formation. Instead, cell-to-cell variability in lipid droplet formation is dependent on the cascade responses of an insulin signaling pathway which includes insulin sensitivity, kinase activity, glucose import, expression of an insulin degradation enzyme, and insulin degradation rate. Increased and prolonged insulin stimulation promotes lipid droplet accumulation in all differentiating cells. Single-cell profiling reveals the kinetics of an insulin signaling cascade as the origin of phenotypic variability in drug-inducible adipogenesis

Infestation of Transgenic Powdery Mildew-Resistant Wheat by Naturally Occurring Insect Herbivores under Different Environmental Conditions

A concern associated with the growing of genetically modified (GM) crops is that they could adversely affect non-target organisms. We assessed the impact of several transgenic powdery mildew-resistant spring wheat lines on insect herbivores. The GM lines carried either the Pm3b gene from hexaploid wheat, which confers race-specific resistance to powdery mildew, or the less specific anti-fungal barley seed chitinase and β-1,3-glucanase. In addition to the non-transformed control lines, several conventional spring wheat varieties and barley and triticale were included for comparison. During two consecutive growing seasons, powdery mildew infection and the abundance of and damage by naturally occurring herbivores were estimated under semi-field conditions in a convertible glasshouse and in the field. Mildew was reduced on the Pm3b-transgenic lines but not on the chitinase/glucanase-expressing lines. Abundance of aphids was negatively correlated with powdery mildew in the convertible glasshouse, with Pm3b wheat plants hosting significantly more aphids than their mildew-susceptible controls. In contrast, aphid densities did not differ between GM plants and their non-transformed controls in the field, probably because of low mildew and aphid pressure at this location. Likewise, the GM wheat lines did not affect the abundance of or damage by the herbivores Oulema melanopus (L.) and Chlorops pumilionis Bjerk. Although a previous study has revealed that some of the GM wheat lines show pleiotropic effects under field conditions, their effect on herbivorous insects appears to be low

Mouse Apolipoprotein B Editing Complex 3 (APOBEC3) Is Expressed in Germ Cells and Interacts with Dead-End (DND1)

encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites.The 3′-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development

Removal of Hepatitis C Virus-Infected Cells by a Zymogenized Bacterial Toxin

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named “zymoxins”. These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the “first generation zymoxins” by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases