A Drosophila Model for Clostridium difficile Toxin CDT Reveals Interactions with Multiple Effector Pathways.

Clostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. We created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways in vivo. When expressed in the midgut, CDTa reduces body weight and fecal output and compromises survival, suggesting severe impairment of digestive functions. At the cellular level, CDTa induces F-actin network collapse, elimination of the intestinal brush border, and disruption of intercellular junctions. We confirm toxin-dependent re-distribution of Rab11 to enterocytes' apical surface and observe suppression of CDTa phenotypes by a Dominant-Negative form of Rab11 or RNAi of the dedicated Rab11GEF Crag (DENND4). We also report that Calmodulin (Cam) is required to mediate CDTa activity. In parallel, chemical inhibition of the Cam/Calcineurin pathway by Cyclosporin A or FK506 also reduces CDTa phenotypes, potentially opening new avenues for treating CDIs

Low-Voltage High-Linearity Wideband Current Differencing Transconductance Amplifier and Its Application on Current-Mode Active Filter

A low-voltage high-linearity wideband current differencing transconductance amplifier (CDTA) is presented in this paper. The CDTA consists of a current differencing circuit and a cross-coupling transconductance circuit. The PSPICE simulations of the proposed CDTA show a good performance: -3dB frequency bandwith is about 900 MHz, low power consumption is 2.48 mW, input current linear range is ±100 µA and low current-input resistance is less than 20 Ω, high current-output resistance is more than 3 MΩ. PSpice simulations for a current-mode universal filter and a proposed high-order filter are also conducted, and the results verify the validity of the proposed CDTA

A wideband linear tunable CDTA and its application in field programmable analogue array

This document is the Accepted Manuscript version of the following article: Hu, Z., Wang, C., Sun, J. et al. ‘A wideband linear tunable CDTA and its application in field programmable analogue array’, Analog Integrated Circuits and Signal Processing, Vol. 88 (3): 465-483, September 2016. Under embargo. Embargo end date: 6 June 2017. The final publication is available at Springer via https://link.springer.com/article/10.1007%2Fs10470-016-0772-7 © Springer Science+Business Media New York 2016In this paper, a NMOS-based wideband low power and linear tunable transconductance current differencing transconductance amplifier (CDTA) is presented. Based on the NMOS CDTA, a novel simple and easily reconfigurable configurable analogue block (CAB) is designed. Moreover, using the novel CAB, a simple and versatile butterfly-shaped FPAA structure is introduced. The FPAA consists of six identical CABs, and it could realize six order current-mode low pass filter, second order current-mode universal filter, current-mode quadrature oscillator, current-mode multi-phase oscillator and current-mode multiplier for analog signal processing. The Cadence IC Design Tools 5.1.41 post-layout simulation and measurement results are included to confirm the theory.Peer reviewedFinal Accepted Versio

Cytolethal distending toxin (CDT)-negative Campylobacter jejuni strains and anti-CDT neutralising antibodies induced during human infection but not chicken colonisation

The cytolethal distending toxin (CDT) of Campylobacter jejuni was detectable, using an in vitro assay, in most but not all of 24 strains tested. The reason for the absence of toxin activity in these naturally occurring CDT-negative C. jejuni strains was then investigated at the genetic level. CDT is encoded by three highly conserved genes, cdtA, -B, and -C. In the CDT-negative strains, two types of mutation were identified. The CDT activities of C. jejuni strains possessing both types of mutation were successfully complemented with the functional genes of C. jejuni 11168. The first type of mutation comprised a 667-bp deletion across cdtA and cdtB and considerable degeneration in the remainder of the cdt locus. Using a PCR technique to screen for this deletion, this mutation occurred in fewer than 3% of 147 human, veterinary, and environmental strains tested. The second type of mutation involved at least four nonsynonymous nucleotide changes, but only the replacement of proline with serine at CdtB position 95 was considered important for CDT activity. This was confirmed by site-directed mutagenesis. This type of mutation also occurred in fewer than 3% of strains as determined using a LightCycler biprobe assay. The detection of two CDT-negative clinical isolates raised questions about the role of CDT in some cases of human campylobacteriosis. To determine if anti-CDT antibodies are produced in human infection, a toxin neutralization assay was developed and validated using rabbit antisera. Pooled human sera from infected patients neutralized the toxin, indicating expression and immunogenicity during infection. However, no neutralizing antibodies were detected in colonized chickens despite the expression of CDT in the avian gut as indicated by reverse transcription-PCR

Realization of Resistorless Lossless Positive and Negative Grounded Inductor Simulators Using Single ZC-CCCITA

This paper is in continuation with the very recent work of Prasad et al. [14], wherein new realizations of grounded and floating positive inductor simulator using current differencing transconductance amplifier (CDTA) are reported. The focus of the paper is to provide alternate realizations of lossless, both positive and negative inductor simulators (PIS and NIS) in grounded form using z-copy current-controlled current inverting transconductance amplifier (ZC-CCCITA), which can be considered as a derivative of CDTA, wherein the current differencing unit (CDU) is reduced to a current-controlled current inverting unit. We demonstrate that only a single ZC-CCCITA and one grounded capacitor are sufficient to realize grounded lossless PIS or NIS. The proposed circuits are resistorless whose parameters can be controlled through the bias currents. The workability of the proposed PIS is validated by SPICE simulations on three RLC prototypes

Let\u27s Ride the Bus: Reverse-Commute Challenges Facing Low-Income Inner City Residents of Onondaga County (2009 Report)

This is the second and final phase of our investigation into the reverse-commute challenges facing low-income inner-city residents in Onondaga County. With lower-wage jobs proliferating outside the city core, our findings confirm that transportation remains one of the greatest obstacles to landing and keeping entry-level work. The current transit system does not meet the needs of low-income workers living in the city or employers based in outlying neighborhoods or the suburbs. Although a majority of manufacturing employers contacted for this study said transportation shortfalls do not affect their ability to hire and retain workers, other stakeholders jobseekers, job developers, service providers, county planners, and transit professionals—insist the problem is real: Jobseekers with few skills and limited access to transportation struggle to find employment while employers in other key sectors, notably hospitality and health services, contend with the consequences in the form of high turnover, tardiness, absences, and vacancies, as noted in our 2008 report, Catch That Bus... Inadequacies in the local transit system will affect the county\u27s longer-term economic vitality. Current concerns about air pollution, environmental conservation, energy costs, and strained municipal budgets add to the urgency of addressing the interrelated issues of employment, transportation, economic development, and sprawl. Collaboration among key stakeholders—the County, Centro, employers, private transit operators, service providers, and town boards--is necessary to advance the parties\u27 mutual interests

Nanostructural changes in cell wall pectins during strawberry fruit ripening assessed by atomic force microscopy

Rapid loss of firmness occurs during strawberry (Fragaria × ananassa Duch) ripening, resulting in a short shelf life and high economic losses. The disassembly of cell walls is considered the main responsible for fruit softening, being pectins extensively modified during strawberry ripening (Paniagua et al. 2014). Atomic force microscopy allows the analysis of individual polymer chains at nanostructural level with a minimal sample preparation (Morris et al., 2001). The main objective of this research was to compare pectins of green and red ripe strawberry fruits at the nanostructural level to shed light on structural changes that could be related to softening. Cell walls from strawberry fruits were extracted and fractionated with different solvents to obtain fractions enriched in a specific component. The yield of cell wall material, as well as the amount of the different fractions, decreased in ripe fruits. CDTA and Na2CO3 fractions underwent the largest decrements, being these fractions enriched in pectins supposedly located in the middle lamella and primary cell wall, respectively. Uronic acid content also decreased significantly during ripening in both pectin fractions, but the amount of soluble pectins, those extracted with phenol:acetic acid:water (PAW) and water increased in ripe fruits. Monosaccharide composition in CDTA and Na2CO3 fractions was determined by gas chromatography. In both pectin fractions, the amount of Ara and Gal, the two most abundant carbohydrates, decreased in ripe fruits. The nanostructural characteristics of CDTA and Na2CO3 pectins were analyzed by AFM. Isolated pectic chains present in the CDTA fraction were significantly longer and more branched in samples from green fruits than those present in samples obtained from red fruit. In spite of slight differences in length distributions, Na2CO3 samples from unripe fruits displayed some longer chains at low frequency that were not detected in ripe fruits. Pectin aggregates were more frequently observed in green fruit samples from both fractions. These results support that pectic chain length and the nanostructural complexity of the pectins present in CDTA and Na2CO3 fractions diminish during strawberry fruit development, and these changes, jointly with the loss of neutral sugars, could contribute to the solubilization of pectins and fruit softening. Paniagua et al. (2014). Ann Bot, 114: 1375-1383 Morris et al. (2001). Food Sci Tech 34: 3-10 This research was supported by FEDER EU Funds and the Ministerio de Educación y Ciencia of Spain (grant reference AGL2011-24814)Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Entwicklung stabiler Mn-Komplexe für die multimodale MR/PET-Bildgebung

Different derivatives of the polyaminocarboxylate ligand $\textit{trans}$-cyclohexyldiaminotetraacetic acid(CDTA) were synthesized to evaluate isotopically radiolabelled manganese-based bimodal contrast agents for hybrid positron emission tomography/magnetic resonance (PET/MR) imaging. First, a bifunctional chelating agent precursor was synthesized in five steps, which can be coupled to a small and rigid central scaffold to obtain a multimeric manganese (II) chelating unit. For this purpose, phosphazene, adamantane, triazine and benzene scaffolds were evaluated. With phosphazene, adamantane and triazine, the synthesis of multimeric ligands was unsuccessful. When trisacetylenebenzene derivatives were used as central units, 1,3-dipolar cycloaddition with the azido-functionalized prochelator allowed obtaining a valeric acid functionalized and an unfunctionalised tristriazole-CDTA ligand (TTB-(CDTA)$_{3}$). Radiolabelling of these ligands with the positron emitting nuclide manganese-52g was first tested using commercially available CDTA, which could be successfully labelled within 30 min at room temperature. Using the non-active reference compound, a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method was established for purification and identification of the radioactive compound [$^{52g}$Mn][Mn(CDTA)]$^{-2}$. The purified complex was incubated in human blood serum to quantify the dissociation behaviour of the radiocomplex under $\textit{in vivo}$ conditions. Size-Exclusion-HPLC allowed the determination of t$_{Diss1/2}$ = 12 h at 37 °C. Applying the established radiolabelling protocol, the TTB-(CDTA)$_{3}$-structures were successfully radiolabelled under the same conditions as before. A suitable HPLC purification method was developed using the inactive reference compounds. Relaxivity measurements performed on the unfunctionalized,trimeric manganese-CDTA-complex showed that coupling of three paramagnetic centers to asmall and rigid scaffold significantly enhances the longitudinal relaxivity (r$_{1}$ = 8.8 mmol$^{-1}$s$^{-1}$ at 20 MHz, 25 °C) as compared to unfunctionalised manganese-CDTA. Having also synthesized a valeric acid functionalised trimeric CDTA-ligand, isotopically radiolabelled manganese-based bimodal PET/MR probes with high relaxivity, stability and possible targeting properties are within reach