An Optimized Protocol for Molecular Screening of Avian Pathogenic Escherichia Coli From Broiler Chickens in South East Queensland, Australia

Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colibacillosis and causes localized and/or systemic infections in poultry. The presence of various virulence genes (VGs) may be a useful marker for the detection of APEC directly from fecal samples. The objectives of this study were to evaluate and compare 3 different DNA extraction methods from cloacal swabs and fecal samples of broiler chickens and determine if APEC can be detected directly from feces. The DNA extraction methods were assessed by measuring DNA yield and purity, absence of DNA shearing, 16S ribosomal DNA amplification, and reproducibility. Repeated bead beating plus column (RBB+C) was the preferred extraction method, as it yielded an adequate amount of quality DNA for PCR directly from feces. The DNA extracted from feces, with RBB+C method and DNA extracted from E. coli isolates of organs and feces, taken from 23 broiler chickens (10 healthy, 9 with colibacillosis, and 4 unhealthy with other infections), were screened with a pentaplex-PCR for the prevalence of APEC-associated VGs: iroN, ompT, iutA, iss, and hlyF. There was a statistically significant correlation between the presence of the 5 VGs in E. coli cultured from the cloaca, fecal, and organs samples from chicken affected with colibacillosis. However, screening extracted DNA from the feces for the selected VGs was not an effective diagnostic tool to detect APEC as all of the VGs were detected in the extracted fecal DNA from all chickens

Holograms to Focus Arbitrary Ultrasonic Fields through the Skull

[EN] We report 3D-printed acoustic holographic lenses for the formation of ultrasonic fields of complex spatial distribution inside the skull. Using holographic lenses, we experimentally, numerically and theoretically produce acoustic beams whose spatial distribution matches target structures of the central nervous system. In particular, we produce three types of targets of increasing complexity. First, a set of points are selected at the center of both right and left human hippocampi. Experiments using a skull phantom and 3D printed acoustic holographic lenses show that the corresponding bi-focal lens simultaneously focuses acoustic energy at the target foci, with good agreement between theory and simulations. Second, an arbitrary curve is set as the target inside the skull phantom. Using time-reversal methods the holographic beam bends following the target path, in a similar way as self-bending beams do in free space. Finally, the right human hippocampus is selected as a target volume. The focus of the corresponding holographic lens overlaps with the target volume in excellent agreement between theory in free-media, and experiments and simulations including the skull phantom. The precise control of focused ultrasound into the central nervous system is mainly limited due to the strong phase aberrations produced by refraction and attenuation of the skull. Using the present method, the ultrasonic beam can be focused not only at a single point but overlapping one or various target structures simultaneously using low-cost 3D-printed acoustic holographic lens. The results open new paths to spread incoming biomedical ultrasound applications including blood-brain barrier opening and neuromodulation.This work is supported by the Spanish Ministry of Economy and Innovation (MINECO) through Project No. TEC2016-80976-R. N.J. and S.J. acknowledge financial support from Generalitat Valenciana through Grants No. APOSTD/2017/042, No. ACIF/2017/045, and No. GV/2018/11. F.C. acknowledges financial support from Agencia Valenciana de la Innovacio through Grant No. INNCON00/18/9 and European Regional Development Fund (Grant No. IDIFEDER/2018/022).Jiménez-Gambín, S.; Jimenez, N.; Benlloch Baviera, JM.; Camarena Femenia, F. (2019). Holograms to Focus Arbitrary Ultrasonic Fields through the Skull. Physical Review Applied. 12(1):014016-1-014016-14. https://doi.org/10.1103/PhysRevApplied.12.014016S014016-1014016-14121GABOR, D. (1948). A New Microscopic Principle. Nature, 161(4098), 777-778. doi:10.1038/161777a0Microscopy by reconstructed wave-fronts. (1949). Proceedings of the Royal Society of London. Series A. Mathematical and Physical Sciences, 197(1051), 454-487. doi:10.1098/rspa.1949.0075Leith, E. N., & Upatnieks, J. (1962). Reconstructed Wavefronts and Communication Theory*. 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Class I major histocompatibility complexes loaded by a periodate trigger

Class I major histocompatibility complexes (MHCs) present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. The unstable nature of unliganded MHC necessitates the production of recombinant class I complexes through in vitro refolding reactions in the presence of an added excess of peptides. This strategy is not amenable to high-throughput production of vast collections of class I complexes. To address this issue, we recently designed photocaged MHC ligands that can be cleaved by a UV light trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure. The results obtained with photocaged MHC ligands demonstrate that conditional MHC ligands can form a generally applicable concept for the creation of defined peptide−MHCs. However, the use of UV exposure to mediate ligand exchange is unsuited for a number of applications, due to the lack of UV penetration through cell culture systems and due to the transfer of heat upon UV irradiation, which can induce evaporation. To overcome these limitations, here, we provide proof-of-concept for the generation of defined peptide−MHCs by chemical trigger-induced ligand exchange. The crystal structure of the MHC with the novel chemosensitive ligand showcases that the ligand occupies the expected binding site, in a conformation where the hydroxyl groups should be reactive to periodate. We proceed to validate this technology by producing peptide−MHCs that can be used for T cell detection. The methodology that we describe here should allow loading of MHCs with defined peptides in cell culture devices, thereby permitting antigen-specific T cell expansion and purification for cell therapy. In addition, this technology will be useful to develop miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands

Phosphoenolpyruvate carboxylase dentified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in thePlasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C,15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery

Spatio-temporal Models of Lymphangiogenesis in Wound Healing

Several studies suggest that one possible cause of impaired wound healing is failed or insufficient lymphangiogenesis, that is the formation of new lymphatic capillaries. Although many mathematical models have been developed to describe the formation of blood capillaries (angiogenesis), very few have been proposed for the regeneration of the lymphatic network. Lymphangiogenesis is a markedly different process from angiogenesis, occurring at different times and in response to different chemical stimuli. Two main hypotheses have been proposed: 1) lymphatic capillaries sprout from existing interrupted ones at the edge of the wound in analogy to the blood angiogenesis case; 2) lymphatic endothelial cells first pool in the wound region following the lymph flow and then, once sufficiently populated, start to form a network. Here we present two PDE models describing lymphangiogenesis according to these two different hypotheses. Further, we include the effect of advection due to interstitial flow and lymph flow coming from open capillaries. The variables represent different cell densities and growth factor concentrations, and where possible the parameters are estimated from biological data. The models are then solved numerically and the results are compared with the available biological literature.Comment: 29 pages, 9 Figures, 6 Tables (39 figure files in total