The changing role of cell culture in the generation of transgenic livestock

Transgenesis may allow the generation of farm animals with altered phenotype, animal models for research and animal bioreactors. Although such animals have been produced, the time and expense involved in generating transgenic livestock and then evaluating the transgene expression pattern is very restrictive. If questions about the ability and efficiency of expression could be asked solely in vitro rapid progress could be achieved. Unfortunately, experiments addressing transcriptional control in vitro have proved unreliable in their ability to indicate whether a transgene will be transcribed or not. However, initial studies suggest that cell culture may be able to predict in vivo post-transcriptional events. We review these issues and propose that strategies which engineer the transgene integration site could enhance the probability for efficient expression. This approach has now become feasible with the development of techniques allowing animals to be generated from somatic cells by nuclear transfer. The important step in this procedure is the use of cells grown in culture as the source of genetic information, allowing the selection of specific transgene integration events. This technology which has dramatically increased the potential use of transgenic livestock for both agricultural and biotechnological applications, is based on standard cell culture methodology. We are now at the start of a new era in large animal transgenics

Velocimetry with refractive index matching for complex flow configurations, phase 1

The feasibility of obtaining detailed velocity field measurements in large Reynolds number flow of the Space Shuttle Main Engine (SSME) main injector bowl was demonstrated using laser velocimetry and the developed refractive-index-matching technique. An experimental system to provide appropriate flow rates and temperature control of refractive-index-matching fluid was designed and tested. Test results are presented to establish the feasibility of obtaining accurate velocity measurements that map the entire field including the flow through the LOX post bundles: sample mean velocity, turbulence intensity, and spectral results are presented. The results indicate that a suitable fluid and control system is feasible for the representation of complex rocket-engine configurations and that measurements of velocity characteristics can be obtained without the optical access restrictions normally associated with laser velocimetry. The refractive-index-matching technique considered needs to be further developed and extended to represent other rocket-engine flows where current methods either cannot measure with adequate accuracy or they fail

Cardiac Specific Gene Expression Changes in Long Term Culture of Murine Mesenchymal Stem Cells

Murine MSCs are a readily available source of adult stem cells enabling extensive in vitro study of this cell population. MSCs have been described as multipotent, and have been proven capable of differentiation into several connective tissue types. Furthermore some studies have suggested an ability to differentiate into non-connective tissue cell types such as the cardiomyocyte. The aim of this study was to differentiate murine MSCs toward cardiac lineage with the commonly used method of culture with 5’ Azacytidine. Critically, baseline analysis of gene expression of passage four MSCs demonstrated expression of key cardiac markers including cardiac troponin T and I, and the ryanodine receptor. Furthermore, expression analysis of these genes changed with time in culture and passage number. However, there was no significant alteration when cells were subjected to a differentiation protocol. This study therefore highlights the importance of analyzing baseline cells extensively, and indicates the limitations in extrapolating data for comparison between species. Furthermore this data brings into question the efficacy of cardiac differentiation using MSCs

The role of the basolateral amygdala in cocaine self-administration and cocaine-seeking behaviour

The experiments reported in this thesis have investigated the role of the basolateral amygdala (BLA) in the process by which conditioned stimuli (CS) acquire motivational salience and, as conditioned reinforcers, direct cocaine-seeking behaviour in the rat. Excitotoxic lesions of the BLA did not interfere with the reinforcing effects of cocaine in rats. Intra-peritoneal injections of cocaine produced similar locomotor responses in both lesioned and control animals and both groups also produced equivalent dose-response functions during a within- session dose-response test. Similarly, lesioned and control animals acquired cocaine self-administration under both continuous and progressive-ratio schedules of reinforcement. However, BLA-lesioned animals were (i) severely impaired in the acquisition of second-order schedules of cocaine self-administration; (ii) more sensitive than control animals to reductions in drug dose under a progressive-ratio schedule of cocaine self-administration and (iii) less sensitive than control animals to the omission of a drug-related CS, under a fixed-interval schedule of selfadministration. In vivo microdialysis showed that lesions of the BLA were associated with an impaired glutamatergic response to intra-nucleus accumbens infusions of cocaine, but that the dopaminergic response of lesioned and control animals of were identical. These findings suggest that drug-seeking behaviour in rats with lesions of the BLA is influenced more by the primary reinforcer and concomitantly less by secondary, conditioned reinforcers. This would indicate that the BLA is significantly involved in the development of cue-elicited drug-seeking behaviour and, by inference, this structure may also play an important role in the development of problem drug-use in humans

Susceptibility of Vitis vinifera 'Semillon' and 'Chardonnay' to the root-knot nematode Meloidogyne javanica

A study to assess the effect of the initial population (Pi) densities (0, 200, 400, 600 and 800 second stage juveniles (J2) kg-1 dry soil) of the root knot nematode, Meloidogyne javanica, on the growth, yield and juice characteristics of two white wine grapevine (Vitis vinifera) cvs. 'Semillon' and 'Chardonnay' was conducted in a vineyard located at the Centre for Irrigated Agriculture, Riverina, NSW, Australia. M. javanica J2 population densities in soil after harvest during 2004-2008 growing seasons increased gradually, year by year, and in most cases were higher where the initial densities were higher. Regression analysis revealed that yield, in general, was reduced significantly with the increase of the nematode population densities·kg-1 soil for both cultivars. After six years, the nematode population had increased by ca. 9.0-22.4 fold for 'Semillon' and 6.7-18.5 fold for 'Chardonnay'. All Pi densities significantly reduced Semillon yields in all years but only the highest level (800 J2·kg-1 dry soil) affected 'Chardonnay' yields. At the end of the experiment, M. javanica decreased yields by 15-20 % in Semillon but only 7-13 % in 'Chardonnay'. The nematode inoculation also caused a decrease in bunch numbers in 'Semillon' but not in 'Chardonnay'. This is the first study showing that 'Chardonnay' is less susceptible to M. javanica than 'Semillon'.

Coupled genomic evolutionary histories as signatures of organismal innovations in cephalopods: co-evolutionary signatures across levels of genome organization may shed light on functional linkage and origin of cephalopod novelties

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Ritschard, E. A., Whitelaw, B., Albertin, C. B., Cooke, I. R., Strugnell, J. M., & Simakov, O. Coupled genomic evolutionary histories as signatures of organismal innovations in cephalopods: co-evolutionary signatures across levels of genome organization may shed light on functional linkage and origin of cephalopod novelties. BioEssays, 41, (2019): 1900073, doi: 10.1002/bies.201900073.How genomic innovation translates into organismal organization remains largely unanswered. Possessing the largest invertebrate nervous system, in conjunction with many species‐specific organs, coleoid cephalopods (octopuses, squids, cuttlefishes) provide exciting model systems to investigate how organismal novelties evolve. However, dissecting these processes requires novel approaches that enable deeper interrogation of genome evolution. Here, the existence of specific sets of genomic co‐evolutionary signatures between expanded gene families, genome reorganization, and novel genes is posited. It is reasoned that their co‐evolution has contributed to the complex organization of cephalopod nervous systems and the emergence of ecologically unique organs. In the course of reviewing this field, how the first cephalopod genomic studies have begun to shed light on the molecular underpinnings of morphological novelty is illustrated and their impact on directing future research is described. It is argued that the application and evolutionary profiling of evolutionary signatures from these studies will help identify and dissect the organismal principles of cephalopod innovations. By providing specific examples, the implications of this approach both within and beyond cephalopod biology are discussed.E.A.R. and O.S. are supported by the Austrian Science Fund (Grant No. P30686‐B29). E.A.R. is supported by Stazione Zoologica Anton Dohrn (Naples, Italy) PhD Program. The authors wish to thank Graziano Fiorito (SZN, Italy), Hannah Schmidbaur (University of Vienna, Austria), Thomas Hummel (University of Vienna, Austria) for many insightful comments and reading of the draft manuscript. The authors would like to apologize to all colleagues whose work has been omitted due to space constraints

High-Density Genetic Linkage Map of the Southern Blue-ringed Octopus (Octopodidae: Hapalochlaena maculosa)

Genetic linkage maps provide a useful resource for non-model genomes and can aid in genome reassembly to form more contiguous pseudo-chromosomes. We present the first linkage map of any cephalopod, H. maculosa, composed of 47 linkage groups (LG). A total of 2166 single nucleotide polymorphisms and 2455 presence–absence variant loci were utilised by Lep-Map3 in linkage map construction. The map length spans 2016.62 cM with an average marker distance of 0.85 cM. Integration of the recent H. maculosa genome allowed 1151 scaffolds comprising 34% of the total genomic sequence to be orientated and/or placed using 1278 markers across all 47 LG. The linkage map generated provides a new perspective on HOX gene distribution in octopods. In the H. maculosa linkage map three (SCR, LOX4 and POST1) of six identified HOX genes (HOX1/LAB, SCR, LOX2, LOX4, LOX5, POST1) were located within the same LG (LG 9). The generation of a linkage map for H. maculosa has provided a valuable resource for understanding the evolution of cephalopod genomes and will provide a base for future work

ICU-Associated Acinetobacter baumannii Colonisation/Infection in a High HIV-Prevalence Resource-Poor Setting

BACKGROUND: There are hardly any data about the incidence, risk factors and outcomes of ICU-associated A.baumannii colonisation/infection in HIV-infected and uninfected persons from resource-poor settings like Africa. METHODS: We reviewed the case records of patients with A.baumannii colonisation/infection admitted into the adult respiratory and surgical ICUs in Cape Town, South Africa, from January 1 to December 31 2008. In contrast to colonisation, infection was defined as isolation of A.baumannii from any biological site in conjunction with a compatible clinical picture warranting treatment with antibiotics effective against A.baumannii . RESULTS: The incidence of A.baumannii colonisation/infection in 268 patients was 15 per 100 person-years, with an in-ICU mortality of 26.5 per 100 person-years. The average length of stay in ICU was 15 days (range 1-150). A.baumannii was most commonly isolated from the respiratory tract followed by the bloodstream. Independent predictors of mortality included older age (p = 0.02), low CD4 count if HIV-infected (p = 0.038), surgical intervention (p = 0.047), co-morbid Gram-negative sepsis (p = 0.01), high APACHE-II score (p = 0.001), multi-organ dysfunction syndrome (p = 0.012), and a positive blood culture for A.baumannii (p = 0.017). Of 21 A.baumannii colonised/infected HIV-positive persons those with clinical AIDS (CD4<200 cells/mm 3 ) had significantly higher in-ICU mortality and were more likely to have a positive blood culture. Conclusion In this resource-poor setting A.baumannii infection in critically ill patients is common and associated with high mortality. HIV co-infected patients with advanced immunosuppression are at higher risk of death