TT2013 meeting report: the transgenic technology meeting visits Asia for the first time

The 11th Transgenic Technology meeting was held in Guangzhou, China on 25th–27th February 2013. Over 300 scientists and students from round the world gathered to hear the latest developments in the technologies underpinning the creation of transgenic and knockout animals and their application to biological sciences in areas such as the modeling human diseases and biotechnology. As well as informative presentations from leading researchers in the field, an excellent selection of short talks selected from abstracts and posters, attendees were also treated to an inspiring talk from Allan Bradley who was awarded the 9th International Society of Transgenic Technologies Prize for outstanding contributions to the field of transgenic technologies

Does angiotensin-1 converting enzyme genotype influence motor or cognitive development after pre-term birth?

BACKGROUND: Raised activity of the renin-angiotensin system (RAS) may both amplify inflammatory and free radical responses and decrease tissue metabolic efficiency and thus enhance cerebral injury in the preterm infant. The angiotensin-converting enzyme (ACE) DD genotype is associated with raised ACE and RAS activity as well as potentially adverse stimuli such as inflammation. The DD genotype has been associated with neurological impairments in the elderly, and thus may be also associated with poorer motor or cognitive development amongst children born preterm prematurely. METHODS: The association of DD genotype with developmental progress amongst 176 Caucasian children born at less than 33 weeks gestation (median birthweight 1475 g, range 645–2480 g; gestation 30 weeks, range 22–32; 108 male) was examined at 2 and 5 1/2 years of age. Measured neuro-cognitive outcomes were cranial ultrasound abnormalities, cerebral palsy, disability, Griffiths Developmental Quotient [DQ] at 2 yrs, and General Cognitive Ability [British Ability Scales-11] and motor performance [ABC Movement], both performed at 5 1/2 yrs. All outcomes were correlated with ACE genotype. RESULTS: The DD genotype was not associated with lower developmental quotients even after accounting for important social variables. CONCLUSION: These data do not support either a role for ACE in the development of cognitive or motor function in surviving infants born preterm or inhibition of ACE as a neuroprotective therapy

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 inhibition of staphylococcal delta-haemolysin by human serum

It is well established that staphylococcal delta-haemolysin is inhibited by normal mammalian sera and forms a precipitin line on gel diffusion with normal sera. The purpose of this thesis was to quantitate inhibitory activity against delta-haemolysin and to identify the serum factor(s) involved. The inhibitory titre of normal human serum assayed using purified delta-haemolysin with cod erythrocytes as indicators of haemolysis was 1/500 - 1/1000. Fractionation of serum by ultrafiltration, gel filtration in Sephadex G-150 and Sepharose 6b and ammonium sulphate precipitation indicated that at least two components of molecular weights approximately 300,000 and > 106 were involved. When affinity chromatography was done using delta-haemolysin covalently coupled to Sepharose-4B the selective removal of g-lipoprotein from whole serum was shown by immunoelectrophoresis. High density (alpha-) and low density (beta-) lipoproteins were prepared by zonal ultracentrifugation of serum using potassium bromide density gradients and characterised by polyacrylamide gel electrophoresis, immunoelectrophoresis and gel diffusion using monospecific antisera. Both alpha- and beta-lipoproteins inhibited delta-haemolysin and gave a precipitin line on gel diffusion, while fractions which were shown by immunoelectrophoresis to be devoid of such lipoproteins were non-inhibitory. Proteolytic digestion of lipoproteins with trypsin or papain did not diminish their inhibitory activity, whereas this was diminished by pretreatment with Bacillus cereus phospholipase C. Delta-haemolysin was inhibited hy phospholipids and pretreatment with phospholipase decreased the inhibitory capacity of such phospholipids and also of a serum lipid extract. The results indicated that binding of delta-haemolysin to serum lipoproteins was via the phospholipid moiety and the implications of such an interaction in terms of in vivo activity of the haemolysin are discussed

Human variants in the Neuronal Basic Helix-Loop-Helix/ Per-Arnt-Sim (bHLH/PAS) transcription factor complex NPAS4/ARNT2 disrupt function

Neuronal Per-Arnt-Sim homology (PAS) Factor 4 (NPAS4) is a neuronal activity-dependent transcription factor which heterodimerises with ARNT2 to regulate genes involved in inhibitory synapse formation. NPAS4 functions to maintain excitatory/inhibitory balance in neurons, while mouse models have shown it to play roles in memory formation, social interaction and neurodegeneration. NPAS4 has therefore been implicated in a number of neuropsychiatric or neurodegenerative diseases which are underpinned by defects in excitatory/inhibitory balance. Here we have explored a broad set of non-synonymous human variants in NPAS4 and ARNT2 for disruption of NPAS4 function. We found two variants in NPAS4 (F147S and E257K) and two variants in ARNT2 (R46W and R107H) which significantly reduced transcriptional activity of the heterodimer on a luciferase reporter gene. Furthermore, we found that NPAS4.F147S was unable to activate expression of the NPAS4 target gene BDNF due to reduced dimerisation with ARNT2. Homology modelling predicts F147 in NPAS4 to lie at the dimer interface, where it appears to directly contribute to protein/protein interaction. We also found that reduced transcriptional activation by ARNT2 R46W was due to disruption of nuclear localisation. These results provide insight into the mechanisms of NPAS4/ARNT dimerisation and transcriptional activation and have potential implications for cognitive phenotypic variation and diseases such as autism, schizophrenia and dementia.David C. Bersten, John B. Bruning, Daniel J. Peet, Murray L. Whitela

Clinical diagnostic utility of IP-10 and LAM antigen levels for the diagnosis of tuberculous pleural effusions in a high burden setting

Background: Current tools for the diagnosis of tuberculosis pleural effusions are sub-optimal. Data about the value of new diagnostic technologies are limited, particularly, in high burden settings. Preliminary case control studies have identified IFN-γ-inducible-10kDa protein (IP-10) as a promising diagnostic marker; however, its diagnostic utility in a day-to-day clinical setting is unclear. Detection of LAM antigen has not previously been evaluated in pleural fluid. Methods: We investigated the comparative diagnostic utility of established (adenosine deaminase [ADA]), more recent (standardized nucleic-acid-amplification-test [NAAT]) and newer technologies (a standardized LAM mycobacterial antigendetection assay and IP-10 levels) for the evaluation of pleural effusions in 78 consecutively recruited South African tuberculosis suspects. All consenting participants underwent pleural biopsy unless contra-indicated or refused. The reference standard comprised culture positivity for M. tuberculosis or histology suggestive of tuberculosis. Principal Findings: Of 74 evaluable subjects 48, 7 and 19 had definite, probable and non-TB, respectively. IP-10 levels were significantly higher in TB vs non-TB participants (p<0.0001). The respective outcomes [sensitivity, specificity, PPV, NPV %] for the different diagnostic modalities were: ADA at the 30 IU/L cut-point [96; 69; 90; 85], NAAT [6; 93; 67; 28], IP-10 at the 28,170 pg/ml ROC-derived cut-point [80; 82; 91; 64], and IP-10 at the 4035 pg/ml cut-point [100; 53; 83; 100]. Thus IP-10, using the ROC-derived cut-point, missed ~20% of TB cases and mis-diagnosed ~20% of non-TB cases. By contrast, when a lower cut-point was used a negative test excluded TB. The NAAT had a poor sensitivity but high specificity. LAM antigendetection was not diagnostically useful. Conclusion: Although IP-10, like ADA, has sub-optimal specificity, it may be a clinically useful rule-out test for tuberculous pleural effusions. Larger multi-centric studies are now required to confirm our findings

The Paluxy River Footprints Revisited

Field research by the authors at various times between 1982 and 1989 helped expose some of the elongate impressions imbedded in alleged 108 million year old Cretaceous ledges along the Paluxy River near Glen Rose, Texas. These human-like footprints were exposed in the same horizon with theropod dinosaur ichnites, as have prints in river itself over the decades, as reported by the local residents (I, 2). In order to thoroughly document such significant discoveries, several excavations were initiated since the 1986 ICC proceedings in the search for pristine ichnites. The results of these excavations plus the observable results of many previous excavations and the aspect ratio studies of many of the footprints strongly support the hypothesis that humans and dinosaurs coexisted. Furthermore, when radiocarbon dating results are combined with the paleoanthropological studies, the most logical conclusions are that: dinosaur extinction 65 million years ago is a myth; the long ages for sedimentary rock strata formation are non-existent; dinosaur extinction could have been caused by a major worldwide catastrophe happening perhaps only thousands of years ago