Multicolour interphase cytogenetics: 24 chromosome probes, 6 colours, 4 layers

From the late 1980s onwards, the use of DNA probes to visualise sequences on individual chromosomes (fluorescent in-situ hybridisation - FISH) revolutionised the study of cytogenetics. Following single colour experiments, more fluorochromes were added, culminating in a 24 colour assay that could distinguish all human chromosomes. Interphase cytogenetics (the detection of chromosome copy number in interphase nuclei) soon followed, however 24 colour experiments are hampered for this application as mixing fluorochromes to produce secondary colours produces images that are not easily distinguishable from overlapping signals. This study reports the development and use of a novel protocol, new fast hybridising FISH probes, and a bespoke image capture system for the assessment of chromosome copy number in interphase nuclei. The multicolour probe sets can be used individually or in sequential hybridisation layers to assess ploidy of all 24 human chromosomes in the same nucleus. Applications of this technique are in the investigation of chromosome copy number and the assessment of nuclear organisation for a range of different cell types including human sperm, cancer cells and preimplantation embryos

Identification of optimal assisted aspiration conditions of oocytes for use in porcine in vitro maturation: a re-evaluation of the relationship between the cumulus oocyte complex and oocyte quality

The quality of porcine oocytes for use in IVF is commonly graded according to the number of layers of cumulus cells surrounding the oocyte; together these form the cumulus oocyte complex (COC). At least three compact layers of cumulus cells is regarded as important for efficient IVP. To test this, oocytes were scored according to cumulus investment, with grade A representing COCs with three or more cumulus layers including granulosa cell-cumulus oocyte complexes, grade B those with an intact corona radiata surrounded by another layer of cumulus cells and grades C and D representing COCs with lower cumulus cell investment. These oocytes were then monitored for in vitro maturation (IVM), as assessed by tubulin immunostaining for meiotic progression, the development of a cortical granule ring, and by glutathione levels. Results indicate that grading correlates closely with nuclear maturation and cytoplasmic maturation, suggesting that grading oocytes by cumulus investment is a reliable method to predict IVM success. Importantly, Grade A and B oocytes showed no significant differences in any measure and hence using a cut-off of two or more cumulus cell layers may be optimal. We also determined the effect of assisted aspiration for oocyte retrieval, comparing the effect of needle size and applied pressure on the retrieval rate. These data indicated that both variables affected oocyte recovery rates and the quality of recovered oocytes. In combination, these experiments indicate that grade A and B oocytes have a similar developmental potential and that the recovery of oocytes of these grades is maximised by use of an 18-gauge needle and 50mmHg aspiration pressure

The demand for primary health care services in the Bicol Region of the Philippines.

Analyzes the demand for primary health care using community and household data from one of the poorest regions of the Philippines. Finds, among other things that the distance is not nearly as important to health service demand as it has often been assumed to be; and that visit prices, drug and transport costs, and waiting times do not greatly affect demand patterns for essential or more optional services

Determinants of infant feeding: a household production approach.

The presentation of the infant-feeding decision as a joint selection of breast milk, breast milk, substitutes, and supplemental foods is an entirely new approach. It represents one of the first efforts to model the structure of household production decisions. The approach provides a plausible statistical 'reenactment' of how one small but important household decision is made: how to feed a baby. Statistically, the work suggests that the omission of correlations among feeding methods may lead to incorrect inferences. The results show that the independent variables appear not to affect breast-feeding behaviour strongly per se but to affect the feeding of breast milk substitutes and the timing of supplementation with solid food, which in turn work in a somewhat diluted fashion on breast-feeding

The use of an e-learning constructivist solution in workplace learning

We wished to investigate whether an e-learning approach which uses constructivist principles can be successfully applied to train employees in a highly specialised skill thought to require expert individuals and extensive prolonged training. The approach involved the development of an e-learning package which included simulations and interactivity, then experimental testing in a case study workplace environment with the collection of both quantitative and qualitative data to assess the effectiveness of the package. Our study shows that this e-learning strategy improved the skills of the inexperienced operator significantly. We therefore propose that such programmes could be used as a work based training aid and used as a model system for the training of employees in complex skilled tasks in the workplace. This research demonstrates that the e-learning can be applied outside the traditional learning environment to train unskilled employees to undertake complex practical tasks which traditionally would involve prohibitively expensive instruction. This work also illustrates that simulations and interactivity are powerful tools in the design of successful e-learning packages in preparing learners for real world practical situations. Finally this study shows that workplace learners can be better served by elearning environments rather than conventional training as they allow asynchronous learning and private study which are valued by employees who have other demands on their time and are more comfortable receiving tuition privately Relevance to industry: E-learning using constructivist principles, and incorporating simulations and interactivity can be used successfully in the training of highly specialised and skilled tasks required in the modern workplace

Analysis of bovine blastocysts indicates ovarian stimulation does not induce chromosome errors, nor discordance between inner-cell mass and trophectoderm lineages

Contemporary systems for oocyte retrieval and culture of both cattle and human embryos are suboptimal with respect to pregnancy outcomes following transfer. In humans, chromosome abnormalities are the leading cause of early pregnancy loss in assisted reproduction. Consequently, pre-implantation genetic testing for aneuploidy (PGT-A) is widespread and there is considerable interest in its application to identify suitable cattle IVP embryos for transfer. Here we report on the nature and extent of chromosomal abnormalities following transvaginal follicular aspiration (OPU) and IVP in cattle. Nine sexually mature Holstein heifers underwent nine sequential cycles of OPU-IVP (six non-stimulated and three stimulated cycles), generating 459 blastocysts from 783 oocytes. We adopted a SNP-array approach normally employed in genomic evaluations but reanalysed (Turner et al., 2019; Theriogenology 125: 249) to detect levels of meiotic aneuploidy. Specifically, we asked whether ovarian stimulation increased the level of aneuploidy in either trophectoderm (TE) or inner-cell mass (ICM) lineages of blastocysts generated from OPU-IVP cycles. The proportion of Day 8 blastocysts of inseminated was greater (P < 0.001) for stimulated than non-stimulated cycles (0.712 ± 0.0288 vs. 0.466 ± 0.0360), but the overall proportion aneuploidy was similar for both groups (0.241 ± 0.0231). Most abnormalities consisted of meiotic trisomies. Twenty in vivo derived blastocysts recovered from the same donors were all euploid, thus indicating that 24 h of maturation is primarily responsible for aneuploidy induction. Chromosomal errors in OPU-IVP blastocysts decreased (P < 0.001) proportionately as stage/grade improved (from 0.373 for expanded Grade 2 to 0.128 for hatching Grade 1 blastocysts). Importantly, there was a high degree of concordance in the incidence of aneuploidy between TE and ICM lineages. Proportionately, 0.94 were “perfectly concordant” (i.e. identical result in both); 0.01 were imperfectly concordant (differing abnormalities detected); 0.05 were discordant; of which 0.03 detected a potentially lethal TE abnormality (false positives), leaving only 0.02 false negatives. These data support the use of TE biopsies for PGT-A in embryos undergoing genomic evaluation in cattle breeding. Finally, we report chromosome-specific errors and a high degree of variability in the incidence of aneuploidy between donors, suggesting a genetic contribution that merits further investigation

Integrating sequence and array data to create an improved 1000 Genomes Project haplotype reference panel

A major use of the 1000 Genomes Project (1000GP) data is genotype imputation in genome-wide association studies (GWAS). Here we develop a method to estimate haplotypes from low-coverage sequencing data that can take advantage of single-nucleotide polymorphism (SNP) microarray genotypes on the same samples. First the SNP array data are phased to build a backbone (or 'scaffold') of haplotypes across each chromosome. We then phase the sequence data 'onto' this haplotype scaffold. This approach can take advantage of relatedness between sequenced and non-sequenced samples to improve accuracy. We use this method to create a new 1000GP haplotype reference set for use by the human genetic community. Using a set of validation genotypes at SNP and bi-allelic indels we show that these haplotypes have lower genotype discordance and improved imputation performance into downstream GWAS samples, especially at low-frequency variants. © 2014 Macmillan Publishers Limited. All rights reserved

Size Doesn't Matter: Towards a More Inclusive Philosophy of Biology

notes: As the primary author, O’Malley drafted the paper, and gathered and analysed data (scientific papers and talks). Conceptual analysis was conducted by both authors.publication-status: Publishedtypes: ArticlePhilosophers of biology, along with everyone else, generally perceive life to fall into two broad categories, the microbes and macrobes, and then pay most of their attention to the latter. ‘Macrobe’ is the word we propose for larger life forms, and we use it as part of an argument for microbial equality. We suggest that taking more notice of microbes – the dominant life form on the planet, both now and throughout evolutionary history – will transform some of the philosophy of biology’s standard ideas on ontology, evolution, taxonomy and biodiversity. We set out a number of recent developments in microbiology – including biofilm formation, chemotaxis, quorum sensing and gene transfer – that highlight microbial capacities for cooperation and communication and break down conventional thinking that microbes are solely or primarily single-celled organisms. These insights also bring new perspectives to the levels of selection debate, as well as to discussions of the evolution and nature of multicellularity, and to neo-Darwinian understandings of evolutionary mechanisms. We show how these revisions lead to further complications for microbial classification and the philosophies of systematics and biodiversity. Incorporating microbial insights into the philosophy of biology will challenge many of its assumptions, but also give greater scope and depth to its investigations

Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses

To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1–11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely

Smoking interventions within the hospital setting: A study of demand for smoking interventions and perceived efficacy of nurses in delivery of services

Issues addressed: This study was motivated by the current need for tobacco interventions for inpatients and nursing staff within the hospital setting. In particular, whether inpatients considered quitting while in hospital, if they expected assistance and/or requested help from ward nurses was investigated. In addition, it sought to establish whether nurses considered they had a role in tobacco intervention and if so, whether they felt adequately prepared. The willingness of nurses to participate in ongoing tobacco intervention education and the type of educational program preferences was also explored in relation to current smoking status. Methods: A cohort of 60 patients and 100 nursing personnel were surveyed in the Princess Alexandra Hospital, Brisbane. Results: Of 569 inpatients, 19 per cent were current smokers and 39 per cent of ward nurses smoked. Of inpatients, 57 per cent considered quitting tobacco during hospital admission but only 5 per cent requested assistance. Sixty per cent of inpatients did not expect any assistance from hospital staff. Although infrequently asked for assistance, 51 per cent of ward nurses saw a "definite" nursing role in tobacco intervention, with a further 30 per cent considering a "probable" role. Non-smokers were more likely to support a nursing role in tobacco intervention than nurses who smoke (pConclusion: There is a need to create a more supportive hospital environment for inpatients to assist those considering quitting to seek help. Their unsatisfied needs require a new approach which could involve ward nursing staff, many of whom see they have a role in tobacco intervention. Nurses recognise a need for further training. The development of additional program components, suitable for nurses who smoke, as a workplace intervention, is warranted. The development of improved hospital-based smoking cessation interventions for both patients and nursing personnel should be considered a priority. So what? Many hospital inpatients consider quitting smoking but few feel encouraged to do so or seek support. The positive attitude of nursing staff to participate in tobacco interventions should be further pursued. Nurse- delivered tobacco interventions should also include an intervention directed at nurses who smoke