Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Neural precursor cells differentiating in the absence of Rb exhibit delayed terminal mitosis and deregulated E2F 1 and 3 activity

The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis we examined the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural stem cells derived from Rb-deficient mice. Although Rb -/- neural precursor cells are able to differentiate in vitro we show that these cells exhibit a significant delay in terminal mitosis relative to wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts with the putative Rb target E2F3 in neural precursor cells, our results indicate that it clearly cannot restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3 activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In these studies we show that Rb is essential for the regulation of E2F 1 and 3 activity as well as the onset of terminal mitosis in neural precursor cells.     

Development and Physical Validation of a Finite Element Model of Total Hip Dislocation

Component-on-component impingement, followed by levering of the femoral head, is a common mode of dislocation in total hip arthroplasty. While there have been many registry-based studies of dislocation incidence, confounding factors and sources of variability in the clinical domain make it difficult to identify specific parameter influences. A three dimensional nonlinear finite element model has been developed for the purpose of studying the dislocation event, to allow determination of how individual factors such as component design and clinical implantation position affect the propensity for dislocation. Also, a laboratory testing apparatus was constructed to provide physical validation of the computational model. The finite element model correctly predicted the range of motion observed in the physical apparatus to within 1%, and predicted the peak resisting moment to within 2.5%. Under even a light joint load of 200 N, the von Mises stresses developed in the polyethylene insert reached 13 MPa, and the contact stresses rose to as high as 30 MPa. These deleterious elevations occurred not only at the site of neck impingement, but also at the site of head egress from the liner.     

Using citizen science to measure recolonisation of birds after the Australian 2019–2020 mega-fires

Large and severe fires (‘mega-fires’) are increasing in frequency across the globe, often pushing into ecosystems that have previously had very long fire return intervals. The 2019–2020 Australian bushfire season was one of the most catastrophic fire events on record. Almost 19 million hectares were burnt across the continent displacing and killing unprecedented numbers of native fauna, including bird species. Some bird species are known to thrive in post-fire environments, while others may be absent for an extended period from the firegrounds until there is sufficient ecosystem recovery. To test for systematic patterns in species use of the post-fire environment, we combined citizen science data from eBird with data on sedentism, body size, range size and the specialisation of diet and habitat. Using generalised additive models, we modelled the responses of 76 bird species to the 2019–2020 Australian mega-fires. Twenty-two species decreased in occurrence after the fire; 30 species increased; and no significant effect was found for the remaining 24 species. Furthermore, diet specialists, and birds with smaller body sizes and range sizes were less likely to be found in burnt areas after the fire event compared to before, a result which generates testable hypotheses for recovery from other mega-fires across the globe. Being displaced from the firegrounds for an event of this geographic magnitude may have severe consequences for population dynamics and thus warrant considerable conservation attention in pre-fire planning and in the post-fire aftermath.     

Hearing disability in people aged 50-65: effectiveness and acceptability of rehabilitative intervention.

OBJECTIVE--To determine the best means of detecting hearing disability in subjects aged 50-65 and whether rehabilitative intervention is acceptable in this age group. DESIGN--Questionnaire survey of patients on general practice age-sex registers. Two types of questionnaire were used, one being based on the closed set approach of the Institute of Hearing Research questionnaire, which had been used in a pilot study, and the other being a simplified version of this questionnaire developed by the Welsh Hearing Institute and based on open set questions. Questionnaires were sent up to three times, and any patients who had not responded two months after the last posting were personally contacted. SETTING--Two general practices in Glyncorrwg and Blaengwynfi in the Afan valley, West Glamorgan. PATIENTS--271 Patients in Glyncorrwg (136 men, 135 women) and 333 patients in Blaengwynfi (173 men, 160 women) aged 50-65. INTERVENTIONS--All patients indicating hearing disability in answering the questionnaires were invited to attend for a evaluative session in their village. After audiometric testing advice and arrangements for fitting a hearing aid were offered as appropriate. MAIN OUTCOME MEASURES--Response rates and prevalence of hearing disability before intervention and of possession of hearing aids before and after intervention. RESULTS--After three postings and personal contact the response rate was 98% (266/271) in Glyncorrwg, where the complex questionnaire was used, and 97% (322/333) in Blaengwynfi. The prevalence of hearing disability was respectively 53% (141/266) and 46% (148/322) and the prevalence of owning a hearing aid 7% (19/266) and 8% (24/322). After intervention the possession of hearing aids rose to 24% (64/266) in Glyncorrwg and 22% (71/322) in Blaengwynfi; six months later the aids were being used regularly. A direct comparison of the two questionnaires in 69 subjects from Blaengwynfi showed no significant differences in the amount of disability detected by each one. The first posting of questionnaires detected 65% (189/289) of the hearing disability in the two villages or 78% (72/92) of those prepared to accept hearing aids for the first time; 96% (88/92) of those who accepted hearing aids were detected by two postings. CONCLUSIONS--Simple questionnaires are effective in detecting hearing disabilities in people aged 50-65, and intervention was acceptable in many of those who reported having difficulties in hearing. The response rates from successive postings suggest that two postings are sufficient in terms of the return in detecting those who will accept intervention.     

Is ATP binding responsible for initiating drug translocation by the multidrug transporter ABCG2?

ABCG2 confers resistance to cancer cells by mediating the ATP-dependent outward efflux of chemotherapeutic compounds. Recent studies have indicated that the protein contains a number of interconnected drug binding sites. The present investigation examines the coupling of drug binding to ATP hydrolysis. Initial drug binding to the protein requires a high-affinity interaction with the drug binding site, followed by transition and reorientation to the low-affinity state to enable dissociation at the extracellular face. [3H]Daunomycin binding to the ABCG2 R482G isoform was examined in the nucleotide-bound and post-hydrolytic conformations. Binding of [3H]daunomycin was displaced by ATP analogues, indicating transition to a low-affinity conformation prior to hydrolysis. The low-affinity state was observed to be retained immediately post-hydrolysis. Therefore, the dissociation of phosphate and/or ADP is likely to be responsible for resetting of the transporter. The data indicate that, like ABCB1 and ABCC1, the 'power stroke' for translocation in ABCG2 R482G is the binding of nucleotide.     

Abdominal muscles dominate contributions to vertebral joint stiffness during the push-up

The goal of this study was to quantify the relative contributions of each muscle group surrounding the spine to vertebral joint rotational stiffness (VJRS) during the push-up exercise. Upper-body kinematics, three-dimensional hand forces and lumbar spine postures, and 14 channels (bilaterally from rectus abdominis, external oblique, internal oblique, latissimus dorsi, thoracic erector spinae, lumbar erector spinae, and multifidus) of trunk electromyographic (EMG) activity were collected from 11 males and used as inputs to a biomechanical model that determined the individual contributions of 10 muscle groups surrounding the lumbar spine to VJRS at five lumbar vertebral joints (L1-L2 to L5-S1). On average, the abdominal muscles contributed 64.32 +/- 8.50%, 86.55 +/- 1.13%, and 83.84 +/- 1.95% to VJRS about the flexion/extension, lateral bend, and axial twist axes, respectively. Rectus abdominis contributed 43.16 +/- 3.44% to VJRS about the flexion/extension axis at each lumbar joint, and external oblique and internal oblique, respectively contributed 52.61 +/- 7.73% and 62.13 +/- 8.71% to VJRS about the lateral bend and axial twist axes, respectively, at all lumbar joints with the exception of L5-S1. Owing to changes in moment arm length, the external oblique and internal oblique, respectively contributed 55.89% and 50.01% to VJRS about the axial twist and lateral bend axes at L5-S1. Transversus abdominis, multifidus, and the spine extensors contributed minimally to VJRS during the push-up exercise. The push-up challenges the abdominal musculature to maintain VJRS. The orientation of the abdominal muscles suggests that each muscle primarily controls the rotational stiffness about a single axis.     

The nucleotide-binding domains of P-glycoprotein. Functional symmetry in the isolated domain demonstrated by N-ethylmaleimide labelling.

The two nucleotide-binding domains (NBDs) of a number of ATP-binding cassette (ABC) transporters have been shown to be functionally dissimilar, playing different roles in the transport process. A high degree of co-operativity has been determined for the NBDs of the human multidrug transporter, P-glycoprotein. However, the issue of functional symmetry in P-glycoprotein remains contentious. To address this, the NBDs of P-glycoprotein were expressed and purified to 95% homogeneity, as fusions to maltose-binding protein. The NBDs were engineered to contain a single cysteine residue in the Walker-A homology motif. Reactivity of this cysteine residue was demonstrated by specific, time-dependent, covalent labelling with N-ethylmaleimide. No differences in the rates of labelling of the two NBDs were observed. The relative affinity of binding to each NBD was determined for a number of nucleotides by measuring their ability to effect a reduction in N-ethylmaleimide labelling. In general, nucleotides bound identically to the two NBDs, suggesting that there is little asymmetry in the initial step of the transport cycle, namely the recognition and binding of nucleotide. Any observed functional asymmetry in the intact transporter presumably reflects different rates of hydrolysis at the two NBDs or interdomain communications.     

Elevated temperature restricts growth potential of the coral reef fish <Emphasis Type="Italic">Acanthochromis polyacanthus</Emphasis>

In order to test the effect of temperature variation on the growth of a common coral-reef fish, Acanthochromis polyacanthus, juveniles, sub-adults and adults were reared on either high or low food rations at temperatures corresponding to the long-term (14 year) minimum, average and maximum summer sea-surface temperatures (26, 28 and 31°C respectively) at Orpheus Island, Great Barrier Reef, Australia. Both temperature and food supply affected the growth of juvenile and adult A. polyacanthus. Individuals grew more on high food rations, but growth declined with increasing temperature. Importantly, at 31°C, the growth of juveniles and adults on the high food ration was nearly identical to growth on the low food ration. This indicates that the capacity for growth is severely limited at higher ocean temperatures that are predicted to become the average for Orpheus Island within the next 100 years as a result of rapid climate change. Communicated by Biology Editor Dr Mark McCormick