Length suppression in histone messenger RNA 3′-end maturation: Processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA

Efficient 3′-end processing of cell cycle-regulated mammalian histone premessenger RNAs (pre-mRNAs) requires an upstream stem–loop and a histone downstream element (HDE) that base pairs with the U7 small ribonuclearprotein. Insertions between these elements have two effects: the site of cleavage moves in concert with the HDE and processing efficiency declines. We used Xenopus oocytes to ask whether compensatory length insertions in the human U7 RNA could restore the fidelity and efficiency of processing of mouse histone insertion pre-mRNAs. An insertion of 5 nt into U7 RNA that extends its complementary to the HDE compensated for both defects in processing of a 5-nt insertion substrate; a noncomplementary insertion into U7 did not. Yet, the noncomplementary insertion mutant U7 was shown to be active on insertion substrates further mutated to allow base pairing. Our results suggest that the histone pre-mRNA becomes rigidified upstream of its HDE, allowing the bound U7 small ribonucleoprotein to measure from the HDE to the cleavage site. Such a mechanism may be common to other RNA measuring systems. To our knowledge, this is the first demonstration of length suppression in an RNA processing system.

Structure of neurolysin reveals a deep channel that limits substrate access

The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 10 residues by the limited size of the active site cavity. Some structural elements of neurolysin, including a five-stranded β-sheet and the two active site helices, are conserved with other metallopeptidases. The connecting loop regions of these elements, however, are much extended in neurolysin, and they, together with other open coil elements, line the active site cavity. These potentially flexible elements may account for the ability of the enzyme to cleave a variety of sequences.

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2′-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XbaI and by mass spectrometry of the PCR products.

Full-length myotonin protein kinase (72 kDa) displays serine kinase activity.

We describe the full-length (72 kDa) myotonin protein kinase (Mt-PK) and demonstrate its kinase activity. The 72-kDa protein corresponds to the translation product from the first in-frame AUG codon. This protein was found in the cytoplasmic fraction, whereas the previously reported 55-kDa protein was observed in nuclear extracts. Only the 72-kDa protein was phosphorylated by [32P]phosphate in normal human fibroblasts. To investigate the putative kinase activity of Mt-PK, a construct containing the full-length open reading frame of Mt-PK was expressed in bacterial cells. The recombinant Mt-PK autophosphorylates a Ser residue and phosphorylates the synthetic peptide Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg, which contains a Ser residue in the phosphorylation site. We examined phosphorylation of the voltage-dependent Ca(2+)-release channel, or dihydropyridine receptor (DHPR), by recombinant Mt-PK. We observed that the beta subunit of DHPR was phosphorylated in vitro by Mt-PK. A beta-subunit DHPR peptide containing some of the Ser residues predicted to be phosphorylated was synthesized and found to be a substrate for Mt-PK in vitro. We conclude that the 72-kDa Mt-PK has a protein kinase activity specific for Ser residues.

'We just make the pictures...?' How work is portrayed in children's feature length films

This article explores how adult paid work is portrayed in 'family' feature length films. The study extends previous critical media literature which has overwhelmingly focused on depictions of gender and violence, exploring the visual content of films that is relevant to adult employment. Forty-two G/PG films were analyzed for relevant themes. Consistent with the exploratory nature of the research, themes emerged inductively from the films' content. Results reveal six major themes: males are more visible in adult work roles than women; the division of labour remains gendered; work and home are not mutually exclusive domains; organizational authority and power is wielded in punitive ways; there are avenues to better employment prospects; and status/money is paramount. The findings of the study reflect a range of subject matters related to occupational characteristics and work-related communication and interactions which are typically viewed by children in contemporary society.

Using Trauma Injury Severity Score (TRISS) variables to predict length of hospital stay following trauma in New Zealand

Aim – To develop and assess the predictive capabilities of a statistical model that relates routinely collected Trauma Injury Severity Score (TRISS) variables to length of hospital stay (LOS) in survivors of traumatic injury. Method – Retrospective cohort study of adults who sustained a serious traumatic injury, and who survived until discharge from Auckland City, Middlemore, Waikato, or North Shore Hospitals between 2002 and 2006. Cubic-root transformed LOS was analysed using two-level mixed-effects regression models. Results – 1498 eligible patients were identified, 1446 (97%) injured from a blunt mechanism and 52 (3%) from a penetrating mechanism. For blunt mechanism trauma, 1096 (76%) were male, average age was 37 years (range: 15-94 years), and LOS and TRISS score information was available for 1362 patients. Spearman’s correlation and the median absolute prediction error between LOS and the original TRISS model was ρ=0.31 and 10.8 days, respectively, and between LOS and the final multivariable two-level mixed-effects regression model was ρ=0.38 and 6.0 days, respectively. Insufficient data were available for the analysis of penetrating mechanism models. Conclusions – Neither the original TRISS model nor the refined model has sufficient ability to accurately or reliably predict LOS. Additional predictor variables for LOS and other indicators for morbidity need to be considered.

Using a longitudinal model to estimate the effect of methicillin-resistant staphylococcus aureus infection on length of stay in an intensive care unit

Healthcare-associated methicillin-resistant Staphylococcus aureus(MRSA) infection may cause increased hospital stay or, sometimes, death. Quantifying this effect is complicated because it is a time-dependent exposure: infection may prolong hospital stay, while longer stays increase the risk of infection. We overcome these problems by using a multinomial longitudinal model for estimating the daily probability of death and discharge. We then extend the basic model to estimate how the effect of MRSA infection varies over time, and to quantify the number of excess ICU days due to infection. We find that infection decreases the relative risk of discharge (relative risk ratio = 0.68, 95% credible interval: 0.54, 0.82), but is only indirectly associated with increased mortality. An infection on the first day of admission resulted in a mean extra stay of 0.3 days (95% CI: 0.1, 0.5) for a patient with an APACHE II score of 10, and 1.2 days (95% CI: 0.5, 2.0) for a patient with an APACHE II score of 30. The decrease in the relative risk of discharge remained fairly constant with day of MRSA infection, but was slightly stronger closer to the start of infection. These results confirm the importance of MRSA infection in increasing ICU stay, but suggest that previous work may have systematically overestimated the effect size.

Minimising speaker verification utterance length through confidence based early verification decisions

This paper presents a novel approach of estimating the confidence interval of speaker verification scores. This approach is utilised to minimise the utterance lengths required in order to produce a confident verification decision. The confidence estimation method is also extended to address both the problem of high correlation in consecutive frame scores, and robustness with very limited training samples. The proposed technique achieves a drastic reduction in the typical data requirements for producing confident decisions in an automatic speaker verification system. When evaluated on the NIST 2005 SRE, the early verification decision method demonstrates that an average of 5–10 seconds of speech is sufficient to produce verification rates approaching those achieved previously using an average in excess of 100 seconds of speech.

A comparison of different methods to determine diffusion length in dye-sensitized solar cells

A new steady state method for determination of the electron diffusion length in dye-sensitized solar cells (DSCs) is described and illustrated with data obtained using cells containing three different types of electrolyte. The method is based on using near-IR absorbance methods to establish pairs of illumination intensity for which the total number of trapped electrons is the same at open circuit (where all electrons are lost by interfacial electron transfer) as at short circuit (where the majority of electrons are collected at the contact). Electron diffusion length values obtained by this method are compared with values derived by intensity modulated methods and by impedance measurements under illumination. The results indicate that the values of electron diffusion length derived from the steady state measurements are consistently lower than the values obtained by the non steady-state methods. For all three electrolytes used in the study, the electron diffusion length was sufficiently high to guarantee electron collection efficiencies greater than 90%. Measurement of the trap distributions by near-IR absorption confirmed earlier observations of much higher electron trap densities for electrolytes containing Li+ ions. It is suggested that the electron trap distributions may not be intrinsic properties of the TiO2 nanoparticles, but may be associated with electron-ion interactions.

Is length of experience an appropriate criterion to identify level of expertise?

Clinical experience plays an important role in the development of expertise, particularly when coupled with reflection on practice. There is debate, however, regarding the amount of clinical experience that is required to become an expert. Various lengths of practice have been suggested as suitable for determining expertise, ranging from five years to 15 years. This study aimed to investigate the association between length of experience and therapists’ level of expertise in the field of cerebral palsy with upper limb hypertonicity using an empirical procedure named Cochrane–Weiss–Shanteau (CWS). The methodology involved re-analysis of quantitative data collected in two previous studies. In Study 1, 18 experienced occupational therapists made hypothetical clinical decisions related to 110 case vignettes, while in Study 2, 29 therapists considered 60 case vignettes drawn randomly from those used in Study 1. A CWS index was calculated for each participant's case decisions. Then, in each study, Spearman's rho was calculated to identify the correlations between the duration of experience and level of expertise. There was no significant association between these two variables in both studies. These analyses corroborated previous findings of no association between length of experience and judgemental performance. Therefore, length of experience may not be an appropriate criterion for determining level of expertise in relation to cerebral palsy practice.