Microtubules in the ovarioles of notonecta and oncopeltus.

Antibodies specific for tyrosinated, detyrosinated and acetylated tubulin were used to investigate the content and distribution of post-translational modifications of tubulin in the ovarioles of Notonecta glauca glauca and Oncopeltus fasciatus. Furthermore, antibodies were raised against tubulin purified from Notonecta ovarioles and characterised on a number of different cellular systems. Using one- and two-dimensional polyacrylamide gel electrophoresis and Western blotting, ovariole extracts were probed with the isotype specific antibodies. This revealed a major and a minor α-tubulin species in Notonecta, both of which were detyrosinated and acetylated. In contrast, Oncopeltus had only one major α-tubulin species which was tyrosinated. The in situ localisation of tyrosinated tubulin in the nutritive tubes of Notonecta ovarioles revealed a restricted distribution at the inner edge of the periphery of the functional tubes only, this distribution suggesting a novel model by which microtubules are inserted and grow in maturing tubes. Detyrosinated and acetylated tubulin were more widespread, having distributions in both the functional and redundant tubes. In Oncopeltus, tyrosinated tubulin could be visualised in all nutritive tubes, detyrosinated and acetylated tubulin being almost totally absent. This lack of post-translational modifications in Oncopeltus was shown to be due to either the lack or near total inactivation of the enzymatic machinery. The production of monoclonal antibodies against tubulin purified from Notonecta ovarioles was a constantly evolving process, each change producing improved results. In the final production one cloned serum contained antibodies specific for a 114kD epitope, suggesting the presence of a kinesin specific antibody, while 4 sera recognised a 54kD epitope, suggesting the presence of α-tubulin specific antibodies. Further characterisation of the sera produced confusing results which suggested the presence of several different antibody producing hybridomas in each clone and, hence, a conclusive analysis of the antibody content of the sera could not be reached

Characterising the tumour morphological response to therapeutic intervention:an ex vivo model

In cancer, morphological assessment of histological tissue samples is a fundamental part of both diagnosis and prognosis. Image analysis offers opportunities to support that assessment through quantitative metrics of morphology. Generally, morphometric analysis is carried out on two dimensional tissue section data and so only represents a small fraction of any tumour. We present a novel application of three-dimensional (3D) morphometrics for 3D imaging data obtained from tumours grown in a culture model. Minkowski functionals, a set of measures that characterise geometry and topology in n-dimensional space, are used to quantify tumour topology in the absence of and in response to therapeutic intervention. These measures are used to stratify the morphological response of tumours to therapeutic intervention. Breast tumours are characterised by estrogen receptor (ER) status, human epidermal growth factor receptor (HER)2 status and tumour grade. Previously, we have shown that ER status is associated with tumour volume in response to tamoxifen treatment ex vivo. Here, HER2 status is found to predict the changes in morphology other than volume as a result of tamoxifen treatment ex vivo. Finally, we show the extent to which Minkowski functionals might be used to predict tumour grade.Minkowski functionals are generalisable to any 3D data set, including in vivo and cellular systems. This quantitative topological analysis can provide a valuable link among biomarkers, drug intervention and tumour morphology that is complementary to existing, non-morphological measures of tumour response to intervention and could ultimately inform patient treatment

Silver nanoparticles embedded in zeolite membranes: release of silver ions and mechanism of antibacterial action

Amber Nagy1, Alistair Harrison2, Supriya Sabbani3, Robert S Munson, Jr2, Prabir K Dutta3, W James Waldman11Department of Pathology, The Ohio State University; 2Center for Microbial Pathogenesis, Research Institute at Nationwide Children's Hospital, 3Department of Chemistry, The Ohio State University, Columbus, OH, USABackground: The focus of this study is on the antibacterial properties of silver nanoparticles embedded within a zeolite membrane (AgNP-ZM).Methods and Results: These membranes were effective in killing Escherichia coli and were bacteriostatic against methicillin-resistant Staphylococcus aureus. E. coli suspended in Luria Bertani (LB) broth and isolated from physical contact with the membrane were also killed. Elemental analysis indicated slow release of Ag+ from the AgNP-ZM into the LB broth. The E. coli killing efficiency of AgNP-ZM was found to decrease with repeated use, and this was correlated with decreased release of silver ions with each use of the support. Gene expression microarrays revealed upregulation of several antioxidant genes as well as genes coding for metal transport, metal reduction, and ATPase pumps in response to silver ions released from AgNP-ZM. Gene expression of iron transporters was reduced, and increased expression of ferrochelatase was observed. In addition, upregulation of multiple antibiotic resistance genes was demonstrated. The expression levels of multicopper oxidase, glutaredoxin, and thioredoxin decreased with each support use, reflecting the lower amounts of Ag+ released from the membrane. The antibacterial mechanism of AgNP-ZM is proposed to be related to the exhaustion of antioxidant capacity.Conclusion: These results indicate that AgNP-ZM provide a novel matrix for gradual release of Ag+.Keywords: silver nanoparticles, zeolite, antibacterial agent, oxidative stres

Assessment and practical science:identifying generalizable characteristics of written assessments that reward and incentivise effective practices in practical science lessons

High-stakes assessments prominently influence what is done in secondary school science lessons (‘washback’ effects). It is therefore important that assessments of knowledge and understanding gained from practical work are constructed to reward and incentivise effective practices in practical work. To do that, they must differentiate between pupils who have experienced practical work in different ways. This empirical, mixed-methods study identifies generalizable characteristics of written assessments that differentially reward pupils who experienced practical activities through hands-on work, teacher demonstration, video demonstration, or reading about the activity. Conclusions are drawn from 1486 post-intervention tests completed by pupils aged 14–15 in England, from lesson observations and teacher interviews. This study also identifies pedagogical practices that were more noticeable in practical work that was most rewarded by the written assessments: the work was teacher-guided; and pupils were encouraged to be active participants. Existing literature describes negative washback effects of high-stakes, written assessments that limit the use and effectiveness of practical work as a pedagogical tool. We describe ways in which written assessments could be constructed to better reward effective practices in practical work (practices that better support learning), with the intention of having positive washback effects on pedagogy by better incentivising these practices

The CATH Domain Structure Database and related resources Gene3D and DHS provide comprehensive domain family information for genome analysis

The CATH database of protein domain structures (http://www.biochem.ucl.ac.uk/bsm/cath/) currently contains 43 229 domains classified into 1467 superfamilies and 5107 sequence families. Each structural family is expanded with sequence relatives from GenBank and completed genomes, using a variety of efficient sequence search protocols and reliable thresholds. This extended CATH protein family database contains 616 470 domain sequences classified into 23 876 sequence families. This results in the significant expansion of the CATHHMMmodel library to include models built from the CATH sequence relatives, giving a10%increase in coveragefor detecting remote homologues. An improved Dictionary of Homologous superfamilies (DHS) (http://www.biochem.ucl.ac.uk/bsm/dhs/) containing specific sequence, structural and functional information for each superfamily in CATH considerably assists manual validation of homologues. Information on sequence relatives in CATH superfamilies, GenBank and completed genomes is presented in the CATH associated DHS and Gene3D resources. Domain partnership information can be obtained from Gene3D (http://www.biochem.ucl.ac.uk/bsm/cath/Gene3D/). A new CATH server has been implemented (http://www.biochem.ucl.ac.uk/cgi-bin/cath/CathServer.pl) providing automatic classification of newly determined sequences and structures using a suite of rapid sequence and structure comparison methods. The statistical significance of matches is assessed and links are provided to the putative superfamily or fold group to which the query sequence or structure is assigned

The effect of blood pressure on mortality following out-of-hospital cardiac arrest : a retrospective cohort study of the United Kingdom Intensive Care National Audit and Research Centre database

Correction: Volume27, Issue 1 Article Number:169 DOI: 10.1186/s13054-023-04458-x Published: MAY 4 2023Background: Hypotension following out-of-hospital cardiac arrest (OHCA) may cause secondary brain injury and increase mortality rates. Current guidelines recommend avoiding hypotension. However, the optimal blood pressure following OHCA is unknown. We hypothesised that exposure to hypotension and hypertension in the first 24 h in ICU would be associated with mortality following OHCA. Methods: We conducted a retrospective analysis of OHCA patients included in the Intensive Care National Audit and Research Centre Case Mix Programme from 1 January 2010 to 31 December 2019. Restricted cubic splines were created following adjustment for important prognostic variables. We report the adjusted odds ratio for associations between lowest and highest mean arterial pressure (MAP) and systolic blood pressure (SBP) in the first 24 h of ICU care and hospital mortality. Results: A total of 32,349 patients were included in the analysis. Hospital mortality was 56.2%. The median lowest and highest MAP and SBP were similar in survivors and non-survivors. Both hypotension and hypertension were associated with increased mortality. Patients who had a lowest recorded MAP in the range 60-63 mmHg had the lowest associated mortality. Patients who had a highest recorded MAP in the range 95-104 mmHg had the lowest associated mortality. The association between SBP and mortality followed a similar pattern to MAP. Conclusions: We found an association between hypotension and hypertension in the first 24 h in ICU and mortality following OHCA. The inability to distinguish between the median blood pressure of survivors and non-survivors indicates the need for research into individualised blood pressure targets for survivors following OHCA.Peer reviewe

A novel nonsense CDK5RAP2 mutation in a Somali child with primary microcephaly and sensorineural hearing loss

Primary microcephaly is a genetically heterogeneous condition characterized by reduced head circumference (-3 SDS or more) and mild-to-moderate learning disability. Here, we describe clinical and molecular investigations of a microcephalic child with sensorineural hearing loss. Although consanguinity was unreported initially, detection of 13.7 Mb of copy neutral loss of heterozygosity (cnLOH) on chromosome 9 implicated the CDK5RAP2 gene. Targeted sequencing identified a homozygous E234X mutation, only the third mutation to be described in CDK5RAP2, the first in an individual of non-Pakistani descent. Sensorineural hearing loss is not generally considered to be consistent with autosomal recessive microcephaly and therefore it seems likely that the deafness in this individual is caused by the co-occurrence of a further gene mutation, independent of CDK5RAP2. Nevertheless, further detailed clinical descriptions of rare CDK5RAP2 patients, including hearing assessments will be needed to resolve fully the phenotypic range associated with mutations in this gene. This study also highlights the utility of SNP-array testing to guide disease gene identification where an autosomal recessive condition is plausible