The role of upstream sequences in selecting the reading frame on tmRNA

<p>Abstract</p> <p>Background</p> <p>tmRNA acts first as a tRNA and then as an mRNA to rescue stalled ribosomes in eubacteria. Two unanswered questions about tmRNA function remain: how does tmRNA, lacking an anticodon, bypass the decoding machinery and enter the ribosome? Secondly, how does the ribosome choose the proper codon to resume translation on tmRNA? According to the -1 triplet hypothesis, the answer to both questions lies in the unique properties of the three nucleotides upstream of the first tmRNA codon. These nucleotides assume an A-form conformation that mimics the codon-anticodon interaction, leading to recognition by the decoding center and choice of the reading frame. The -1 triplet hypothesis is important because it is the most credible model in which direct binding and recognition by the ribosome sets the reading frame on tmRNA.</p> <p>Results</p> <p>Conformational analysis predicts that 18 triplets cannot form the correct structure to function as the -1 triplet of tmRNA. We tested the tmRNA activity of all possible -1 triplet mutants using a genetic assay in <it>Escherichia coli</it>. While many mutants displayed reduced activity, our findings do not match the predictions of this model. Additional mutagenesis identified sequences further upstream that are required for tmRNA function. An immunoblot assay for translation of the tmRNA tag revealed that certain mutations in U85, A86, and the -1 triplet sequence result in improper selection of the first codon and translation in the wrong frame (-1 or +1) <it>in vivo</it>.</p> <p>Conclusion</p> <p>Our findings disprove the -1 triplet hypothesis. The -1 triplet is not required for accommodation of tmRNA into the ribosome, although it plays a minor role in frame selection. Our results strongly disfavor direct ribosomal recognition of the upstream sequence, instead supporting a model in which the binding of a separate ligand to A86 is primarily responsible for frame selection.</p

Impact of sex, MHC, and age of recipients on the therapeutic effect of transferred leukocytes from cancer-resistant SR/CR mice

<p>Abstract</p> <p>Background</p> <p>Spontaneous Regression/Complete Resistant (SR/CR) mice are resistant to cancer through a mechanism that is mediated entirely by leukocytes of innate immunity. Transfer of leukocytes from SR/CR mice can confer cancer resistance in wild-type (WT) recipients in both preventative and therapeutic settings. In the current studies, we investigated factors that may impact the efficacy and functionality of SR/CR donor leukocytes in recipients.</p> <p>Results</p> <p>In sex-mismatched transfers, functionality of female donor leukocytes was not affected in male recipients. In contrast, male donor leukocytes were greatly affected in the female recipients. In MHC-mismatches, recipients of different MHC backgrounds, or mice of different strains, showed a greater negative impact on donor leukocytes than sex-mismatches. The negative effects of sex-mismatch and MHC-mismatch on donor leukocytes were additive. Old donor leukocytes performed worse than young donor leukocytes in all settings including in young recipients. Young recipients were not able to revive the declining function of old donor leukocytes. However, the function of young donor leukocytes declined gradually in old recipients, suggesting that an aged environment may contain factors that are deleterious to cellular functions. The irradiation of donor leukocytes prior to transfers had a profound suppressive effect on donor leukocyte functions, possibly as a result of impaired transcription. The cryopreserving of donor leukocytes in liquid nitrogen had no apparent effect on donor leukocyte functions, except for a small loss of cell number after revival from freezing.</p> <p>Conclusion</p> <p>Despite the functional suppression of donor leukocytes in sex- and MHC-mismatched recipients, as well as old recipients, there was a therapeutic time period during the initial few weeks during which donor leukocytes were functional before their eventual rejection or functional decline. The eventual rejection of donor leukocytes will likely prevent donor leukocyte engraftment which would help minimize the risk of transfusion-associated graft-versus-host disease. Therefore, using leukocytes from healthy donors with high anti-cancer activity may be a feasible therapeutic concept for treating malignant diseases.</p

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Methods for specifying the target difference in a randomised controlled trial : the Difference ELicitation in TriAls (DELTA) systematic review

Peer reviewedPublisher PD

Defining dental operative skills curricula: an ADEE consensus paper

This paper reports on the recent activity of the pan‐European consensus of the ADEE Special Interest Group for Pre‐Clinical Operative Skills. Following the previous recommendations from the group, and in order to support teachers and to harmonise the delivery of skills training across Europe, a more formal curriculum relating to pre‐clinical operative skills needs to be created. This paper reports European consensus surrounding the categorisation (level of importance, and difficulty) of basic operative dental clinical skills within the undergraduate curriculum and provides recommendations relating to session structure and timing of curricular elements for basic operative dental clinical skills teaching

Anemia of Inflammation Is Related to Cognitive Impairment among Children in Leyte, The Philippines

Past studies have demonstrated that iron deficiency anemia is related to deficits in cognitive fucntioning in children, and treating iron deficiency anemia with iron supplementation can improve cognition. Anemia of inflammation is another type of anemia caused by many diseases of lesser-developed countries including bacterial and parasitic infections. Anemia of inflammation is characterized by disordered iron metabolism, such that iron is sequestered in storage forms, preventing its use from tissues that require it. We hypothesized that decreased iron delivery to the brain in the context of anemia of inflammation might lead to decreased cognitive performance. This study found that children with anemia of inflammation had decreased cognitive performance in specific domains, compared to subjects with no anemia. True total body iron deficiency anemia was related to lower performance in the same domains. The only treatment option for anemia of inflammation is treatment of the underlying disease. Iron supplementation will not prevent cognitive deficits in children with anemia of inflammation. Interventions aimed towards maximizing the cognitive development of children in lesser-developed countries will need to focus on the prevention and treatment of bacterial and parasitic infections

Clinical pre-test probability for obstructive coronary artery disease: insights from the European DISCHARGE pilot study.

To test the accuracy of clinical pre-test probability (PTP) for prediction of obstructive coronary artery disease (CAD) in a pan-European setting. Patients with suspected CAD and stable chest pain who were clinically referred for invasive coronary angiography (ICA) or computed tomography (CT) were included by clinical sites participating in the pilot study of the European multi-centre DISCHARGE trial. PTP of CAD was determined using the Diamond-Forrester (D+F) prediction model initially introduced in 1979 and the updated D+F model from 2011. Obstructive coronary artery disease (CAD) was defined by one at least 50% diameter coronary stenosis by both CT and ICA. In total, 1440 patients (654 female, 786 male) were included at 25 clinical sites from May 2014 until July 2017. Of these patients, 725 underwent CT, while 715 underwent ICA. Both prediction models overestimated the prevalence of obstructive CAD (31.7%, 456 of 1440 patients, PTP: initial D+F 58.9% (28.1-90.6%), updated D+F 47.3% (34.2-59.9%), both p < 0.001), but overestimation of disease prevalence was higher for the initial D+F (p < 0.001). The discriminative ability was higher for the updated D+F 2011 (AUC of 0.73 95% confidence interval [CI] 0.70-0.76 versus AUC of 0.70 CI 0.67-0.73 for the initial D+F; p < 0.001; odds ratio (or) 1.55 CI 1.29-1.86, net reclassification index 0.11 CI 0.05-0.16, p < 0.001). Clinical PTP calculation using the initial and updated D+F prediction models relevantly overestimates the actual prevalence of obstructive CAD in patients with stable chest pain clinically referred for ICA and CT suggesting that further refinements to improve clinical decision-making are needed. https://www.clinicaltrials.gov/ct2/show/NCT02400229 KEY POINTS: • Clinical pre-test probability calculation using the initial and updated D+F model overestimates the prevalence of obstructive CAD identified by ICA and CT. • Overestimation of disease prevalence is higher for the initial D+F compared with the updated D+F. • Diagnostic accuracy of PTP assessment varies strongly between different clinical sites throughout Europe

Health-related qualify of life, angina type and coronary artery disease in patients with stable chest pain.

Health-related quality of life (HRQoL) is impaired in patients with stable angina but patients often present with other forms of chest pain. The aim of this study was to compare the pre-diagnostic HRQoL in patients with suspected coronary artery disease (CAD) according to angina type, gender, and presence of obstructive CAD. From the pilot study for the European DISCHARGE trial, we analysed data from 24 sites including 1263 patients (45.9% women, 61.1 ± 11.3 years) who were clinically referred for invasive coronary angiography (ICA; 617 patients) or coronary computed tomography angiography (CTA; 646 patients). Prior to the procedures, patients completed HRQoL questionnaires: the Short Form (SF)-12v2, the EuroQoL (EQ-5D-3 L) and the Hospital Anxiety and Depression Scale. Fifty-five percent of ICA and 35% of CTA patients had typical angina, 23 and 33% had atypical angina, 18 and 28% had non-anginal chest discomfort and 5 and 5% had other chest discomfort, respectively. Patients with typical angina had the poorest physical functioning compared to the other angina groups (SF-12 physical component score; 41.2 ± 8.8, 43.3 ± 9.1, 46.2 ± 9.0, 46.4 ± 11.4, respectively, all age and gender-adjusted p < 0.01), and highest anxiety levels (8.3 ± 4.1, 7.5 ± 4.1, 6.5 ± 4.0, 4.7 ± 4.5, respectively, all adjusted p < 0.01). On all other measures, patients with typical or atypical angina had lower HRQoL compared to the two other groups (all adjusted p < 0.05). HRQoL did not differ between patients with and without obstructive CAD while women had worse HRQoL compared with men, irrespective of age and angina type. Prior to a diagnostic procedure for stable chest pain, HRQoL is associated with chest pain characteristics, but not with obstructive CAD, and is significantly lower in women. Clinicaltrials.gov, NCT02400229