Comparison of SIV and HIV-1 Genomic RNA Structures Reveals Impact of Sequence Evolution on Conserved and Non-Conserved Structural Motifs

RNA secondary structure plays a central role in the replication and metabolism of all RNA viruses, including retroviruses like HIV-1. However, structures with known function represent only a fraction of the secondary structure reported for HIV-1NL4-3. One tool to assess the importance of RNA structures is to examine their conservation over evolutionary time. To this end, we used SHAPE to model the secondary structure of a second primate lentiviral genome, SIVmac239, which shares only 50% sequence identity at the nucleotide level with HIV-1NL4-3. Only about half of the paired nucleotides are paired in both genomic RNAs and, across the genome, just 71 base pairs form with the same pairing partner in both genomes. On average the RNA secondary structure is thus evolving at a much faster rate than the sequence. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form, while the impact of selection for maintaining a protein binding interaction can be seen in the conservation of pairing partners in the small RRE stems where Rev binds. Structures that are conserved between SIVmac239 and HIV-1NL4-3 also occur at the 5′ polyadenylation sequence, in the plus strand primer sites, PPT and cPPT, and in the stem-loop structure that includes the first splice acceptor site. The two genomes are adenosine-rich and cytidine-poor. The structured regions are enriched in guanosines, while unpaired regions are enriched in adenosines, and functionaly important structures have stronger base pairing than nonconserved structures. We conclude that much of the secondary structure is the result of fortuitous pairing in a metastable state that reforms during sequence evolution. However, secondary structure elements with important function are stabilized by higher guanosine content that allows regions of structure to persist as sequence evolution proceeds, and, within the confines of selective pressure, allows structures to evolve

Older adult experience of care and staffing on hospital and community wards: a cross-sectional study

BACKGROUND: Recent major concerns about the quality of healthcare delivered to older adults have been linked to inadequate staffing and a lack of patient-centred care. Patient experience is a key component of quality care - yet there has been little research on whether and how staffing levels and staffing types affect satisfaction amongst older adult hospital inpatients. This study aimed to evaluate the association between registered nurse and healthcare assistant staffing levels and satisfaction with care amongst older adult hospital inpatients, and to test whether any positive effect of higher staffing levels is mediated by staff feeling they have more time to care for patients. METHODS: Survey data from 4928 inpatients aged 65 years and older and 2237 medical and nursing staff from 123 acute and community medical wards in England, United Kingdom (UK) was collected through the Royal College of Psychiatrist's Elder Care Quality Mark. The cross-sectional association between staffing ratios and older adult patient satisfaction, and mediation by staff perceived time to care, was evaluated using multi-level modelling, adjusted for ward type and with a random effect for ward identity. RESULTS: Higher numbers of patients per healthcare assistant were associated with poorer patient satisfaction (adjusted β = - 0.32, 95% CI - 0.55 to 0.10, p < 0.01), and this was found to be partially mediated by all ward staff reporting less time to care for patients (adjusted β = - 0.10, bias-corrected 95% CI - 1.16 to - 0.02). By contrast, in both unadjusted and adjusted models, the number of patients per registered nurse was not associated with patient satisfaction. CONCLUSIONS: Older adult hospital patients may particularly value the type of care provided by healthcare assistants, such as basic personal care and supportive communication. Additionally, higher availability of healthcare assistants may contribute to all ward staff feeling more able to spend time with patients. However, high availability of registered nurses has been shown in other research to be vital for ensuring quality and safety of patient care. Future research should seek to identify the ideal balance of registered nurses and healthcare assistants for optimising a range of outcomes amongst older adult patients

Evaluation of high-throughput genomic assays for the Fc gamma receptor locus

Cancer immunotherapy has been revolutionised by the use of monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics

When do past events require explanation? Insights from social psychology

Some past events incite more wonder about their causes than do others. For example, negative events require explanation more than positive events. We review social psychologists’ theoretical and empirical insights on what kinds of past events “beg explanation.” We draw on attribution theory that became popular among psychologists from the 1960s onward, on research on counterfactual reasoning, and on conversational and discursive critiques of attribution theory. We argue that factors predicting what is or is not perceived as requiring explanation are culturally and historically grounded, and that accordingly, what begs explanation varies between contexts and can change over time. Yet, drawing on the distinction between content and process, we argue that there are recognizable patterns across time and space. Specifically, we propose the relationship between events and background expectations as a rather stable predictor of what begs explanation—and as a level of analysis that can unite seemingly disparate approaches

Feasibility of preoperative chemotherapy for locally advanced, operable colon cancer: The pilot phase of a randomised controlled trial

Summary: Background Preoperative (neoadjuvant) chemotherapy and radiotherapy are more eff ective than similar postoperative treatment for oesophageal, gastric, and rectal cancers, perhaps because of more eff ective micrometastasis eradication and reduced risk of incomplete excision and tumour cell shedding during surgery. The FOxTROT trial aims to investigate the feasibility, safety, and effi cacy of preoperative chemotherapy for colon cancer. Methods In the pilot stage of this randomised controlled trial, 150 patients with radiologically staged locally advanced (T3 with ≥5 mm invasion beyond the muscularis propria or T4) tumours from 35 UK centres were randomly assigned (2:1) to preoperative (three cycles of OxMdG [oxaliplatin 85 mg/m², l-folinic acid 175 mg, fl uorouracil 400 mg/m² bolus, then 2400 mg/m² by 46 h infusion] repeated at 2-weekly intervals followed by surgery and a further nine cycles of OxMdG) or standard postoperative chemotherapy (12 cycles of OxMdG). Patients with KRAS wild-type tumours were randomly assigned (1:1) to receive panitumumab (6 mg/kg; every 2 weeks with the fi rst 6 weeks of chemotherapy) or not. Treatment allocation was through a central randomisation service using a minimised randomisation procedure including age, radiological T and N stage, site of tumour, and presence of defunctioning colostomy as stratifi cation variables. Primary outcome measures of the pilot phase were feasibility, safety, and tolerance of preoperative therapy, and accuracy of radiological staging. Analysis was by intention to treat. This trial is registered, number ISRCTN 87163246. Findings 96% (95 of 99) of patients started and 89% (85 of 95) completed preoperative chemotherapy with grade 3–4 gastrointestinal toxicity in 7% (seven of 94) of patients. All 99 tumours in the preoperative group were resected, with no signifi cant diff erences in postoperative morbidity between the preoperative and control groups: 14% (14 of 99) versus 12% (six of 51) had complications prolonging hospital stay (p=0·81). 98% (50 of 51) of postoperative chemotherapy patients had T3 or more advanced tumours confi rmed at post-resection pathology compared with 91% (90 of 99) of patients following preoperative chemotherapy (p=0·10). Preoperative therapy resulted in signifi cant downstaging of TNM5 compared with the postoperative group (p=0·04), including two pathological complete responses, apical node involvement (1% [one of 98] vs 20% [ten of 50], p<0·0001), resection margin involvement (4% [ four of 99] vs 20% [ten of 50], p=0·002), and blinded centrally scored tumour regression grading: 31% (29 of 94) vs 2% (one of 46) moderate or greater regression (p=0·0001). Interpretation Preoperative chemotherapy for radiologically staged, locally advanced operable primary colon cancer is feasible with acceptable toxicity and perioperative morbidity. Proceeding to the phase 3 trial, to establish whether the encouraging pathological responses seen with preoperative therapy translates into improved long-term oncological outcome, is appropriate

Codon alignment and predicted pairing partners in the stem-loop surrounding SA1 of SIVmac239 and HIV-1_NL4-3.

<p>(A) Structures for the conserved stem in SIVmac239 (left) and HIV-1_NL4-3 (right). Blue lines indicate the base pairs that are exactly conserved between the two viruses. (B) The sequences of SIVmac239 (top) and HIV-1_NL4-3 are aligned horizontally. Curved lines indicate base-pairing partners. Gray boxes indicate regions of amino acid alignment. Bold letters represent the bases that are involved in the conserved pairing interactions.</p

G minus A comparison for genomic sequences of various primate lentiviruses.

<p>(A) Organization of the SIVmac239 genome. Gray boxes indicate protein coding regions, dark lines indicate the boundaries of the mature viral proteins. (B) The number of adenosines was subtracted from the number of guanosines (G minus A) in a 75 nucleotide sliding window for selected primate lentiviruses. Dotted lines indicate the point where the difference between the number of G and A is zero for each individual virus. Values above the dotted line indicate higher guanosine concentration than adenosine. Viruses were aligned to SIVmac239 based on protein-coding regions. GenBank accession numbers for each virus are the following: SIVL'hoest, AF075269; SIVsyk173, L06042; SIVagm, M30931; HIV2BEN, M30502; HIV-1 MVP, L20571. Domain junctions on polyproteins are indicated by yellow bars. Other landmarks within the genome are indicated by gray bars.</p