The barriers and facilitators for recognising distress in people with severe dementia on general hospital wards.

Introduction: psychological symptoms and delirium are common, but underreported in people with dementia on hospital wards. Unrecognised and untreated symptoms can manifest as distress. Identifying distress accurately therefore could act as a trigger for better investigation and treatment of the underlying causes. The challenges faced by healthcare professionals to recognise and report distress are poorly understood. Methods: semi-structured interviews with a purposive sample of 25 healthcare professionals working with older people in general hospitals were conducted. Interviews were analysed generating themes that describe the facilitators and barriers of recognising and caring for distress in dementia. Results: regardless of training or experience all participants had a similar understanding of distress, and identified it as a term that is easily understood and communicated. All participants believed they recognised distress innately. However, the majority also believed it was facilitated by experience, being familiar with their patients and listening to the concerns of the person's usual carers. Barriers to distress recognition included busy ward environments, and that some people may lack the skill to identify distress in hypoactive patients. Conclusion: distress may be a simple and easily identified marker of unmet need in people with dementia in hospital. However, modifiable and unmodifiable barriers are suggested that reduce the chance of distress being identified or acted on. Improving our understanding of how distress is identified in this environment, and in turn developing systems that overcome these barriers, may improve the accuracy with which distress is identified on hospital wards.

Characterization of the Vibrio fischeri Fatty Acid Chemoreceptors, VfcB and VfcB2.

Bacteria use a wide variety of methyl-accepting chemotaxis proteins (MCPs) to mediate their attraction to or repulsion from different chemical signals in their environment. The bioluminescent marine bacterium Vibrio fischeri is the monospecific symbiont of the Hawaiian bobtail squid, Euprymna scolopes, and encodes a large repertoire of MCPs that are hypothesized to be used during different parts of its complex, multistage lifestyle. Here, we report the initial characterization of two such MCPs from V. fischeri that are responsible for mediating migration toward short- and medium-chain aliphatic (or fatty) acids. These receptors appear to be distributed among only members of the family Vibrionaceae and are likely descended from a receptor that has been lost by the majority of the members of this family. While chemotaxis greatly enhances the efficiency of host colonization by V. fischeri, fatty acids do not appear to be used as a chemical cue during this stage of the symbiosis. This study presents an example of straight-chain fatty acid chemoattraction and contributes to the growing body of characterized MCP-ligand interactions.

Randomised controlled trial of a psychotherapeutic intervention to improve quality of life and other outcomes in people who repeatedly self-harm: FReSH START study protocol.

BACKGROUND: Self-harm is a major public health challenge, and repeated self-harm is common in those attending hospital following an episode. Evidence suggests psychological interventions could help people who self-harm, but few definitive studies have assessed their clinical and cost-effectiveness. Repeated self-harm is associated with poor quality of life, depression, suicide and increased health service costs which justify the development of psychotherapeutic interventions tailored for people with repeated self-harm. METHODS: FReSH START is a multicentre individually 1:1 randomised controlled trial evaluating the clinical and cost-effectiveness of standard care plus psychological therapy or standard care alone for adults (≥ 18 years) presenting at an emergency department (ED) with repeated self-harm. Recruiting 630 participants, it includes an internal pilot, economic evaluation and process evaluation. The intervention will be delivered by mental health staff working in acute settings, with experience of assessing and managing risk in people presenting to emergency services with self-harm. Staff will be trained and supervised to deliver one of three specially adapted therapies: psychodynamic interpersonal therapy, cognitive behavioural therapy or acceptance and commitment therapy. Participants allocated to the intervention will receive one of the adapted therapies according to therapist allocation for up to 6 months via 12 weekly, one to one, 45-50-min sessions. The primary outcome is quality of life measured by the Clinical Outcomes in Routine Evaluation Outcome Measure at 12 months post-randomisation. Secondary outcomes include suicidal intent, depression and cost-effectiveness. Data are collected using hospital attendance records and online/postal/telephone questionnaires at 6 and 12 months post-randomisation, with resource use additionally collected at 3 and 9 months. DISCUSSION: This protocol outlines a randomised controlled trial to investigate whether modified therapies are cost-effective and improve quality of life for people who repeatedly self-harm. Few interventions are proven to be deliverable in the NHS for this population. This study is strengthened by the involvement of qualified mental health workers experienced in managing risk as therapists. TRIAL REGISTRATION: Registered on August 03, 2021. IRAS number: 297939. ISRCTN: https://doi.org/10.1186/ISRCTN73357210 . REC reference: 21/EE/0145. SPONSOR: University of Leeds.

Molecular analyses of HIV-1 group O and HIV-2 variants from Africa.

Genetic variation among HIV isolates creates challenges for their detection by serologic and genetic techniques. To characterize the sequence variation and its correlation to serologic diversity of HIV-1 Group O and HIV-2 isolates, samples were identified by differential reactivity in selected commercial and research assays. Analysis of sera from Equatorial Guinea (EG) led to identification of 4 HIV-1 Group O variants. Viral RNA, extracted from these samples was used to PCR amplify overlapping sequences of the entire envelope gene using multiple primer pairs. Sequence analysis indicated that the V3 loop nucleotide and protein sequences aligned more closely with HIVANT70 compared to other Group O sequences. The amino acid sequences at the octameric tip of the V3 loop were RIGPLAWY, RIGPMAWY, or GLGPLAVY. The tetrameric tip GPLA is represented only once in the published 1994 HIV database (Los Alamos) but was present in 2 of 4 of EG samples. The immuno-dominant region (IDR) sequences derived from EG sera were unique in that none of the sequences were completely homologous to other HIV-1 group O variants. Further, the HIV-1 group O sequence variation could be correlated with differential serologic reactivity using IDR peptides. Compared to HIV-1, the sequence information on HIV-2 isolates is relatively limited, though the HIV-2 isolates also show genetic variation similar to HIV-1. To further establish a correlation between the genetic diversity and serologic detection of HIV-2, plasma samples from Western Africa were evaluated. Eight samples were selected based on weak serologic reactivity to env proteins. PCR amplification and sequence analysis of the gag, env V3 loop, and env IDR regions indicated that the samples could be classified as subtypes A (4 samples), B (3 samples) and D (1 sample). Across the subtypes, there was conservation in the IDR region of the sequence WGCAFRQVCHT. This region is absolutely conserved among the majority of currently known HIV-2 and related SIV viruses (1994 HIV database). One subtype B sample had a unique sequence immediately adjacent to the IDR, however, this did not change the serologic detection using a HIV-2 IDR specific monoclonal antibody.

Transgenic mice expressing either bovine growth hormone (bGH) or human GH releasing hormone (hGRH) have increased splenic progenitor cell colony formation and DNA synthesis in vitro and in vivo.

To investigate the potential effects of growth hormone (GH) on the hematopoietic system, mice transgenic for bovine GH (bGH) or human growth hormone releasing hormone (hGRH) genes, each of which can result in the constitutive overproduction of GH, were analyzed for splenic and bone marrow (BM) localized hematopoietic progenitor cells. These transgenic mice had splenic hyperplasia with increased absolute numbers of splenic erythroid and megakaryocytic progenitor cells as assessed by in vitro assay and megakaryocyte development as seen in spleens. As an in vivo indication of multilineage progenitor cell effects in hGRH mice, the number of day-10 CFU-S colonies derived from the donor spleen was significantly higher than in nontransgenic littermate controls. A high proportion (54-71%) of splenic erythroid, granulocyte-macrophage, and megakaryocyte progenitors were in cycle in transgenic mice in contrast to < or = 30% in nontransgenic control littermates. Compared to controls, splenocytes from hGRH mice had a significantly higher proliferative index when infused into irradiated nontransgenic controls. With the exception of the megakaryocyte colony assay and in vivo proliferative index, none of these findings were evident when identical assays were performed on the BM from the same mice. Consistent with the BM data, peripheral blood leukocyte, erythroid, and platelet numbers were comparable in transgenic and nontransgenic control littermates. We conclude that the constitutive expression of bGH or hGRH leads largely to splenic hematopoietic effects involving progenitor cell populations from at least two lineages.

Serologic and phylogenetic characterization of HIV-1 subtypes in Uganda.

OBJECTIVES: To determine the HIV genetic subtypes present in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate serologic detection of infection by commercial immunoassays; to evaluate samples for HIV-1 group O infections. METHODS: Sixty-four HIV-seropositive plasma samples were collected from the Nakasero Blood Bank, Kampala, Uganda. The plasma were evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. HIV-1 group M and O infections were identified on the basis of discordant seroreactivity in EIA and reactivity to group M and O antigens on the immunoblot. Regions of gag p24 and env gp41 were amplified using reverse transcriptase polymerase chain reaction, and genetic subtypes were determined by phylogenetic analysis. RESULTS: Serologic testing confirmed that 63 out of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of HIV-1 group O infections. Genetic subtyping determined that 25 samples were subtype A, three subtype C, 22 subtype D, and nine were heterogeneous for subtypes A and D. CONCLUSIONS: Despite the sequence variation observed in Uganda, commercial EIA based on HIV-1 subtype B proteins detected all the infections. In contrast, a peptide-based assay failed to detect three infections by subtype D viruses. This emphasizes the negative impact of HIV genetic variation on assays that rely on peptides to detect HIV infections. The number of infections with heterogeneous subtype (due to mixed infections or recombinant viruses) is high and reflects the growing complexity of the HIV epidemic in endemic regions where multiple subtypes are present in the population.

PIP: Extensive sequence heterogeneity between HIV-1 isolates has led to the classification of HIV-1 into group M (major) subtypes A-J, and group O (outlier). Some isolates have also been found to be the result of recombination between different group M subtypes. Findings are reported from a study conducted to determine the various HIV genetic subtypes in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate the serologic detection of infection by commercial immunoassays. 64 HIV-seropositive plasma samples were collected from the Nakasero Blood Bank in Kampala and evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. 63 of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of group O infections. According to phylogenetic analysis, 25 samples were subtype A, 3 subtype C, 22 subtype D, and 9 heterogenous for subtypes A and D. Despite the sequence variation observed in this study population, commercial EIA based upon HIV-1 subtype B proteins detected all of the infections. A peptide-based assay failed to detect 3 infections by subtype D viruses.

Identification of a new HIV-2 subtype based on phylogenetic analysis of full-length genomic sequence.

Human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus from sooty mangabey (SIV(SM) form one of the six primate lentivirus lineages. The close phylogenetic relationship and geographic coincidence indicate that HIV-2 originated from cross-species transmission of SIV(SM) to humans. HIV-2 exhibits considerable genetic diversity, with subtypes A-F identified. Previously, we reported the partial gag and env sequences of an unusual HIV-2 isolate, Abt96. Abt96 was collected in Ivory Coast from an asymptomatic blood donor. Here we describe the near full-length genomic sequence of Abt96. The genome was assembled from overlapping PCR fragments amplified from viral RNA isolated from plasma. Phylogenetic analysis of sequences derived from segments of the Abt96 genome demonstrate that the Abt96 isolate branches independently of all other characterized HIV-2 isolates. On the basis of the phylogenetic data being presented, we propose that Abt96 is a new HIV-2 subtype and designate it subtype G.

Sequence of gp41env immunodominant region of HIV type 1 group O from west central Africa.

PIP: HIV-1 group O is endemic in the west central region of Africa, where the frequency of infection is estimated to be 3-10% of all HIV-1-infected individuals. However, international travel and immigration have led to group O cases being identified in France, Germany, Belgium, Spain, and the US. With the exception of an infected French woman, all reported group O-infected individuals originate from or have a connection to west central Africa. Since most immunoassay reagents are based upon HIV-1 group M, many HIV immunoassays have lower sensitivity for the detection of group O infections. Serum samples were collected from patients at hospitals, tuberculosis (TB) clinics, and STD clinics in endemic regions of Cameroon and Equatorial Guinea in a study of the sequence divergence with group O isolate infections. Screening of the 1086 samples using a range of research and commercial immunoassays found 255 to be HIV-1 seropositive. On the basis of differential reactivity in the various immunoassays, 8 individuals were identified as potentially being infected with group O virus, of which 4 were drawn from TB patients. 7 of the group-O samples were then subjected to polymerase chain reaction (PCR) amplification to verify group O infection. The gp41(env) immunodominant region was successfully amplified and sequenced from 4 of the 7 samples, 2 of which were from the TB patients; 4 of 1086 samples were definitely infected with HIV-1 group O.^ieng

Mutations in an RNP1 consensus sequence of Rho protein reduce RNA binding affinity but facilitate helicase turnover.

Escherichia coli rho protein facilitates transcription termination by a mechanism that involves rho binding to the nascent RNA, activation of rho's RNA-dependent ATPase activity, and release of the mRNA from the DNA template. The initial step, formation of a rho-RNA complex, is mediated primarily by an RNA binding domain included within the amino-terminal 151 amino acids of rho protein. We have now identified one specific portion of this region that is involved in RNA binding, by photocross-linking and by site-directed mutagenesis. UV irradiation of rho-RNA complexes results in covalent attachment of the RNA to a single peptide in rho that apparently spans amino acids 45-100. Within this peptide is a ribonucleoprotein (RNP1) consensus sequence, Gly-Phe-Gly-Phe, that is present in many RNA-binding proteins. Mutagenesis of the phenylalanine residues in this consensus to leucine or alanine results in mutant proteins that are defective for RNA binding and have altered ATPase and RNA-DNA helicase activities. The weakened affinity but increased salt sensitivity of RNA binding by the mutant proteins suggests that they have lost more than just a set of nonionic interactions and are consistent with a change in the conformation of the RNA binding site. Whatever the changes, they appear localized primarily to the RNA binding domain because the mutants retain much of their RNA-dependent ATPase activity. We infer that the Phe residues themselves do not play a substantial role in the activation of ATP hydrolysis. Our results indicate that several different components of RNA interaction are required for rho activity and support a role for the RNP1 consensus region of rho in at least one specific aspect of RNA binding.

T4 RNA ligase catalyzed synthesis of base analogue-containing oligodeoxyribonucleotides and a characterization of their thermal stabilities.

Self-complementary oligodeoxyribonucleotides containing the base analogues 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, uracil, and 5-bromouracil were synthesized by a general method that allows incorporation of the analogues at specific positions. The method uses chemically synthesized partial sequences but circumvents the need for protected base analogues by incorporating their unprotected 3',5'-bisphosphate derivatives enzymatically. T4 RNA ligase was used to add the analogues to the oligodeoxyribonucleotides with yields from 54 to greater than 95 percent. Oligodeoxyribonucleotides were joined to the oligodeoxyribonucleotides containing the analogues at their 3'-termini in yields from 22 to 81 percent. The high yields obtained in these joinings suggest that RNA ligase should be of general use for the specific incorporation of other deoxyribonucleotide analogues into oligodeoxyribonucleotides. The oligodeoxyribonucleotides containing the base analogues were characterized by their mobilities during HPLC, nucleoside compositions, sequences, and thermal stabilities.

Determination of Drosophila photoreceptors: timing is everything.

This review covers recent findings concerning the specification of the photoreceptor subtypes in the Drosophila eye. Particular attention is paid to aspects of retinal patterning and differentiation where relative timing of events seems to be tightly controlled and essential for proper assembly of the compound eye. For example, specification of the founding photoreceptors of each cluster requires sequential positive and negative signaling through the Notch pathway, and reiterated signaling through the epidermal growth factor receptor leads to the pairwise recruitment of the distinct types of photoreceptors in discrete zones across the eye. Results suggest that different signaling environments for these two receptors may exist across the disc, and that receiving cells may constantly shift their predisposition to respond to such signals by adopting given fates. In addition, considerable data exist that the rate of expansion of retinal patterning across the disc is restricted to allow the orderly patterning of retinal precursors, and that one mechanism for controlling this rate may be the co-ordinated expression anterior to the furrow of factors which both inhibit and promote the expansion of retinal patterning. Finally, this review considers the possibility that the morphogenetic furrow serves as a moving source of morphogens which supply spatial information to both anterior and posterior tissue, providing temporal cues that regulate the many events involved in orderly assembly of the precise array of retinal cell types in the compound eye.

The preparative synthesis of oligodeoxyribonucleotides using RNA ligase.

The synthesis of nmol quantities of defined sequences of oligodeoxyribonucleotides using T4 RNA ligase has been demonstrated. Reacting using from 18 to 200 nmol of substrates in which a single 2'-deoxyribonucleoside 3',5'-bisphosphate was added to an oligodeoxyribonucleotide resulted in yields from 13 to 95%. When two oligodeoxyribonucleotides were similarly joined using RNA ligase, the yields ranged from 10 to 50%. Although the reactions contained high concentrations of enzyme and were incubated from 5 to 21 days, there was little degradation of either substrates or products. We have also characterized an unusual product which arises when 3'-phosphate terminated oligodeoxyribonucleotides are incubated with RNA ligase and high concentrations of ATP. This product has an adenylyl group linked to the 3'-phosphate by an anhydride bond. The mechanistic and synthetic implications of forming this product are discussed.

Transcription termination factor rho is an RNA-DNA helicase.

E. coli rho factor can unwind a short RNA-DNA duplex in vitro. The duplex is formed between a polylinker sequence at the 3' end of RNA derived from the rho-dependent terminator trp t' and the complementary sequence in a single-strand DNA molecule. Release of trp t' RNA from the duplex requires nucleoside triphosphate hydrolysis by rho's NTPase activity and is dependent on rho recognition of the RNA that is 5' to the RNA-DNA duplex region. The direction of helix unwinding appears to be 5' to 3' along the RNA molecule. These characteristics now account for how the RNA-binding and RNA-dependent NTP hydrolysis activities of rho may participate directly in transcription termination. Our results suggest that NTP hydrolysis is utilized to help unwind the RNA-DNA duplex at the 3' end of a nascent transcript, facilitating RNA release from the DNA template.

Ecdysone pathway is required for furrow progression in the developing Drosophila eye.

In Drosophila, secretion of the steroid hormone ecdysone from the prothoracic ring gland coordinates and triggers events such as molting and metamorphosis. In the developing Drosophila compound eye, pattern formation and cell-type specification initiate at a moving boundary known as the morphogenetic furrow. We have investigated the role of ecdysone in eye development and report here that the ecdysone signaling pathway is required for progression of the morphogenetic furrow in the eye imaginal disc of Drosophila. Genetic disruption both of the ecdysone signal in vivo with the ecdysoneless1 (ecd1) mutant and of ecdysone response with a Broad-Complex mutant result in disruption of morphogenetic furrow progression. In addition, we show that ecdysone-dependent gene expression, both of a reporter of transcriptional activity of the Ecdysone Receptor and of the Z1 isoform of the Broad Complex, are localized in and close to the furrow. These results suggest that, in the morphogenetic furrow, temporal hormonal signals are integrated into genetic pathways specifying spatial pattern.

A short intervening structure can block rho factor helicase action at a distance.

We have characterized the helicase activity of transcription termination factor rho on a variety of substrates. Helicase activity requires specific recognition of a single-stranded region of RNA upstream (5') of the nucleic acid duplex on which rho acts. Spacer sequences of at least 450 nucleotides can be inserted between the rho-binding signals and the duplex region with little effect on activity. RNA-DNA helices of up to 120 base pairs, but not as long as 210 base pairs, can be disrupted efficiently by rho. The stoichiometry of release of substrates with long spacer sequences, as with the standard substrate, approaches a value of one RNA released per rho hexamer; thus cooperative binding by rho does not account for action at a distance. Instead, these results are consistent with a model in which a single rho hexamer binds initially to terminator sequences and then either loops out or tracks along the intervening RNA to reach the duplex region. Results with complex substrates are inconsistent with looping and support the tracking model: under conditions that allow disruption of RNA-DNA, but not RNA-RNA helices (0.4 mM Mg2+), the presence of a short RNA-RNA helix acts as a block to the disruption of an RNA-DNA helix downstream. These findings are discussed in relation to the mechanism of the helicase activity as well as its role in rho-dependent transcription termination.

The incidence and mortality for meningococcal disease associated with area deprivation: an ecological study of hospital episode statistics.

AIMS: To determine whether incidence, mortality, and case fatality for meningococcal disease (MD) differs by area deprivation, and if this has changed over time. METHODS: The population of children aged less than 5 years with MD was analysed as quintiles of area deprivation scores over two time periods, 1995-99 and 1991-94. Annual age standardised rates were calculated and the association between incidence, mortality, and area deprivation quintiles assessed using Poisson regression and the risk ratios determined. Case fatality was calculated from the odds ratio of mortality by area deprivation score for the two time periods. RESULTS: There were 10,524 cases of MD and 441 deaths (4.2%). Incidence rates were higher for 1995-99 (45.4 per 100,000) compared to 1991-94 (27.4 per 100,000). Mortality rates remained stable over time, indicating a decline in risk of death of around 40%. The incidence rates for the most deprived quintile were around twice those for the most affluent quintile, but this gradient declined over time. A threefold gradient was seen for mortality rates across the top and bottom quintiles, which was constant over time. The odds of mortality did not show a linear pattern, with mortality being lowest in the first and highest in the second and fifth area deprivation quintiles. CONCLUSIONS: These data show that MD incidence and mortality are socially patterned. The determinants of case fatality are more complex and require further investigation.

The effects of base analogue substitutions on the methylation by the EcoRI modification methylase of octadeoxyribonucleotides containing modified EcoRI recognition sequences.

We have examined the DNA-protein interactions involved in the recognition of a specific hexameric sequence, GAATTC, by the EcoRI modification methylase by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues 2-aminopurine, 5-bromocytosine, 5-bromouracil, 2,6-diaminopurine, hypoxanthine, 5-methylcytosine, N6-methyladenine, and uracil were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the ability of the enzyme to methylate the modified substrates were monitored by determining the steady state kinetic values of the reaction in the presence of the cosubstrate, S-adenosylmethionine. The substitutions resulted in effects ranging from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those analogues that were active, whereas the octanucleotides containing hypoxanthine at the guanine site, N6-methyladenine at the first or 2-aminopurine at the second adenine site, or uracil at the second thymine site were completely inactive. The results are discussed in terms of the possible interactions between the methylase and its recognition sequence. In addition, the interactions are compared to those of the EcoRI restriction endonuclease, which has been similarly tested with the same analogue oligonucleotides. The results of that study are reported in an accompanying paper. Although both enzymes recognize the same sequence, they do so in different ways.