Evaluation of a point-of-care blood test for identification of Ebola virus disease at Ebola holding units, Western Area, Sierra Leone, January to February 2015.

Current Ebola virus disease (EVD) diagnosis relies on reverse transcription-PCR (RT-PCR) technology, requiring skilled laboratory personnel and technical infrastructure. Lack of laboratory diagnostic capacity has led to diagnostic delays in the current West African EVD outbreak of 2014 and 2015, compromising outbreak control. We evaluated the diagnostic accuracy of the EVD bedside rapid diagnostic antigen test (RDT) developed by the United Kingdom's Defence Science and Technology Laboratory, compared with Ebola virus RT-PCR, in an operational setting for EVD diagnosis of suspected cases admitted to Ebola holding units in the Western Area of Sierra Leone. From 22 January to 16 February 2015, 138 participants were enrolled. EVD prevalence was 11.5%. All EVD cases were identified by a positive RDT with a test line score of 6 or more, giving a sensitivity of 100% (95% confidence interval (CI): 78.2-100). The corresponding specificity was high (96.6%, 95% CI: 91.3-99.1). The positive and negative predictive values for the population prevalence were 79.0% (95% CI: 54.4-93.8) and 100% (95% CI: 96.7-100), respectively. These results, if confirmed in a larger study, suggest that this RDT could be used as a 'rule-out' screening test for EVD to improve rapid case identification and resource allocation.

Duplication of ATR inhibits MyoD, induces aneuploidy and eliminates radiation-induced G1 arrest.

Chromosome 3q alterations occur frequently in many types of tumours. In a genetic screen for loci present in rhabdomyosarcomas, we identified an isochromosome 3q [i(3q)], which inhibits muscle differentiation when transferred into myoblasts. The i(3q) inhibits MyoD function, resulting in a non-differentiating phenotype. Furthermore, the i(3q) induces a 'cut' phenotype, abnormal centrosome amplification, aneuploidy and loss of G1 arrest following gamma-irradiation. Testing candidate genes within this region reveals that forced expression of ataxia-telangiectasia and rad3-related (ATR) results in a phenocopy of the i(3q). Thus, genetic alteration of ATR leads to loss of differentiation as well as cell-cycle abnormalities.

A Rad3-Rad26 complex responds to DNA damage independently of other checkpoint proteins.

The conserved PIK-related kinase Rad3 is required for all DNA-integrity-checkpoint responses in fission yeast. Here we report a stable association between Rad3 and Rad26 in soluble protein extracts. Rad26 shows Rad3-dependent phosphorylation after DNA damage. Unlike phosphorylation of Hus1, Crb2/Rhp9, Cds1 and Chk1, phosphorylation of Rad26 does not require other known checkpoint proteins. Rad26 phosphorylation is the first biochemical marker of Rad3 function, indicating that Rad3-related checkpoint kinases may have a direct role in DNA-damage recognition.

The chk1 pathway is required to prevent mitosis following cell-cycle arrest at 'start'.

BACKGROUND: The G2-M-phase transition is controlled by cell-cycle checkpoint pathways which inhibit mitosis if previous events are incomplete or if the DNA is damaged. Genetic analyses in yeast have defined two related, but distinct, pathways which prevent mitosis--one which acts when S phase is inhibited, and one which acts when the DNA is damaged. In the fission yeast Schizosaccharomyces pombe, many of the gene products involved have been identified. Six 'radiation checkpoint' (rad) gene products are required for both the S-M and DNA-damage checkpoints, whereas Chk1, a putative protein kinase, is required only for the DNA-damage checkpoint and not for the S-M checkpoint following the inhibition of DNA synthesis. RESULTS: We have genetically defined a third mitotic control checkpoint pathway in fission yeast which prevents mitosis when passage through 'start' (the commitment point in G1) is compromized. In cycling cells arrested at start, mitosis is prevented by a Chk1-dependent pathway. In the absence of Chk1, G1 cells attempt an abortive mitosis with a 1C DNA content without entering S phase. Similar results are seen in the absence of Rad17, a typical example of a rad gene product. CONCLUSIONS: Genetic dissection of checkpoints in logarithmically growing fission yeast has identified a pathway that couples mitosis to correct passage through start. This pathway is related to the DNA-structure check-points which ensure that mitosis is dependent on the completion of replication and the integrity of the DNA. We propose that all three mitotic control checkpoints monitor distinct DNA or protein structures at different stages in the cell cycle.

Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator.

The tyrosinase gene is expressed specifically in melanocytes and the cells of the retinal pigment epithelium, which together are responsible for skin, hair, and eye color. By using a combination of DNase I footprinting and band shift assays coupled with mutagenesis of specific DNA elements, we examined the requirements for melanocyte-specific expression of the human tyrosinase promoter. We found that as little as 115 bp of the upstream sequence was sufficient to direct tissue-specific expression. This 115-bp stretch contains three positive elements: the M box, a conserved element found in other melanocyte-specific promoters; an Sp1 site; and a highly evolutionarily conserved element located between -14 and +1 comprising an E-box motif and an overlapping octamer element. In addition, two further elements, one positive and one negative, are located between positions -185 and -150 and positions -150 and -115, respectively. We also found that the basic helix-loop-helix factor encoded by the microphthalmia gene, which is essential for melanocyte differentiation, can transactivate the tyrosinase promoter via the M box and the conserved E box located close to the initiator. Since in vitro assays failed to identify any melanocyte-specific DNA-binding activity, the possibility that the specific arrangement of elements within the basal tyrosinase promoter determines melanocyte-specific expression is discussed.

Platelet and coagulation function in patients with abnormal cardiac valves treated with sulphinpyrazone.

Eight patients on warfarin with rheumatic heart disease and prosthetic cardiac valves were selected for study on the basis of persistently elevated plasma beta-thromboglobulin (beta-tg) and platelet factor 4 (PF4) concentrations. Platelet mean lifespan and fibrinogen half life were short, and positively correlated, and both were inversely related to the plasma concentration of the platelet specific proteins. Antithrombin III (ATIII) levels were also reduced. Treatment with sulphinpyrazone resulted in lengthening of both platelet and fibrinogen survival, a rise in ATIII but no change in the beta tg or PF4 concentrations. It is concluded that patients with abnormal cardiac valves and raised plasma levels of beta tg or PF4 have, despite warfarin, a consumption coagulopathy that can be inhibited by sulphinpyrazone.

Comparison of a modified thiazole orange technique with a fully automated analyser for reticulocyte counting.

Two independent methods for quantitating reticulocyte counts were compared. One used a modified thiazole orange technique and a flow cytometer (Becton Dickinson FACS); the other was a fully automated whole blood analyser (Sysmex R1000). Both methods gave comparable results with a coefficient of variation of less than 5%. Samples measured using the R1000 showed a negligible decrease in the reticulocyte count over five days at room temperature, although there was evidence of continuing intracellular maturation: with thiazole orange there was an apparent increase. A practical reference range of 20-70 x 10(9)/l was established from 89 normal subjects. The close correlation between the two independent estimates indicates the validity of the quantitation of the reticulocyte count and shows that automation allows significant changes within and below the normal range to be detected with a degree of reliability which was not previously possible.

Real-time PCR investigation into the importance of Fusobacterium necrophorum as a cause of acute pharyngitis in general practice.

Fusobacterium necrophorum is recognized as the cause of a severe life-threatening illness characterized by bacteraemia with metastatic abscesses following an acute sore throat (Lemierre's disease). However, the importance of F. necrophorum as a cause of simple sore throat in the community is unknown. Using quantitative real-time PCR with primers targeting the rpoB gene, 100 routine throat swabs collected from patients presenting to general practitioners with pharyngitis were analysed for the presence of F. necrophorum-specific DNA. The results were compared with those obtained from throat swabs collected from 100 healthy subjects. Ten clinical samples were positive for F. necrophorum DNA, identified as F. necrophorum subspecies funduliforme, using a haemagglutinin-related protein gene-specific PCR assay. All the healthy controls were negative (two-tailed P value = 0.0015; Fisher exact test). These findings suggest that F. necrophorum may play a more important role as a cause of simple sore throat in the community than has been previously appreciated.

Prevalence, clinical features and quantification of genital non-viral infections.

We conducted a study of the prevalence, clinical features and microscopy findings, by retrospective case-notes survey, of six non-viral organisms, among 1718 attendees at a genitourinary (GU) medicine clinic in England. An in-house assay for six non-viral infections was used and quantitation of ureaplasmas performed. The prevalences of the six organisms were: Chlamydia trachomatis (CT), 7.1%; Neisseria gonorrhoeae (NG), 0.6%; Mycoplasma genitalium (MG), 1.0%; Trichomonas vaginalis, 0.2%; Ureaplasma urealyticum, 16.1%; Ureaplasma parvum, 35.6%. Among men (but not women) there were significantly raised odds ratios compared with that for U. parvum, for the symptom of discharge with CT, 7.30; MG, 6.43; NG 19.29; dysuria with CT, 5.89 and MG, 5.95; and the microscopy finding of >4 pus cells per high power field with: CT, 7.22; MG, 4.58 and NG 22.31. Evaluation of a possible link between quantitation of U. urealyticum and urethritis did not confirm research findings elsewhere.

The role of the midwife in family planning.

PIP: There are many midwives working in the family planning clinic who have been given the impression that because they are members of the RCM they are no longer eligible for membership of the Family Planning Nurses Forum. This has caused much upset, and they now feel that their opinions on family planning matters are being ignored. The purpose of the Rcn draft document is to attempt to persuade doctors to allow the family planning nurse more scope, but midwives who are trained to work in the family planning clinics do not appear to suffer from the same problem. In fact, any midwife worthy of the title should be capable of carrying out every duty that she is trained to do. The Forum document indicates that the Rcn Family Planning Nurses Forum has become more and more aware that nurses engaged in family planning are extending their role and taking on duties that they were not taught as part of the Family Planning Association course and which are not included in the Joint Board of Clinical Nursing Studies Course 900. Some contradiction appears to exist between the "Nursing Times" article and the Forum document, allowing a nurse more scope within her existing role and undertaking duties that have not been taught are 2 different matters. No responsible nurse would undertake duties for which she has not been trained. It is incumbent on any family planning midwife or nurse to know the policy of the employing authority regarding her duties within the family planning team. The only duties which would appear to come within the terms of the extended role of the nurse would be the prescribing of oral contraception (OC) as opposed to dispensing it. This extension of a nurse's role would constitute a need to change the JBCNS Course 900 syllabus to include a module of training that would equip the family planning nurse to function effectively. Midwives feel strongly that any nurse undergoing family planning training must be taught every part of the syllabus and should not be given the JBCNS Course 900 Certificate of Competence unless they are able to carry out all the duties required of a family planning nurse. Regarding midwives and nurses, it seems an appropriate start for members of the RCM and Rcn to be able to participate fully in the Family Planning Nurses Forum and for the RCM to be seen as active regarding the midvwives' involvement in family planning.^ieng

DNA structure-dependent checkpoints in model systems.

DNA structure dependent checkpoints require a number of proteins which function to arrest the cell cycle in response to DNA damage (such as UV induced lesions) or blocks to DNA replication. Analogous to a signal transduction pathway, checkpoints communicate information between a DNA lesion and the cell cycle machinery. This brief review will focus on yeast model systems which have been instrumental in identifying the various components (initiating signal, detection, signal transduction and cell cycle effector) of the checkpoint pathways. The biological significance of these pathways in mammalian cells is illustrated in patients with ataxia telangiectasia (AT), a multi-system cancer-prone disorder in which DNA damage checkpoints affecting both DNA replication and mitosis are lost. ATM, the gene mutated in this disorder is structurally related to the yeast rad3/MEC1 checkpoint genes. This demonstrates the high degree of evolutionary conservation of checkpoints amongst eukaryotic organisms.

Studies in red blood cell preservation. 8. Liquid storage of red cells in a glycerol-containing additive solution.

The purpose of the present study was to determine whether a hypotonic additive containing a low concentration of glycerol as a membrane permeable solute would improve the liquid storage of red blood cells (RBCs). Packed RBCs were stored either with 200 ml of an experimental additive solution, EAS 25, containing (mM): glycerol 150, adenine 2, glucose 110, mannitol 55, and NaCl 50, or with 100 ml/unit of a conventional additive solution Adsol. The results show that the adenosine triphosphate values, hemolysis, potassium leakage, and the morphology scores of RBCs were significantly better with EAS 25 than with Adsol up to 84 days of storage. The ATP values were significantly different only after the first 42 days of storage. The mean corpuscular volumes (MCVs) of the RBCs were significantly higher throughout in the experimental additive accompanied by decreased microvesiculation as compared to Adsol. The total microvesicle membrane protein shed by 100 ml of RBCs was 47.92 +/- 12.31 mg in Adsol and 18.96 +/- 5.49 mg in EAS 25 (p < 0.001). The larger MCVs of the RBCs in EAS 25 may have a favorable effect on maintaining membrane integrity by decreasing the loss of membrane by microvesiculation.

The small heat-shock protein Hsp26 of Saccharomyces cerevisiae assembles into a high molecular weight aggregate.

Hsp26 is one of the major small heat-shock proteins (Hsp) of the yeast Saccharomyces cerevisiae, yet its cellular role remains to be discovered. To examine the cellular consequences of overexpression of Hsp26, the gene encoding this protein (HSP26) was overexpressed from a multicopy plasmid using either its own promoter or by coupling it to the efficient constitutive PGK promoter. The PGK promoter provided the opportunity to overexpress Hsp26 under non-stress conditions and such high level synthesis, prior to a lethal heat shock (50 degrees C), gave a small but reproducible elevation in thermotolerance. In transformed strains overexpressing Hsp26 under either stressed or non-stress conditions, the Hsp26 polypeptide was recovered almost exclusively as a high molecular weight aggregate. This high molecular weight aggregate (or heat-shock granule; HSG) was purified by differential centrifugation and sucrose gradient density centrifugation and shown, by electron microscopic analysis, to be of a uniform size (15-25 nm diameter). Analysis of the purified HSG demonstrated that it had a molecular weight of 550 kDa, yet contained no other integral polypeptides or other macromolecules.

Mik1 levels accumulate in S phase and may mediate an intrinsic link between S phase and mitosis.

Two paradigms exist for maintaining order during cell-cycle progression: intrinsic controls, where passage through one part of the cell cycle directly affects the ability to execute another, and checkpoint controls, where external pathways impose order in response to aberrant structures. By studying the mitotic inhibitor Mik1, we have identified evidence for an intrinsic link between unperturbed S phase and mitosis. We propose a model in which S/M linkage can be generated by the production and stabilization of Mik1 protein during S phase. The production of Mik1 during unperturbed S phase is independent of the Rad3- and Cds1-dependent checkpoint controls. In response to perturbed S phase, Rad3-Cds1 checkpoint controls are required to maintain high levels of Mik1, probably indirectly by extending the S phase period, where Mik1 is stable. In addition, we find that Mik1 protein can be moderately induced in response to irradiation of G(2) cells in a Chk1-dependent manner.

Evaluation of erythropoiesis after bone marrow transplantation: quantitative reticulocyte counting.

Erythroid regeneration is an important and separate element in the engraftment process in allogeneic and autologous bone marrow transplantation (alloBMT, autoBMT). Qualitative visual reticulocyte counting has proved inadequate in the evaluation of erythropoiesis after BMT but automated flow cytometry now allows the reliable quantitation of reticulocytes even to very low levels. Reticulocyte counts and highly fluorescent reticulocyte (HFR) counts (very early reticulocytes) were estimated daily in recipients of 22 autoBMT and 14 alloBMT using a Sysmex R-1000 automated reticulocyte counter. Marrow ablation caused an immediate and rapid fall in both the reticulocyte count and the HFR. Measurable numbers of reticulocytes persisted throughout the hypoplastic period, but HFR fell to zero in the majority of both the autoBMT and alloBMT. HFR rose significantly after a median time of 14 d post-autoBMT, and 12 d post-alloBMT. Attainment of 15 x 10(9)/l reticulocytes and 0.5 x 10(9)/l HFR at day 21 post-transplant was associated with ultimate engraftment in 100% cases. Inadequate engraftment was seen in the majority of patients whose responses fell below these levels. Graft-versus-host disease was associated with a transient slight reduction in reticulocyte count. Neither episodes of infection nor blood transfusions had any significant impact on trends of reticulocytes or HFR. Automated flow cytometric reticulocyte counting has been shown to provide an accessible measure of erythroid activity which may be of predictive value in the management of patients following bone marrow transplantation.

The Schizosaccharomyces pombe rad3 checkpoint gene.

The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei-41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the 'lipid kinase' subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei-41. ATR can functionally complement esr1-1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution.

Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control.

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia-telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.