NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNW NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

SeqDoC: rapid SNP and mutation detection by direct comparison of DNA sequence chromatograms

Background: This paper describes SeqDoC, a simple, web-based tool to carry out direct comparison of ABI sequence chromatograms. This allows the rapid identification of single nucleotide polymorphisms (SNPs) and point mutations without the need to install or learn more complicated analysis software. Results: SeqDoC produces a subtracted trace showing differences between a reference and test chromatogram, and is optimised to emphasise those characteristic of single base changes. It automatically aligns sequences, and produces straightforward graphical output. The use of direct comparison of the sequence chromatograms means that artefacts introduced by automatic base-calling software are avoided. Homozygous and heterozygous substitutions and insertion/deletion events are all readily identified. SeqDoC successfully highlights nucleotide changes missed by the Staden package 'tracediff' program. Conclusion: SeqDoC is ideal for small-scale SNP identification, for identification of changes in random mutagenesis screens, and for verification of PCR amplification fidelity. Differences are highlighted, not interpreted, allowing the investigator to make the ultimate decision on the nature of the change.

Introduction to the practice of telemedicine

Telemedicine is the delivery of health care and the exchange of health-care information across distances. It is not a technology or a separate or new branch of medicine. Telemedicine episodes may be classified on the basis of: (I) the interaction between the client and the expert (i.e. realtime or prerecorded), and (2) the type of information being transmitted (e.g. text, audio, video). Much of the telemedicine which is now practised is performed in industrialized countries, such as the USA, but there is increasing interest in the use of telemedicine in developing countries. There are basically two conditions under which telemedicine should be considered: (I) when there is no alternative (e.g. in emergencies in remote environments), and (2) when it is better than existing conventional services (e.g. teleradiology for rural hospitals). For example, telemedicine can be expected to improve equity of access to health care, the quality of that care, and the efficiency by which it is delivered. Research in telemedicine increased steadily in the late 1990s, although the quality of the research could be improved - there have been few randomized controlled trials to date.

Australian and New Zealand practice in intensive care (ANZPIC II): Predictors of physician confidence to diagnose pneumonia and determine illness severity in ventilated patients

The manner in which elements of clinical history, physical examination and investigations influence subjectively assessed illness severity and outcome prediction is poorly understood. This study investigates the relationship between clinician and objectively assessed illness severity and the factors influencing clinician's diagnostic confidence and illness severity rating for ventilated patients with suspected pneumonia in the intensive care unit (ICU). A prospective study of fourteen ICUs included all ventilated admissions with a clinical diagnosis of pneumonia. Data collection included pneumonia type - community-acquired (CAP), hospital-acquired (HAP) and ventilator-associated (VAP), clinician determined illness severity (CDIS), diagnostic methods, clinical diagnostic confidence (CDC), microbiological isolates and antibiotic use. For 476 episodes of pneumonia (48% CAP, 24% HAP, 28% VAP), CDC was greatest for CAP (64% CAP, 50% HAP and 49% VAP, P < 0.01) or when pneumonia was considered life-threatening (84% high CDC, 13% medium CDC and 3% low CDC, P < 0.001). Life-threatening pneumonia was predicted by worsening gas exchange (OR 4.8, CI 95% 2.3-10.2, P < 0.001), clinical signs of consolidation (OR 2.0, CI 95% 1.2-3.2, P < 0.01) and the Sepsis-Related Organ Failure Assessment (SOFA) Score (OR 1.1, CI 95% 1.1-1.2, P < 0.001). Diagnostic confidence increased with CDIS (OR 163, CI 95% 8.4-31.4, P < 0.001), definite pathogen isolation (OR 3.3, CI 95% 2.0-5.6) and clinical signs of consolidation (OR 2.1, CI 95% 1.3-3.3, P = 0.001). Although the CDIS, SOFA Score and the Simplified Acute Physiologic Score (SAPS II) were all associated with mortality, the SAPS II Score was the best predictor of mortality (P = 0.02). Diagnostic confidence for pneumonia is moderate but increases with more classical presentations. A small set of clinical parameters influence subjective assessment. Objective assessment using SAPS II Scoring is a better predictor of mortality.

Porous functionalised silica particles: a potential platform for biomolecular screening

Novel, porous, functionalised silica particles have been developed with controlled morphology, which promote covalent attachment of fluorescent dyes which can act as an optical barcode.

Prostanoids as friends, not foes: Further evidence from the interference by cycloxygenase-inhibitory drugs when inducing tolerance to experimental arthritigens in rats

Pharmacologists have generally been prejudiced against prostanoids, uncritically accepting their suppression as desirable therapy, especially for ‘quick-fix’ analgesia. This myopic perception for a long time ignored (a) the essentiality of prostanoid precursors in nutrition, (b) the physiological protective functions of natural prostaglandins (PGs) (vasculature, stomach, kidney), (c) resolution of inflammation after the expression of COX-2 and (d) increasing therapeutic use of either synthetic PGs (for erectile dysfunction, opthalmic disorders, inducing parturition, etc) or their natural precursors, e.g., ω3-rich polyunsaturated oils, to treat arthritis. Experimental studies in rats have indicated that prostaglandins (E series) are (i) useful, perhaps auto-regulators of established immunoreactivity and (ii) able to amplify (or even induce) anti-inflammatory activity with other agents. Furthermore, anti-prostanoid therapy (APT) can be arthritigenic!!, interfering with the acquisition of tolerance to some arthritigens. For patients with rheumatoid arthritis this additional side-effect of APT, barely recognised to date, may actually perpetuate their arthritis by impairing prostanoid-mediated remission processes. Hopefully, recent adverse publicity about COX-2 inhibitory drugs might stimulate serious re-assessment of some traditional anti-inflammatory therapies with low APT activity for the management of both acute pain (non-addictive cannabinoids, celery seed, etc.) and chronic inflammation, e.g., Lyprinol® (a mussel lipid extract).

Assessing the cellular transmembrane electrical potential difference on the hepatic uptake of palmitate

Understanding the driving forces for the hepatic uptake of endogenous and exogenous substrates in isolated cells and organs is fundamental to describing the underlying hepatic physiology/pharmacology. In this study we investigated whether uptake of plasma protein-bound [H-3]-palmitate across the hepatocyte wall is governed by the transmembrane electrical potential difference (PD). Uptake was studied in isolated hepatocytes and isolated perfused rat livers (IPL). Protein-binding and vasoactive properties of the different perfusates were determined using in vitro heptane/buffer partitioning studies and the multiple indicator dilution (MID) technique in the IPL, respectively. Altering hepatocyte PD by perfusate ion substitution resulted in either a substantial depolarization (-14 +/- 1 mV, n = 12, mean +/- S.E., substituting choline for Na+) or hyperpolarization (-46 +/- 3 mV, n = 12, mean +/- S.E., substituting nitrate for Cl-). Perfusate ion substitution also affected the equilibrium binding constant for the palmitate-albumin complex. IPL studies suggested that, other than with gluconate buffer, hepatic [H-3]-palmitate extraction was not affected by the buffer used, implying PD was not a determinant of extraction. [H-3]-Palmitate extraction was much lower (p < 0.05) when gluconate was substituted for Cl- ion. This work contrasts with that for the extraction of [H-3]-alanine where hepatic extraction fraction was significantly reduced during depolarization. Changing the albumin concentration did not affect hepatocyte PD, and [H-3]-palmitate clearance into isolated hepatocytes was not affected by the buffers used. MID studies with vascular and extravascular references revealed that, with the gluconate substituted buffer, the extravascular volume possibly increased the diffusional path length thus explaining reduced [H-3]-palmitate extraction fraction in the IPL.

The design of planar gradient coils. Part ll: A weighted superposition method

In this work a superposition technique for designing gradient coils for the purpose of magnetic resonance imaging is outlined, which uses an optimized weight function superimposed upon an initial winding similar to that obtained from the target field method to generate the final wire winding. This work builds on the preliminary work performed in Part I on designing planar insertable gradient coils for high resolution imaging. The proposed superposition method for designing gradient coils results in coil patterns with relatively low inductances and the gradient coils can be used as inserts into existing magnetic resonance imaging hardware. The new scheme has the capacity to obtain images faster with more detail due to the deliver of greater magnetic held gradients. The proposed method for designing gradient coils is compared with a variant of the state-of-the-art target field method for planar gradient coils designs, and it is shown that the weighted superposition approach outperforms the well-known the classical method.

FRAP analysis of nucleocytoplasmic dynamics of the vitamin D receptor splice variant VDRB1: preferential targeting to nuclear speckles

Although the key components of the cellular nuclear transport machinery have largely been characterized through extensive efforts in recent years, in vivo measurements of the kinetics of nuclear protein import/export are patently few. The present study applies the approach of FRAP (fluorescence recovery after photobleaching) to examine the nucleocytoplasmic flux of a novel human VDRB1 (vitamin D receptor B I) isoform in living cells. Through an N-terminal extension containing a consensus nuclear targeting sequence, VDRB1 is capable of localizing in nuclear speckles adjacent to SC-35 (35 kDa splicing component)containing speckles as well as in the nucleoplasm, dependent on ligand. Investigation of VDRB1 nucleocytoplasmic transport using FRAP indicates for the first time that the VDRB1 has a serum-modulated, active nuclear-import mechanism. There is no evidence of an efficient, active export mechanism for VDRB1, probably as a result of nuclear retention. VDRB1 nuclear import in the absence of serum occurred more rapidly and to a greater extent to nuclear speckles compared with import to other nuclear sites. This preferential transport from the cytoplasm to and accumulation within nuclear speckles is consistent with the idea that the latter represent dynamic centres of VDRB1 interaction with other nuclear proteins. The results are consistent with the existence of specialized pathways to target proteins to nuclear subdomains.