AAV ancestral reconstruction library enables selection of broadly infectious viral variants.

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV's evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.

Upgrades to the ion cyclotron emission diagnostic on the DIII-D tokamak.

The ion cyclotron emission diagnostic on the DIII-D tokamak comprises seven single-turn loops that measure high-frequency (1-100 MHz) magnetic field fluctuations that are often excited by energetic particles in the plasma. The raw voltage signals induced in the loops in response to these fluctuations travel through a series of cables, isolation transformer DC blocks, low-pass filters, and finally a digitizer before being analyzed in frequency space. The diagnostic has been recently upgraded, most notably to include four additional graphite tile loops and a new eight-channel digitizer. The previous three loops are all on the low-field side of the tokamak. The measurement capabilities of the system have been expanded by the addition of a new horizontally oriented loop on the low-field side, an additional toroidal loop on the low-field side, and two toroidal loops on the high-field side. These loops will be used to provide approximate mode polarization, improved toroidal mode number calculations, and information on modes in inward-shifted plasmas, respectively.

Perceptions of anthrax in livestock from Victorian dairy farmers in the Goulburn-Murray region of Victoria, Australia.

To inform future anthrax surveillance and response activities and to revitalise the communication strategy for producers and their communities, seven dairy farmers in the Goulburn-Murray region of Victoria participated in a Design Thinking process to create a better method to share information about the annual probability of anthrax in their region. Design Thinking is a structured, user-centric design process that begins with intentionally un-structured interviews. Following each interview, transcripts are disassembled into common themes identified by clustering similar statements from these interviews. This short contribution presents these themes re-framed into eight core statements. These statements provide a framework for the remainder of the Design Thinking process but in isolation provide a reference for stake-holding agencies seeking to maximise farmer participation in surveillance programs for early anthrax detection, to encourage active farmer participation during a response and to minimise any anthrax-associated stigma by affected farmers post-response.

Cholesterol-lowering effect of mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme a reductase, in healthy volunteers.

Mevinolin reduces cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase. The safety and effectiveness of this agent was evaluated in a double-blind, placebo-controlled study in 59 healthy men (serum cholesterol 3.88--7.76 mmol/liter) in five centers. Subjects maintained their usual diet and activities. Doses of 6.25, 12.5, 25, or 50 mg twice daily for 4 wk produced mean reductions of total serum cholesterol fo 23--27% [vs. placebo (4%), P less than 0.01]. Mean low density lipoprotein cholesterol fell 35--45%, while high density lipoprotein and very low density lipoprotein cholesterol, and triglycerides were not significantly affected. Mean apolipoprotein B fell 27--34%. 50 mg was not significantly more effective than 6.25 mg. Mevinolin was generally well tolerated, and no serious clinical or laboratory abnormalities occurred. One subject (12.5 mg) was withdrawn because of abdominal pain and diarrhea. These results suggest that if long-term safety can be demonstrated, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase are likely to prove useful in the treatment of hypercholesterolemia.

No evidence of Mycobacterium avium subsp. paratuberculosis in in vitro produced cryopreserved embryos derived from subclinically infected cows.

The aim of the project was to ascertain if Mycobacterium avium subsp. paratuberculosis (Map) could be cultured from frozen-thawed in vitro produced (IVP) embryos derived from cows with subclinical Johne's disease (JD). Straws of 109 IVP embryos were obtained from 267 cumulus-oocyte-complexes (COCs) collected from 12 clinically normal cows in which antibodies against Map were detected in blood by an enzyme-linked immunosorbent assay (ELISA). These embryos were processed, washed using the standard protocol as described by the International Embryo Transfer Society (IETS) and frozen in a commercial IVP embryo laboratory. Of the 12 donor cows, 11 had histopathological or bacteriological evidence of infection at post-mortem inspection. The frozen embryos were thawed and the contents of the straws were cultured using the radiometric mycobacterial culture method. No Map was detected in any of the 109 embryos or freezing media. This suggests that the use of in vitro produced and cryopreserved embryos derived from cows with subclinical JD poses very low, if any, risk of spreading infection to susceptible animals.

An expectation maximization algorithm for training hidden substitution models.

We derive an expectation maximization algorithm for maximum-likelihood training of substitution rate matrices from multiple sequence alignments. The algorithm can be used to train hidden substitution models, where the structural context of a residue is treated as a hidden variable that can evolve over time. We used the algorithm to train hidden substitution matrices on protein alignments in the Pfam database. Measuring the accuracy of multiple alignment algorithms with reference to BAliBASE (a database of structural reference alignments) our substitution matrices consistently outperform the PAM series, with the improvement steadily increasing as up to four hidden site classes are added. We discuss several applications of this algorithm in bioinformatics.

Successful pregnancy after high dose chemotherapy and marrow transplantation for treatment of aplastic anemia.

A 29-year-old woman with severe idiopathic aplastic anemia was given immunosuppressive therapy with procarbazine, 37.5 mg/kg, antithymocyte globulin, 36 mg IgG/kg, and cyclophosphamide, 200 mg/kg. This was followed by a marrow transplant from her HLA identical sister, immunosuppressive therapy with intermittent methotrexate for 3 months postgrafting and ultimate restoration of hematopoiesis. Two years after transplantation the patient delivered a healthy male infant. This is the first successful pregnancy after a high dose chemotherapy and marrow transplantation for treatment of aplastic anemia.

Enhancement of uricosuric properties of indacrinone by manipulation of the enantiomer ratio.

Racemic indacrinone is a high-ceiling, relatively long-acting diuretic. Both enantiomers have uricosuric activity, but the diuretic activity resides predominantly in the (-) enantiomer. Usual therapeutic doses of racemic indacrinone have only transient uricosuric activity, so that, as with other diuretics, hyperuricemia occurs. Sixty-five healthy men participated in a multicenter, double-blind, randomized, balanced, incomplete-block study comparing the effects on plasma urate and urate clearance of indacrinone (-) enantiomer 10 mg given concomitantly with 0, 10, 20, 40, and 80 mg (+) enantiomer (10/0, 10/10, 10/20, 10/40, 10/80), as single daily doses for 7 days. Hydrochlorothiazide (HCTZ) 50 mg daily and ticrynafen (T) 250 mg daily were controls. Each subject received two of the seven treatments, so that there were 18 subjects per treatment. On days 7 to 8, morning (mean of 0-hr values on days 7 and 8), HCTZ, 10/0, 10/10, and 10/20 elevated plasma urate by 8% to 16%. 10/40 was approximately isouricemic, and 10/80 and T lowered plasma urate by 13% and 41%. There were corresponding changes in urate clearance.

Periplasmic expression of part of the major rotavirus capsid protein VP7 containing all the three antigenic regions in Escherichia coli.

Part of the porcine rotavirus outer capsid protein VP7 containing all the three antigenic regions was expressed as a chimeric protein with bacterial alkaline phosphatase (AP) in E. coli. The construct contains an ompF promoter, the DNA encoding the signal sequence and the first 12 amino acids of mature OmpF, part of vp7 and the DNA encoding mature AP. The chimeric protein is stable, retains the biological property of AP and ability to react with polyclonal antiserum against the virus, and can be exported through the bacterial inner membrane into the periplasm.

Rapid and substantial lowering of human serum cholesterol by mevinolin (MK-803), an inhibitor of hydroxymethylglutaryl-coenzyme A reductase.

Mevinolin (MK-803) is a potent inhibitor of HMG-CoA reductase. After a placebo run-in period, mevinolin 5,15 or 50 mg, or placebo was given twice daily for 7-11 days under double -blind conditions ot 4 groups of 6 normocholesterolemic male volunteers. After 7 days, mean serum cholesterol fell 14%, 25% and 24% on 5, 15 and 50 mg, respectively, which was significantly greater than the fall on placebo (4%) in the case of the two higher doses (P less than 0.01). Serum triglycerides did not change significantly. Mevinolin was generally well-tolerated and there were no serious adverse effects.

Dynamic programming alignment accuracy.

Algorithms for generating alignments of biological sequences have inherent statistical limitations when it comes to the accuracy of the alignments they produce. Using simulations, we measure the accuracy of the standard global dynamic programming method and show that it can be reasonably well modelled by an "edge wander" approximation to the distribution of the optimal scoring path around the correct path in the vicinity of a gap. We also give a table from which accuracy values can be predicted for commonly used scoring schemes and sequence divergences (the PAM and BLOSUM series). Finally we describe how to calculate the expected accuracy of a given alignment, and show how this can be used to construct an optimal accuracy alignment algorithm which generates significantly more accurate alignments than standard dynamic programming methods in simulated experiments.

Synthesis and biological evaluation of sialylmimetics as rotavirus inhibitors.

Rotaviruses cause severe gastroenteritis in infants and are estimated to be responsible for over 600 000 deaths annually, primarily in developing countries. The development of potential inhibitors of this virus is therefore of great interest, particularly since the safety and efficacy of rotaviral vaccines has recently been questioned. This study describes the synthesis of a variety of compounds that can be considered as mimetics of N-acetylneuraminic acid thioglycosides and the subsequent in vitro biological evaluation of these sialylmimetics as inhibitors of rotaviral infection. Our results show that readily accessible carbohydrate-based compounds have the potential to act as inhibitors of rotaviral replication in vitro, presumably through inhibition of the rotaviral adhesion process.

Synthesis and biological evaluation of N-acetylneuraminic acid-based rotavirus inhibitors.

Rotavirus can cause severe gastrointestinal disease, especially in infants and young children, and is particularly prevalent in Third-World countries. Therefore, the development of potential inhibitors of this virus is of great interest. The present study describes the synthesis and in vitro biological evaluation of a number of N-acetylneuraminic acid-based compounds as potential rotavirus inhibitors. Our data suggests that it is indeed possible to inhibit adhesion of the virus, and hence in vitro replication, with carbohydrate-based molecules, although this inhibition does appear to be strain dependent.

Potentiating effect of adrenaline on adenosine diphosphate-induced reduction in rabbit circulating platelet count: inhibition by dihydroergotoxine.

The number of circulating platelets was monitored in anaesthetized rabbits by a continuous flow technique, using a Technicon Autocounter. Transient reductions in circulating platelet count induced by a submaximal dose of adenosine diphosphate (ADP) were potentiated by concomitant infusion of adrenaline at doses (1-25 microgram/kg) i.v.) which did not influence platelet count when infused alone. The adrenaline effect was dose-dependent. Repeated infusions of adrenaline at 15 min intervals resulted in reproducible reductions in circulating platelet count during an observation period of at least 105 min. Dihydroergotoxine (DHET), administered either i.v. (2.5-10 microgram/kg) or intraduodenally (i.d.; 25-100 microgram/kg), inhibited adrenaline-induced potentiation dose-dependently; ADP-induced effects were not influenced. Duration of action was relatively long, and significant inhibitory activity was still apparent 50 (i.v.) and 115 (i.d.) min after drug administration. DHET doses inhibiting adrenaline-induced potentiation of platelet aggregation in the rabbit are similar to doses used in the treatment of impaired human cerebral function. It is conceivable that DHET could prevent activation of human platelets by catecholamines released into the blood stream in clinical stress situations.

The effect of intravenous adenosine diphosphate on the number of circulating platelets in experimental animals: inhibition by prostaglandin E1, dipyridamole, SH-869 and VK-774.

The number of circulating platelets was monitored in anaesthetized animals by a continuous flow technique, using a Technicon Autocounter. Intravenous infusions of adenosine diphosphate (ADP) produced transient, dose-dependent falls in circulating platelet numbers in rabbits, dogs, rats, pigs and squirrel monkeys. The rat was the most sensitive of the species investigated. In the rabbit, the effect of a submaximal dose of ADP was inhibited in a dose-dependent manner by intravenous infusions of prostaglandin E1 (PGE1), dipyridamole, and two derivatives of dipyridamole (SH-869 and VK-774). The dose-response curves for PGE1, SH-869 and VK-774 were approximately parallel, whereas that for dipyridamole was considerably less steep. PGE1 was the most potent inhibitor, but the duration of action was very short. Dipyridamole and SH-869 produced inhibition of long duration. The duration of action of VK-774 was intermediate. All inhibitors produced marked and often long-lasting hypotension. The fact no inhibition of ADP effects could be demonstrated with dibenzyline and hexamethonium, which also produced marked hypotension of long duration, indicated that inhibition of the ADP effect by the four antagonists studied was not due to changes in blood pressure.

Expression of rotavirus VP7 antigens in fusions with bacterial proteins.

We have cloned antigenic regions of the VP7 gene from rotavirus RV-5 into the lamB gene. The insertions discussed in this paper comprise 250bp of rotavirus DNA and cover the A and B antigenic regions of the protein. The fusion proteins are expressed and are present in the outer membrane: they react with anti-rotavirus antibody. However, as yet we have not been able to demonstrate that the fusion has LamB protein functions with regard to maltodextrins or L phage, and the fusion protein appears to exist in the outer membrane as a monomer rather than as a trimer.

Self-injection with olive oil. A cause of lipoid pneumonia.

A 48-year-old man with unipolar depression and a psychosexual problem concerning his body image was injecting his scrotum repeatedly with olive oil to increase the size of his genitals. He developed respiratory failure following accidental intravenous injection of olive oil and was found to have lipogranulomatous lesions in the lung and the scrotum.

Immunofluorescence in duodenal mucosa of children with acute enteritis due to a new virus.

Electron microscopy od duodenal mucosa from children with acute non-bacterial enteritis has shown virus particles in epithelial cells. Indirect immunofluorescent techniques applied to the same tissue showed virus antigen localized in the cytoplasm of epithelial cells of the villi. Specific IgM antibody was present in sera from infected patients as early as two days after the onset of symptoms. Virus particles from different patients appeared to share a common antigen. The evidence presented supports our belief that this new virus was the cause of acute enteritis in the children studied.

An epidemic of diarrhoea in human neonates involving a reovirus-like agent and 'enteropathogenic' serotypes of Escherichia coli.

During December 1974, an epidemic of diarrhoea occurred in the Royal Children's Hospital, Melbourne, in a ward caring for neonates with acute or chronic medical and surgical problems. Electron microscopy of diarrhoeal faeces revealed a reovirus-like particle ('duovirus' or 'rotavirus') known to cause acute enteritis in older children. This virus is considered to have been primarily involved in the aetiology of the epidemic. In addition, three 'enteropathogenic' serotypes of Escherichia coli were isolated from babies during the epidemic, but none produced enterotoxin when tested in ligated ileal loops of rabbits or in monolayers of Y1 adrenal cells. Further epidemics of neonatal diarrhoea must be studied using culture and electron microscopy of faeces to determine the relative importance of this virus and of E. coli in the aetiology of diarrhoea in this age-group.

Development of rotavirus molecular epidemiology: electropherotyping.

Early in the era of rotavirology it was realized that the characteristic patterns of bands produced in polyacrylamide gels following electrophoresis of genomic dsRNA were useful for checking the identity of rotavirus isolates. However it was Romilio Espejo who first proposed the use of this technique for epidemiology, although most others did not take the suggestion seriously because the technique was then rather specialized and RNA staining methods were not very sensitive. Using samples collected by Ruth Bishop in Melbourne following the original identification of human rotaviruses, Sue Rodger recorded the "electropherotypes" of all samples available to 1979 and painstakingly compared them, side by side (since minor variations in conditions, especially temperature, alter the relative migration distances of dsRNA bands). These efforts produced the first longitudinal, extensive study of human rotavirus strain variation. Since then, technical improvements have greatly increased the sensitivity of the procedures, and electropherotyping has been recognized as a powerful and economical method for epidemiological studies of rotaviruses.