A strange family, or how a new pleolipovirus reveals its friends and relatives.

A new virus of halophilic Archaea is reported by Liu et al., and is remarkable in many ways. SNJ2 is the first temperate, pleomorphic virus (pleolipovirus) that integrates into the genome of its host. Analyses of the virus structure and its genome have provided an unexpected puzzle while at the same time solving another. On the one hand, the study shows a curious relationship exists between SNJ2 and an unrelated provirus (SNJ1) found as a plasmid in the same cell. The presence of SNJ1 appears to allow much higher levels of SNJ2 virus to be produced, although the mechanism involved remains unclear. On the other hand, the curious occurrence of a conserved cluster of pleolipovirus-related genes found widely distributed among haloarchaeal genomes and known for almost 10 years, now appears to correspond to SNJ2-related proviruses.

Prodrugs of imidazotriazine and pyrrolotriazine C-nucleosides can increase anti-HCV activity and enhance nucleotide triphosphate concentrations in vitro.

A number of prodrugs of HCV-active purine nucleoside analogues 2'-C-methyl 4-aza-9-deaza adenosine 1, 2'-C-methyl 4-aza-7,9-dideaza adenosine 2, 2'-C-methyl 4-aza-9-deaza guanosine 3 and 2'-C-methyl 4-aza-7,9-dideaza guanosine 4 were prepared and evaluated to improve potency, selectivity and liver targeting. Phosphoramidate guanosine prodrugs (3a-3k and 4a, b) showed insufficient cell activity for further profiling. Striking enhancement in replicon activity relative to the parent was observed for phosphoramidate imidazo[2,1-f][1,2,4]triazine-4-amine adenosine prodrugs (1a-1p), but this was accompanied by an increase in cytotoxicity. Improved or similar potency without a concomitant increase in toxicity relative to the parent was demonstrated for phosphoramidate pyrrolo[2,1-f][1,2,4]triazine-4-amine adenosine prodrugs (2a-2k). Carbamate, ester and mixed prodrugs of 2 showed mixed results. Selected prodrugs of 2 were analysed for activation to the triphosphate, with most demonstrating much better activation in hepatocytes over replicon cells. The best activation was observed for a mixed phosphoramidate-3'ester (11) followed by a simple 3'-ester (10).

Derivatives of imidazotriazine and pyrrolotriazine C-nucleosides as potential new anti-HCV agents.

Previous investigations identified 2'-C-Me-branched ribo-C-nucleoside adenosine analogues, 1, which contains a pyrrolo[2,1-f][1,2,4]triazin-4-amine heterocyclic base, and 2, which contains an imidazo[2,1-f][1,2,4]triazin-4-amine heterocyclic base as two compounds with promising anti-HCV in vitro activity. This Letter describes the synthesis and evaluation of a series of novel analogues of these compounds substituted at the 2-, 7-, and 8-positions of the heterocyclic bases. A number of active new HCV inhibitors were identified but most compounds also demonstrated unacceptable cytotoxicity. However, the 7-fluoro analogue of 1 displayed good potency with a promising cytotherapeutic margin.

PH1: an archaeovirus of Haloarcula hispanica related to SH1 and HHIV-2.

Halovirus PH1 infects Haloarcula hispanica and was isolated from an Australian salt lake. The burst size in single-step growth conditions was 50-100 PFU/cell, but cell density did not decrease until well after the rise (4-6 hr p.i.), indicating that the virus could exit without cell lysis. Virions were round, 51 nm in diameter, displayed a layered capsid structure, and were sensitive to chloroform and lowered salt concentration. The genome is linear dsDNA, 28,064 bp in length, with 337 bp terminal repeats and terminal proteins, and could transfect haloarchaeal species belonging to five different genera. The genome is predicted to carry 49 ORFs, including those for structural proteins, several of which were identified by mass spectroscopy. The close similarity of PH1 to SH1 (74% nucleotide identity) allowed a detailed description and analysis of the differences (divergent regions) between the two genomes, including the detection of repeat-mediated deletions. The relationship of SH1-like and pleolipoviruses to previously described genomic loci of virus and plasmid-related elements (ViPREs) of haloarchaea revealed an extensive level of recombination between the known haloviruses. PH1 is a member of the same virus group as SH1 and HHIV-2, and we propose the name halosphaerovirus to accommodate these viruses.

The Royal Army Dental Corps today.

The Royal Army Dental Corps (RADC) provides dental care to the British Army both in barracks and on deployment While a significant proportion of most RADC dental officers' careers is spent working in permanent military primary care dental clinics, it is the ability of all RADC dental officers to provide dental care when deployed that provides unique challenges only seen in the military environment. All RADC dental officers are able to deliver high quality care in temporary facilities. During a career the majority of RADC dental officers will have at least one job in a medical regiment providing the dental component of the British Army's rapidly deployable medical capability. This article covers some of the issues faced when delivering dental care outside the UK using portable equipment on a recent exercise.As RADC officers gain experience they are expected to lead increasingly large teams, both within primary care dentistry and some in the wider Army Medical Services, with the appropriate professional and military training to support their development.Working under pressure can be developed through the use of adventurous training and sport and the authors have pushed themselves well beyond their comfort zones in the air, on land and under the sea.

Determination of selected water-soluble vitamins (thiamine, riboflavin, nicotinamide and pyridoxine) from a food matrix using hydrophilic interaction liquid chromatography coupled with mass spectroscopy.

Water-soluble vitamins are essential dietary components with a multitude of important functions that require quantification from food sources to characterise the nutritional status of food. In this study, we have developed a hydrophilic interaction chromatography (HILIC) based method coupled to single-quadrupole mass spectrometry (MS) for the analysis of selected water-soluble vitamins. Due to their involvement in energy release from macronutrients, the quantification of thiamine (B1), riboflavin (B2), nicotinamide (B3) and pyridoxine (B6) offers significant value in food analysis. A commercially available vegetable soup was selected as the food matrix for this study and utilised to develop an efficient extraction procedure for the vitamins of interest. Vitamins were extracted using meta-phosphoric acid coupled with a reducing agent, DL-dithiothreitol (DTT) to produce the parent compound. The extracted vitamins were then analysed using an LC-MS system with electrospray - atmospheric pressure ionization (ES-API) source, operated in positive single ion monitoring (SIM) mode. The MS provided good linearity within the investigated range from 5 to 400 ng/mL with coefficient of determination (r2) ranging from 0.98 to 0.99. Retention times (0.65-9.04 min) were reproducible and no coelution between vitamins was observed. Limit of detection (LOD) varied from 2.4 to 9.0 ng/mL and limit of quantification (LOQ) was from 8 to 30 ng/mL, comparable to previously published studies. The extraction method provided good intra-day (%CV 1.56-6.56) and inter-day precision (%CV 8.07-10.97). Standard injections were used as part of quality control measures and provided excellent reproducibility (%CV 0.9-3.4). The overall runtime of this method was 19 min, including column reconditioning. Using this method, the quantity of thiamine (67 ±â€¯7 ng/g), riboflavin (423 ±â€¯39 ng/g), nicotinamide (856 ±â€¯77 ng/g) and pyridoxine (133 ±â€¯11 ng/g) was determined from a complex food matrix. In conclusion, we have developed a rapid and reliable, HILIC-single quad MS method utilising SIM for the low-level quantification of four B vitamins in a vegetable soup matrix in under 20 min. This method has shown excellent linearity, intra- and inter-day reproducibility and is directly applicable to other plant-based food matrices.

Diversity of Haloquadratum and other haloarchaea in three, geographically distant, Australian saltern crystallizer ponds.

Haloquadratum walsbyi is frequently a dominant member of the microbial communities in hypersaline waters. 16S rRNA gene sequences indicate that divergence within this species is very low but relatively few sites have been examined, particularly in the southern hemisphere. The diversity of Haloquadratum was examined in three coastal, but geographically distant saltern crystallizer ponds in Australia, using both culture-independent and culture-dependent methods. Two 97%-OTU, comprising Haloquadratum- and Halorubrum-related sequences, were shared by all three sites, with the former OTU representing about 40% of the sequences recovered at each site. Sequences 99.5% identical to that of Hqr. walsbyi C23(T) were present at all three sites and, overall, 98% of the Haloquadratum-related sequences displayed

Idronoxil as an Anticancer Agent: Activity and Mechanisms.

Idronoxil has been the subject of more than 50 peer-reviewed publications over the last two decades. This isoflavone is an intriguing regulator of multiple signal transduction pathways, capable of causing a range of biological effects, including cell cycle arrest, apoptosis, an ability to stimulate the immune system, and inhibition of angiogenesis. These multifaceted actions suggest that idronoxil has the potential to synergize with, or complement, a wide range of cancer therapies. Whilst clinically tested in the past, idronoxil's journey was discontinued as a result of its low bioavailability in humans when administered either intravenously or orally, though strategies to overcome this issue are currently being explored. Here, we summarize the current literature regarding the key cellular targets of idronoxil and the mechanisms by which idronoxil exerts its anticancer effects, laying a new foundation toward giving this unique molecule a second chance of contributing to the future of cancer treatment.

Transfection of haloarchaea by the DNAs of spindle and round haloviruses and the use of transposon mutagenesis to identify non-essential regions.

Spindle-shaped halovirus His2 and spherical halovirus SH1 represent ecologically dominant virus morphotypes in high-salt environments. Both have linear dsDNA genomes with inverted terminal repeat sequences and terminal proteins, and probably replicate using protein priming. As a first step towards conventional genetic analyses on these viruses, we show that purified viral DNAs can transfect host cells. Intact terminal proteins were essential for this process. Despite the narrow host ranges of these viruses, at least under laboratory conditions, their DNAs were able to transfect a wide range of haloarchaeal species, demonstrating that the cytoplasms of diverse haloarchaea possess all the factors necessary for viral DNA synthesis and virion assembly. Transposon mutagenesis of viral DNAs was then used in conjunction with transfection to produce recombinant viruses, and to then map the insertion sites to identify non-essential genes. The inserts in 34 His2 mutants were mapped precisely, and most clustered in a few, specific regions, particularly in the inverted terminal repeats and near the ends of ORFs. The results are consistent with the small genome size and densely packed, often overlapping ORFs that are transcribed as long operons. This study is the first demonstration of transfection and transposon mutagenesis in protein-primed archaeal viruses.

Extracting nucleic acids from activated sludge which reflect community population diversity.

Critical to most studies in molecular microbial ecology is the application of DNA/RNA extraction methods which can reveal the true level of population biodiversity present in samples from the community under investigation. Activated sludge communities have been studied extensively using molecular methods, but rarely have the nucleic acid isolation methods applied been assessed for their ability to achieve this. This study compares eight published RNA and DNA extraction protocols and one commercially available DNA isolation kit for their capacity to provide high quality nucleic acids that reflect the community composition. Each method was assessed on the basis of nucleic acid yield, purity and integrity, and the ability to provide PCR amplifiable RNA and DNA from known marker populations that varied in their resistance to nucleic acid extraction. Only three consistently provided DNA from each of the marker populations known to be present in the samples from fluorescence in situ hybridisation analysis. The failure of the other methods emphasises the need to validate all DNA/RNA extraction protocols. It is recommended that several validated extraction methods be used and the extracts pooled to further minimise any risk of bias.

Virus-host interactions in salt lakes.

Natural hypersaline waters are widely distributed around the globe, as both continental surface waters and sea floor lakes, the latter being maintained by the large density difference between the hypersaline and overlying marine water. Owing to the extreme salt concentrations, close to or at saturation (approximately 35%, w/v), such waters might be expected to be devoid of life but, in fact, maintain dense populations of microbes. The majority of these microorganisms are halophilic prokaryotes belonging to the Domain Archaea, 'haloarchaea'. Viruses infecting haloarchaea are a vital part of hypersaline ecosystems, in many circumstances outnumbering cells by 10-100-fold. However, few of these 'haloviruses' have been isolated and even fewer have been characterised in molecular detail. In this review, we explore the methods used by haloviruses to replicate within their hosts and consider the implications of haloviral-haloarchaeal interactions for salt lake ecology.

Simple and safe method for simultaneous isolation of microbial RNA and DNA from problematic populations.

We describe a novel, rapid, and safe method for extracting RNA and DNA from refractory microbes, which avoids the use of phenol or chloroform. It has been used successfully to isolate high-quality nucleic acids from pure cultures and environmental populations known to resist widely used extraction protocols.

Discovery and Synthesis of C-Nucleosides as Potential New Anti-HCV Agents.

Nucleoside analogues have long been recognized as prospects for the discovery of direct acting antivirals (DAAs) to treat hepatitis C virus because they have generally exhibited cross-genotype activity and a high barrier to resistance. C-Nucleosides have the potential for improved metabolism and pharmacokinetic properties over their N-nucleoside counterparts due to the presence of a strong carbon-carbon glycosidic bond and a non-natural heterocyclic base. Three 2'CMe-C-adenosine analogues and two 2'CMe-guanosine analogues were synthesized and evaluated for their anti-HCV efficacy. The nucleotide triphosphates of four of these analogues were found to inhibit the NS5B polymerase, and adenosine analogue 1 was discovered to have excellent pharmacokinetic properties demonstrating the potential of this drug class.

The transcription programme of the protein-primed halovirus SH1.

SH1 is the only reported isolate of a spherical halovirus, a dominant morphotype in hypersaline lakes. The virus lytically infects the haloarchaeon Haloarcula hispanica, and carries a 30.9 kb linear dsDNA genome that, in a previous study, was proposed to contain 56 protein-coding genes, probably organized into between four and eight operons. In the present study, these predictions were directly tested by determining the orientations and lengths of virus transcripts using systematic RT-PCR and primer extension. Seven major transcripts were observed that together covered most of the genome. Six transcripts were synthesized from early in infection (1 h post-infection; p.i.) onwards, while transcript T6 was only detected late in infection (5-6 h p.i.). No transcripts were detected in the inverted terminal repeat sequences or at the extreme right end of the genome (ORFs 55-56). Start points for the major transcripts were mapped by primer extension and corresponded closely to the 5' termini determined by RT-PCR. Between 1 and 4 h p.i., transcripts usually terminated not far beyond the end of their last coding ORF, but late in infection, transcripts from the same promoters often terminated at more distal points, resulting in much of the genome being transcribed from both strands. Since many of these transcripts are complementary, RNA-RNA interactions are likely, and may play a role in regulating viral gene expression. Puromycin blockage of post-infection protein synthesis significantly altered the levels of certain virus transcripts, indicating that de novo protein synthesis is essential for the correct regulation of SH1 gene expression.

His1 and His2 are distantly related, spindle-shaped haloviruses belonging to the novel virus group, Salterprovirus.

Spindle-shaped viruses are a dominant morphotype in hypersaline waters but their molecular characteristics and their relationship to other archaeal viruses have not been determined. Here, we describe the isolation, characteristics and genome sequence of His2, a spindle-shaped halovirus, and compare it to the previously reported halovirus His1. Their particle dimensions, host-ranges and buoyant densities were found to be similar but they differed in their stabilities to raised temperature, low salinity and chloroform. The genomes of both viruses were linear dsDNA, of similar size (His1, 14,464 bp; His2, 16,067 bp) and mol% G+C (approximately 40%), with long, inverted terminal repeat sequences. The genomic termini of both viruses are likely to possess bound proteins. They shared little nucleotide similarity and, except for their putative DNA polymerase ORFs, no significant similarity at the predicted protein level. A few of the 35 predicted ORFs of both viruses showed significant matches to sequences in GenBank, and these were always to proteins of haloarchaea. Their DNA polymerases showed 42% aa identity, and belonged to the type B group of replicases that use protein-priming. Purified His2 particles were composed of four main proteins (62, 36, 28 and 21 kDa) and the gene for the major capsid protein was identified. Hypothetical proteins similar to His2 VP1 are present in four haloarchaeal genomes but are not part of complete prophages. This, and other evidence, suggests a high frequency of recombination between haloviruses and their hosts. His1 and His2 are unlike fuselloviruses and have been placed in a new virus group, Salterprovirus.

SH1: A novel, spherical halovirus isolated from an Australian hypersaline lake.

A novel halovirus, SH1, with a spherical morphology is described. Isolated from a hypersaline lake, SH1 is divalent, producing clear plaques on Haloarcula hispanica and a natural Halorubrum isolate. Single-step growth curves gave a latent period of 5-6 h and a burst size of around 200 PFU/cell. The host can differentiate to form tight clusters of thick cell-walled forms, and these were shown to be resistant to infection. Purified virions had no visible tail, were about 70 nm in diameter, and displayed a fragile outer capsid layer, possibly with an underlying membrane component. The structural proteins of the virion were analyzed by SDS-PAGE and several were found to be cross-linked, forming protein complexes. The genome was linear, dsDNA, of approximately 30 kb in length. This morphology and linear genome are features not observed in any other euryarchaeal viruses, but have properties similar to the bacterial virus PRD1.