Analysis of intrahost variation in Venezuelan equine encephalitis virus reveals repeated deletions in the 6-kilodalton protein gene

RNA viruses exist as a spectrum of mutants that is generated and maintained during replication within the host. Consensus sequencing overlooks minority genotypes present in the viral sample that may impact the population's phenotype. In-depth sequencing of an original field isolate of subtype IE Venezuelan equine encephalitis virus (VEEV) demonstrated the presence of multiple deletions within the 6,000-molecular-weight (6K) protein gene. Using in vitro and in vivo experiments, similar deletions were generated in an additional VEEV strain originating from an infectious cDNA clone. Time course experiments demonstrated that the deletions are produced during acute infection although not until 24 h postinfection. Molecular clones containing some of these deletions were generated, and although the larger deletions appear to be noninfectious, viruses with the smaller deletions were viable and formed small plaques. Serial passages provided no evidence that these deletion mutants function as defective interfering particles. Furthermore, since wild-type infections generally occur at a low multiplicity of infection, it is unlikely that these deletions are propagated in natural transmission cycles. However, they could affect pathogenesis at later stages of infection. Because they are ubiquitously generated both in vivo and in vitro, further investigation is warranted to understand the generation of these deletions and their significance for disease.     

An isotopic approach to measuring nitrogen balance in caribou

Nutritional restrictions in winter may reduce the availability of protein for reproduction and survival in northern ungulates. We refined a technique that uses recently voided excreta on snow to assess protein status in wild caribou (Rangifer tarandus) in late winter. Our study was the first application of this non-invasive, isotopic approach to assess protein status of wild caribou by determining dietary and endogenous contributions of nitrogen (N) to urinary urea. We used isotopic ratios of N (δ¹⁵N) in urine and fecal samples to estimate the proportion of urea N derived from body N (p-UN) in pregnant, adult females of the Chisana Herd, a small population that ranged across the Alaska-Yukon border. We took advantage of a predator-exclosure project to examine N status of penned caribou in April 2006. Lichens were the primary forage (>40%) consumed by caribou in the pen and δ¹⁵N of fiber tracked the major forages in their diets. The δ¹⁵N of urinary urea for females in the pen was depleted relative (−1.3 ± 1.0 parts per thousand [‰], ) to the δ¹⁵N of body N (2.7 ± 0.7‰). A similar proportion of animals in the exclosure lost core body mass (excluding estimates of fetal and uterine tissues; 55%) and body protein (estimated by isotope ratios; 54%). This non-invasive technique could be applied at various spatial and temporal scales to assess trends in protein status of free-ranging populations of northern ungulates. Intra- and inter-annual estimates of protein status could help managers monitor effects of foraging conditions on nutritional constraints in ungulates, increase the efficiency and efficacy of management actions, and help prepare stakeholders for potential changes in population trends. © 2010 The Wildlife Society.     

Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027-0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes.     

Defective S phase chromatin assembly causes DNA damage,activation of the S phase checkpoint,and S phase arrest

The S phase checkpoint protects the genome from spontaneous damage during DNA replication, although the cause of damage has been unknown. We used a dominant-negative mutant of a subunit of CAF-I, a complex that assembles newly synthesized DNA into nucleosomes, to inhibit S phase chromatin assembly and found that this induced S phase arrest. Arrest was accompanied by DNA damage and S phase checkpoint activation and required ATR or ATM kinase activity. These results show that in human cells CAF-I activity is required for completion of S phase and that a defect in chromatin assembly can itself induce DNA damage. We propose that errors in chromatin assembly, occurring spontaneously or caused by genetic mutations or environmental agents, contribute to genome instability.     

Enhanced locomotion caused by loss of the Drosophila DEG/ENaC protein Pickpocket1

Coordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain. ppk1 null mutant larvae had normal external touch sensation and md neuron morphology but displayed striking alterations in crawling behavior. Loss of PPK1 function caused an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion resulted from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype was rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the PPK1 channel plays an essential role in controlling rhythmic locomotion and provide a powerful genetic model system for further analysis of central and peripheral control mechanisms and their role in movement disorders.     

The antimitotic effect of the neem terpenoid azadirachtin on cultured insect cells

When cultured insect cells (Sf9) were grown in the presence of 5 x 10(-6) M azadirachtin, there was a rapid increase in the mitotic index, with the appearance of many aberrant mitotic figures. Flow cytometry established that cells accumulated in the G2/M phase of the cell cycle, and that the effect was concentration-dependent. At 10(-8) M a period of 20 h was necessary to raise the proportion in G2/M to 42% above the control values, but at 5 x 10(-6) M more than 90% of the cells were in this phase. Azadirachtin had the same effect on C6/36 mosquito cells, but failed to affect L929 murine fibroblast cells even at a concentration of 10(-4) M over 72 h. Experiments with colchcine and taxol showed similarities of action between azadirachtin and colchicine, and azadirachtin was apparently able to displace colchicine-fluorescein from binding-sites in living insect cells. Another similarity between azdirachtin and colchicine was that both phytochemicals prevented the polymerisatrion in vitro of mammalian tubulin, although the azadirachtin was much less effective.     

New antibacterial tetrahydro-4(2H)-thiopyran and thiomorpholine S-oxide and S,S-dioxide phenyloxazolidinones

Combinatorial libraries of N-acylated 5-(S)-aminomethyloxazolidinone derivatives of S-oxide and S,S-dioxide tetrahydro-4(2H)-thiopyranyl and thiomorpholine phenyloxazolidinone series have been synthesized on a solid phase and evaluated for antimicrobial activity. Several novel potent leads have been identified, including orally active oxazolidinones with enhanced activity against respiratory tract infection pathogens Haemophilus influenzae and Moraxella catarrhalis.     

Migration patterns and navigation cues of Atlantic salmon post-smolts migrating from 12 rivers through the coastal zones around the Irish Sea

The freshwater phase of the first seaward migration of juvenile Atlantic salmon (Salmo salar) is relatively well understood when compared with our understanding of the marine phase of their migration. In 2021, 1008 wild and 60 ranched Atlantic salmon smolts were tagged with acoustic transmitters in 12 rivers in England, Scotland, Northern Ireland and Ireland. Large marine receiver arrays were deployed in the Irish Sea at two locations: at the transition of the Irish Sea into the North Atlantic between Ireland and Scotland, and between southern Scotland and Northern Ireland, to examine the early phase of the marine migration of Atlantic salmon smolts. After leaving their natal rivers' post-smolt migration through the Irish Sea was rapid with minimum speeds ranging from 14.03 to 38.56 km.day⁻¹ for Atlantic salmon smolts that entered the Irish Sea directly from their natal river, to 9.69–39.94 km.day⁻¹ for Atlantic salmon smolts that entered the Irish Sea directly from their natal estuary. Population minimum migration success through the study area was strongly correlated with the distance of travel, populations further away from the point of entry to the open North Atlantic exhibited lower migration success. Post-smolts from different populations experienced different water temperatures on entering the North Atlantic. This was largely driven by the timing of their migration and may have significant consequences for feeding and ultimately survivorship. The influence of water currents on post-smolt movement was investigated using data from previously constructed numerical hydrodynamic models. Modeled water current data in the northern Irish Sea showed that post-smolts had a strong preference for migrating when the current direction was at around 283° (west-north-west) but did not migrate when exposed to strong currents in other directions. This is the most favorable direction for onward passage from the Irish Sea to the continental shelf edge current, a known accumulation point for migrating post-smolts. These results strongly indicate that post-smolts migrating through the coastal marine environment are: (1) not simply migrating by current following (2) engage in active directional swimming (3) have an intrinsic sense of their migration direction and (4) can use cues other than water current direction to orientate during this part of their migration.     

Effects of different protein and carbohydrate levels on growth performance and feed utilisation of brook trout,Salvelinus fontinalis (Mitchill, 1814), at two temperatures

A 12‐week feeding trial was conducted to determine the optimum dietary protein requirement of brook trout, Salvelinus fontinalis, at 15 and 19°C. Twelve iso‐energetic (22 MJ · kg^?1) and iso‐lipidic (23%) diets (36–58% protein at 2% increments) were prepared. Fish (29.45 ± 3.25 g · fish^?1) were fed 2% of body weight per day, divided into two equal rations. The specific growth rate (SGR, % · day^?1), feed efficiency ratio (FER), productive protein value (PPV), productive lipid value (PLV) and productive energy value (PEV), apparent digestibility of diet (AD_DM) and protein (AD_CP) were significantly higher at optimum temperature (15°C). Increasing PPV with increasing dietary carbohydrate and with decreasing dietary protein content was due to the protein‐sparing effect of carbohydrates. A piecewise regression (broken line) model between the SGR and digestible dietary protein level revealed that the digestible dietary protein requirement of brook trout was 44 and 40% at 15 and 19°C, respectively. When PPV (digestible protein retention basis) was modelled with a broken line, the digestible protein requirement of brook trout was 39 and 35% at 15 and 19°C, respectively. A reduction in dietary protein content balanced by increased gelatinised carbohydrate might be useful for improving the protein utilization efficiency for growth at 15 and 19°C; however, the growth and feed efficiency was lower at the elevated temperature.