Identification of a new genotype of Torque Teno Mini virus

Background

Since we were able to isolate viable virus from brain and lung of H7N1 low pathogenic avian influenza virus (LPAIV) infected chickens, we here examined the distribution of different LPAIV strains in chickens by measuring the viral AI RNA load in multiple organs. Subtypes of H5 (H5N1, H5N2), H7 (H7N1, H7N7) and H9 (H9N2), of chicken (H5N2, H7N1, H7N7, H9N2), or mallard (H5N1) origin were tested. The actual presence of viable virus was evaluated with virus isolation in organs of H7N7 inoculated chickens.

Findings

Viral RNA was found by PCR in lung, brain, intestine, peripheral blood mononuclear cells, heart, liver, kidney and spleen from chickens infected with chicken isolated LPAIV H5N2, H7N1, H7N7 or H9N2. H7N7 virus could be isolated from lung, ileum, heart, liver, kidney and spleen, but not from brain, which was in agreement with the data from the PCR. Infection with mallard isolated H5N1 LPAIV resulted in viral RNA detection in lung and peripheral blood mononuclear cells only.

Conclusion

We speculate that chicken isolated LPAI viruses are spreading systemically in chicken, independently of the strain.     

The not-so-humble worm

A report of the Wellcome Trust meeting "Caenorhabditis elegans past, present and future: The not-so-humble worm", Hinxton, UK, 10 September 2003.     

COMT polymorphism modulates the resting‐state EEG alpha oscillatory response to acute nicotine in male non‐smokers

Performance improvements in cognitive tasks requiring executive functions are evident with nicotinic acetylcholine receptor (nAChR) agonists, and activation of the underlying neural circuitry supporting these cognitive effects is thought to involve dopamine neurotransmission. As individual difference in response to nicotine may be related to a functional polymorphism in the gene encoding catechol‐O‐methyltransferase (COMT), an enzyme that strongly influences cortical dopamine metabolism, this study examined the modulatory effects of the COMT Val158Met polymorphism on the neural response to acute nicotine as measured with resting‐state electroencephalographic (EEG) oscillations. In a sample of 62 healthy non‐smoking adult males, a single dose (6 mg) of nicotine gum administered in a randomized, double‐blind, placebo‐controlled design was shown to affect α oscillatory activity, increasing power of upper α oscillations in frontocentral regions of Met/Met homozygotes and in parietal/occipital regions of Val/Met heterozygotes. Peak α frequency was also found to be faster with nicotine (vs. placebo) treatment in Val/Met heterozygotes, who exhibited a slower α frequency compared to Val/Val homozygotes. The data tentatively suggest that interindividual differences in brain α oscillations and their response to nicotinic agonist treatment are influenced by genetic mechanisms involving COMT.     

How healthy are clones and their progeny: 5 years of field experience

There is considerable concern regarding the health of cloned cattle and their safety as a source of food. The objective was to summarize 5 years of commercial experience with cloning in three countries (United States, Argentina and Brazil). Overall, only 9% of transferred embryos resulted in calves; efficiency ranged from 0 to 45% (most were from 1 to 10%, but 24% of cell lines never produced live calves). There was no significant difference in pregnancy rate following transfer of one versus two embryos. Before 90 days of gestation, two ultrasound markers for embryo death were found, either crown rump length (CRL) or heart beat less than 7.5mm and 150bpm, respectively, were observed alone or together in 27% of clones that died. In addition, after 100 days of pregnancy, placental edema, hydrops fetalis and increased abdominal circumference size were used as ultrasound findings of a fetus at risk of loss. At 114 days of gestation, abdominal circumference in clones that died was statistically larger than in clones that survived alive to term and from MOET- and IVF-derived pregnancies (P<0.05). Since elective cesarean section (C-section) was partially replaced by natural or assisted parturition, C-section rates decreased from 100% in 2000 to 54% in 2005. On average, 42% of cloned calves died between delivery and 150 days of life; the most common abnormalities were: enlarged umbilical cord (37%), respiratory problems (19%), calves depressed/prolonged recumbency (20%) and contracted flexor tendons (21%). From 11 blood parameters evaluated during the first week of life, lactate decreased twice and glucose doubled its original value from 24h to 7 days. Adult cloned females had normal breeding and calving rates and cloned bulls produced good quality semen and had normal fertility when used for AI or natural mating. In conclusion, cloning had no risks qualitatively different from those encountered in animals involved in modern agricultural practices, although the frequency of the risks appeared to be increased in cattle during the early portions of the life cycle of cattle clones.     

T47D breast cancer cell growth is inhibited by expression of VACM-1, a cul-5 gene

Vasopressin-activated calcium-mobilizing (VACM-1), a cul-5 gene, is localized on chromosome 11q22-23 close to the gene for Ataxia Telangiectasia in a region associated with a loss of heterozygosity in breast cancer tumor samples. To examine the biological role of VACM-1, we studied the effect of VACM-1 expression on cellular growth and gene expression in T47D breast cancer cells. Immunocytochemistry studies demonstrated that VACM-1 was expressed in 0.6-6% of the T47D cells and localized to the nucleus of mitotic cells. Overexpressing VACM-1 significantly attenuated cellular proliferation and MAPK phosphorylation when compared to the control cells. In addition, VACM-1 decreased egr-1 and increased Fas-L mRNA levels. Further, egr-1 protein levels were significantly lower in the nuclear fraction from VACM-1 transfected cells when compared to controls. These data indicate that VACM-1 is involved in the regulation of cellular growth.     

Pre- and post-hatching effects of corticosterone treatment on behavior of the domestic chick

We investigated the effect of 60 ìg of corticosterone administered to domestic chicks either before or after hatching on the behavioral response to isolation in a novel arena and performance in a task involving the simultaneous identification of food and detection of a predator (overhead silhouette of a hawk moving overhead). Following release into a novel arena, chicks treated with corticosterone at 18 days of incubation emitted more distress vocalizations. In contrast, no difference in the number of vocalizations was found between chicks treated with corticosterone at day 1 post-hatching and controls. Behavior in the home cages was generally similar across treatments, though chicks treated with corticosterone at 18 days of incubation slept more than control chicks. While searching for grain against a background of pebbles, chicks treated with corticosterone at embryonic day 18, but not chicks treated on day 1 post-hatching, took longer to detect the overhead image of a predator than did controls. Corticosterone treatment at both ages increased the rate of pecking at grains and pebbles. Our findings support work on other birds indicating that corticosterone treatment during incubation influences stress reactivity. The impairment in predator detection in chicks treated with corticosterone on day 18 of incubation appears to be caused by the known effects of corticosterone treatment at this age in preventing the development of lateralization of the thalamofugal visual projections. This further supports the hypothesis that brain lateralization provides an advantage in performing more than one task simultaneously.     

The effect of Trim5 polymorphisms on the clinical course of HIV-1 infection

The antiviral factor tripartite interaction motif 5alpha (Trim5alpha) restricts a broad range of retroviruses in a species-specific manner. Although human Trim5alpha is unable to block HIV-1 infection in human cells, a modest inhibition of HIV-1 replication has been reported. Recently two polymorphisms in the Trim5 gene (H43Y and R136Q) were shown to affect the antiviral activity of Trim5alpha in vitro. In this study, participants of the Amsterdam Cohort studies were screened for polymorphisms at amino acid residue 43 and 136 of the Trim5 gene, and the potential effects of these polymorphisms on the clinical course of HIV-1 infection were analyzed. In agreement with the reported decreased antiviral activity of Trim5alpha that contains a Y at amino acid residue 43 in vitro, an accelerated disease progression was observed for individuals who were homozygous for the 43Y genotype as compared to individuals who were heterozygous or homozygous for the 43H genotype. A protective effect of the 136Q genotype was observed but only after the emergence of CXCR4-using (X4) HIV-1 variants and when a viral load of 10(4.5) copies per ml plasma was used as an endpoint in survival analysis. Interestingly, naive CD4 T cells, which are selectively targeted by X4 HIV-1, revealed a significantly higher expression of Trim5alpha than memory CD4 T cells. In addition, we observed that the 136Q allele in combination with the -2GG genotype in the 5'UTR was associated with an accelerated disease progression. Thus, polymorphisms in the Trim5 gene may influence the clinical course of HIV-1 infection also underscoring the antiviral effect of Trim5alpha on HIV-1 in vivo.     

Isolation,leishmanicidal evaluation and molecular docking simulations of piperidine alkaloids from Senna spectabilis

Leishmaniasis is one of the most important neglected tropical diseases (NTDs) that are especially common among low-income populations in developing regions of Africa, Asia, and the Americas. Many natural products, particularly alkaloids, have been reported to have inhibitory activity against arginase, the key enzyme in the pathology caused by Leishmania sp. In this way, piperidine alkaloids (–)-cassine (1), (–)-spectaline (2), (–)-3-O-acetylcassine (3), and (–)-3-O-acetylspectaline (4) were isolated from Senna spectabilis flowers. These compounds (1/2 and 3/4) initially present as homologous mixtures were separated by high performance liquid chromatography and evaluated against the promastigote phase of Leishmania amazonensis. In addition, molecular docking simulations were implemented in order to probe the binding modes of the ligands 1–4 to the amino acids in the active site of L. amazonensis arginase. Alkaloid 2 (IC₅₀ 15.81?μg?mL^?1) was the most effective against L. amazonensis. Compounds 2 and 4, with larger side chain, were more effective against the parasite than compounds 1 and 3. The cell viability test on Vero cells revealed that compound 2 (CC₅₀ 66.67?μg?mL^?1) was the most toxic. The acetyl group in the 3-O position of the parent structures reduced the leishmanicidal activity and the toxicity of the alkaloids. Further, molecular docking suggested that Asn143 is essential for arginase to interact with (–)-spectaline-derived compounds, which agreed with the IC₅₀ measurements. Our findings revealed that S. spectabilis is an important source of piperidine alkaloids with leishmanicidal activity. Moreover, the natural compound 3 has been isolated for the first time. Experimental investigation combined with theoretical study advances knowledge about the enzyme binding site mode of interaction and contributes to the design of new bioactive drugs against Leishmania infection.     

Inhibition of Furin/Proprotein Convertase-catalyzed Surface and Intracellular Processing by Small Molecules

Furin is a ubiquitously expressed proprotein convertase (PC) that plays a vital role in numerous disease processes including cancer metastasis, bacterial toxin activation (e.g. anthrax and Pseudomonas), and viral propagation (e.g. avian influenza and human immunodeficiency virus). To identify small molecule inhibitors of furin and related processing enzymes, we performed high-throughput screens of chemical diversity libraries utilizing both enzyme-based and cell-based assays. The screens identified partially overlapping sets of compounds that were further characterized for affinity, mechanism, and efficacy in additional cellular processing assays. Dicoumarols were identified as a class of compounds that inhibited furin non-competitively and reversibly with K_i values in the micromolar range. These compounds inhibited furin/furin-like activity both at the cell surface (protecting against anthrax toxin) and in the secretory pathway (blocking processing of the metastasis factor membrane-type 1 matrix metalloproteinase/MT1-MMP) at concentrations close to K_i values. Compounds tested exhibited distinct patterns of inhibition of other furin-family PCs (rat PACE4, human PC5/6 and human PC7), showing that dicoumarol derivatives might be developed as either generic or selective inhibitors of the PCs. The extensive clinical use, high bioavailability and relatively low toxicity of dicoumarols suggests that the dicoumarol structure will be a good starting point for development of drug-like inhibitors of furin and other PCs that can act both intracellularly and at the cell surface.Furin, is a subtilisin-related serine protease and member of the proprotein convertase (PCs)4 family that functions within the secretory and endocytic pathways and at the cell surface, cleaving proproteins at clusters of basic residues, typically of the form RX(K/R)R↓ (for reviews see Refs. 1–3). The specificity of furin and its yeast homologue Kex2 correlate well with the three-dimensional structures of their catalytic domains (4, 5). Ubiquitously expressed, furin has numerous known or suspected physiological substrates that include growth factors, receptors, coagulation proteins, plasma proteins (e.g. pro-von Willebrand factor), extracellular matrix components, and protease precursors (e.g. matrix metalloproteases) (2). Although the homozygous furin knock-out mouse exhibits embryonic lethality (6), analysis of liver-specific ablation suggests functional overlap with other PCs, such as PACE4, PC5/6, and PC7, that are also widely expressed and act in the constitutive secretory pathway (7). Furin activity contributes to numerous chronic pathological conditions, including Alzheimer disease (8), other non-Alzheimer cerebral amyloidoses (9), osteoarthritis (10), atherosclerosis (11), and tumor progression and malignancy (12). Moreover, activation by host cells of bacterial toxins such as anthrax toxin, Pseudomonas exotoxin A, diphtheria toxin (13), Shiga toxin (14), and Bordetella dermonecrotic toxin (15), requires cleavage by furin or other PCs. Furin or furin-like cleavage of viral envelope glycoproteins is necessary for propagation of many lipid-enveloped viral pathogens including H5N1 avian influenza (16), human immunodeficiency virus-1 (17), ebola (18), measles (19), cytomegalovirus (20), and flaviviruses (21). Even non-enveloped viruses, such as human papillomavirus, can require furin-type processing for entry into the cyotsol after endocytosis (22).The multiple roles for furin in human pathophysiology have made it a target of interest for development of therapeutic agents. Numerous protein- and peptide-based furin inhibitors have been devised (23). For the most part, these are not drug-like and their use as pharmaceutical agents is hampered by large size, instability, toxicity, and/or low cell permeability. Recently, 2,5-dideoxystreptamine derivatives have shown promise (24), although these molecules have yet to be examined for inhibition of intracellular processing. Important pathophysiological roles exist for furin at the cell surface, such as in the processing of anthrax protective antigen. However, maturation of other bacterial toxins, viral envelope glycoproteins, and metalloprotease precursors such as membrane-type 1 matrix metalloproteinase (MT1-MMP), a matrix metalloprotease whose activity contributes directly to degradation of extracellular matrix components and is important for angiogenesis, tumor invasion, and metastasis (25), require processing by furin in the trans Golgi network and endosomal compartments (2, 26).Here we report identification of drug-like small molecule inhibitors through simultaneous high-throughput screening (HTS) of chemical diversity libraries with both enzyme-based and cell-based assays for furin and furin-like activities. A preliminary report of the cell-based assay has been published elsewhere (27). Combining the results of the enzymatic screen with the cellular screen allowed identification of small molecule lead compounds with the desired properties of high affinity, high cell permeability, and low toxicity. Dicoumarols, which have an extensive pharmacological history (28), were identified in this study as a family of compounds that inhibited furin reversibly and non-competitively, also inhibited rat PACE4 (rPACE4), human PC5/6 (hPC5/6), and hPC7 and blocked both extracellular maturation of anthrax protective antigen (PA) and intracellular processing of MT1-MMP and other substrates.