Molecular basis of organ-specific selection of viral variants during chronic infection.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

High-yield expression and purification of human interferon alpha-1 in Pichia pastoris

For several years, interferon alpha-1, also known as interferon alpha-D, has been studied for treatment of various viral diseases, such as hepatic fibrosis caused by hepatitis B, herpes simplex virus keratitis, and bovine respiratory diseases in calves. Currently, recombinant human interferon alpha-D (rHuIFNalphaD) is expressed intracellularly in Escherichia coli or secreted by Bacillus subtilis and Saccharomyces cerevisiae. In this report, we describe the process of obtaining a relatively high-yield secretion of biologically active recombinant rHuIFNalphaD using the Pichia pastoris system. The process produced as high as 0.7 mg of purified protein per 20 ml of shake culture of rHuIFNalphaD with better bioactivity than the commercially available rHuIFNalphaD molecule produced in E. coli.     

High-Yield Expression and Purification of Human Interferon α-1 in Pichia pastoris

For several years, interferon α-1, also known as interferon α-D, has been studied for treatment of various viral diseases, such as hepatic fibrosis caused by hepatitis B, herpes simplex virus keratitis, and bovine respiratory diseases in calves. Currently, recombinant human interferon α-D (rHuIFNαD) is expressed intracellularly in Escherichia coli or secreted by Bacillus subtilis and Saccharomyces cerevisiae. In this report, we describe the process of obtaining a relatively high-yield secretion of biologically active recombinant rHuIFNαD using the Pichia pastoris system. The process produced as high as 0.7 mg of purified protein per 20 ml of shake culture of rHuIFNαD with better bioactivity than the commercially available rHuIFNαD molecule produced in E. coli.     

Neuropsychopharmacology and Neurogenetic Aspects of Executive Functioning: Should Reward Gene Polymorphisms Constitute a Diagnostic Tool to Identify Individuals at Risk for Impaired Judgment?

Executive functions are processes that act in harmony to control behaviors necessary for maintaining focus and achieving outcomes. Executive dysfunction in neuropsychiatric disorders is attributed to structural or functional pathology of brain networks involving prefrontal cortex (PFC) and its connections with other brain regions. The PFC receives innervations from different neurons associated with a number of neurotransmitters, especially dopamine (DA). Here we review findings on the contribution of PFC DA to higher-order cognitive and emotional behaviors. We suggest that examination of multifactorial interactions of an individual's genetic history, along with environmental risk factors, can assist in the characterization of executive functioning for that individual. Based upon the results of genetic studies, we also propose genetic mapping as a probable diagnostic tool serving as a therapeutic adjunct for augmenting executive functioning capabilities. We conclude that preservation of the neurological underpinnings of executive functions requires the integrity of complex neural systems including the influence of specific genes and associated polymorphisms to provide adequate neurotransmission.     

Regulation of TRH release by the cultured neonate rat pancreas

The TRH secretory responsiveness of the pancreatic islet cell clusters from newborn rat in organ culture was studied. Basal TRH secretion was stable over a 9-day period. The response to various secretagogues was tested on day 4. TRH secretion was stimulated by high potassium-induced depolarization and also through both cAMP and protein kinase-C dependent pathways. Like insulin, TRH release was stimulated by glucose and arginine and inhibited by somatostatin. These data suggest the existence of a common mechanism for TRH and insulin secretion by the pancreatic β-cells.     

Tissue-mediated selection of viral variants: correlation between glycoprotein mutation and growth in neuronal cells.

Viral variants with different biological properties predominate in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates have the same phenotype as the parental strain and cause acute infections in adult mice, while the spleen-derived isolates cause chronic infections associated with suppressed T-cell responses and susceptibility to opportunistic infections. Our previous studies have identified a single amino acid change in the viral glycoprotein, a phenylalanine-to-leucine (F-->L) mutation at residue 260, that correlates with the tissue-specific selection and the persistent and immunosuppressive phenotype of the spleen isolates (R. Ahmed, C.S. Hahn, T. Somasundaram, L. Villarete, M. Matloubian, and J. H. Strauss, J. Virol. 65:4242-4247, 1991). In this study, we screened viral isolates obtained from the spleen, liver, kidney, and brain of carrier mice for the presence of this mutation and determined the temporal selection of variants as they appear in these organs. We found that this F-->L amino acid change is common to > 90% of the spleen and liver isolates and is selected for rapidly by day 32 postinfection (p.i.). Although the kinetics observed in the kidney are relatively slower than in the spleen and liver, this F-->L mutation predominates in the kidney-derived isolates by 250 days p.i. In contrast, the majority of the CNS isolates retain the parental sequence up to 250 days p.i. In addition, most of the brain isolates replicated efficiently in a neuronal cell line, and this enhanced growth phenotype in neurons correlated with the parental F genotype. This linkage with neurotropism, along with our earlier finding that the F-->L mutation is necessary for enhanced infection of macrophages (M. Matloubian, S. R. Kolhekar, T. Somasundaram, and R. Ahmed, J. Virol. 67:7340-7349, 1993), provides a cellular basis for the molecular changes associated with tissue-specific selection. Taken together, these results suggest that tropism for macrophages is a critical determinant in selection of variants with the F-->L mutation in tissues such as spleen and liver, and tropism for neurons is important in retention of the F genotype in the CNS.     

Elimination of Mange Mites <Emphasis Type="Italic">Sarcoptes scabiei</Emphasis> var. <Emphasis Type="Italic">suis</Emphasis>from Two Naturally Infested Danish Sow Herds Using a Single Injection Regime with Doramectin

Attempts to eliminate Sarcoptes scabiei var. suis were made in 2 naturally infested sow herds, by intramuscular (IM) injection of doramectin (Dectomax^®, Pfizer, New York, USA). A single injection strategy was used. In one of the herds, the environment was treated with an acaricide following dry cleaning of floors, walls and equipment. In the second herd, no environmental treatment was performed. Results were measured by skin lesion scoring, ear scrapings to show Sarcoptes scabiei var. suis, and calculating rubbing index throughout the observation period of 20 months following treatment. Skin lesion scores decreased and stayed low following treatment for the entire observation period. Live Sarcoptes scabiei var. suis mites were isolated prior to treatment from both herds, but not following treatment. Rubbing index decreased following treatment, but was occasionally at or above 0.4. The results of these studies indicate that elimination of Sarcoptes scabiei var. suis from 2 naturally infested herds was successful, using doramectin in a single injection strategy. Precautions must be taken to ensure adequate dosing of every pig, and to avoid reinfestation due to poor biosecurity.     

Spatio-temporal distribution of sperm whales (Physeter macrocephalus) off Kaikoura,New Zealand,in relation to bathymetric features

Using a shore-based station we monitored the position of sperm whales (Physeter macrocephalus) within the Kaikoura submarine canyon from 2010 to 2012. We tracked sperm whales using a theodolite station for a total of 290 days. We extracted the distance from the nearest coast, the depth and the bathymetric slope using ArcGIS 10.1. We estimated the seasonal spatial distribution of sperm whales using general additive models. The distribution varied significantly between seasons; individuals were found in deeper water and further offshore in the spring than in winter. This study improved our understanding of the variability of sperm whale distribution patterns off Kaikoura. We determined that the distribution was linked to the bathymetric features and we hypothesized that whales adapted their use of the submarine canyon in relation to food aggregation. We would encourage further studies to evaluate the sperm whale relationship with oceanographic variables off Kaikoura.