Dynamic profiling of double-stranded RNA binding proteins

Double-stranded (ds) RNA is a key player in numerous biological activities in cells, including RNA interference, anti-viral immunity and mRNA transport. The class of proteins responsible for recognizing dsRNA is termed double-stranded RNA binding proteins (dsRBP). However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 on six dsRNA substrates that vary in length and secondary structure. We combined single molecule pull-down (SiMPull), single molecule protein-induced fluorescence enhancement (smPIFE) and molecular dynamics (MD) simulations to investigate the dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificities and dynamic properties when in contact with different RNA substrates. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to highly structured RNA substrates. Upon interaction with RNA substrates, TRBP and Staufen1 exhibit dynamic sliding whereas two deaminases ADAR1 and ADAD2 mostly remain immobile when bound. MD simulations provide a detailed atomic interaction map that is largely consistent with the affinity differences observed experimentally. Collectively, our study highlights the diverse nature of substrate specificity and mobility exhibited by dsRBPs that may be critical for their cellular function.     

Differential Susceptibility of SD and CD Rats to a Novel Rat Theilovirus

Antibodies to rat theilovirus (RTV) have been detected in rats for many years because of their serologic crossreactivity with strains of Theiler murine encephalomyelitis virus (TMEV) of mice. Little information exists regarding this pathogen, yet it is among the most common viruses detected in serologic surveys of rats used in research. In the study reported here, a novel isolate of RTV, designated RTV1, was cultured from the feces of infected rats. The RTV1 genome contained 8094 nucleotides and had approximately 95% identity with another rat theilovirus, NSG910, and 73% identity with TMEV strains. In addition, the genome size of RTV1 was similar to those of TMEV strains but larger than that reported for NSG910. Oral inoculation of Sprague–Dawley (SD) and CD male rats (n = 10 each group) with RTV1 revealed that SD rats were more susceptible than CD rats to RTV1 infection. At 14 d postinoculation, 100% of SD rats shed virus in the feces, and 70% were positive for RTV serum antibodies. By 56 d postinoculation 30% of SD rats continued to have detectable virus in the feces, and 90% had seroconverted. In contrast, in inoculated CD rats RTV was detected only in the feces at 14 d postinoculation, at which time 40% of CD rats were fecal positive. By 56 d postinoculation only 20% of CD rats had detectable RTV serum antibodies. Our data provide additional sequence information regarding a rat-specific Cardiovirus and indicate that SD rats are more susceptible than CD rats to RTV1 infection.Abbreviations: RACE, rapid amplification of cDNA ends; RTV, rat theilovirus; SD, Sprague Dawley; TMEV, Theiler murine encephalomyelitis virusFor decades it has been known that rats used in research can develop antibodies to a Cardiovirus that is antigenically similar to Theiler murine encephalomyelitis virus (TMEV) of mice.^{4,6,10,12,13,20} Recent reports on the prevalence of antibodies in rats to this Cardiovirus vary from approximately 0.6% of sera tested from research rats in North America¹⁰ to 54.4% in a survey of 18 Brazilian research facilities.^3,6,20 Multiple designations have been used to identify the Cardiovirus that infects rats, including Theiler-like virus of rats,¹³ Theiler murine encephalomyelitis virus (TMEV),²⁰ rat enterovirus,¹ rat encephalomyelitis virus,⁷ rat cardiovirus,¹⁵ and recently rat theilovirus.² We have elected to refer to the virus as rat theilovirus (RTV), consistent with 1 of the cited references,² to indicate the relation of the rat virus to TMEV of mice and to identify it as a rat-specific agent.The first report of natural infection of rats with a Cardiovirus was in 1964 with the discovery of MHG virus.¹² The finding resulted from an isolated observation in which a few rats in a large research colony displayed clinical signs indicative of central nervous system deficits, including incoordination, torticollis, circling, and tremors. The MHG virus recovered from infected rats was antigenically crossreactive with TMEV strain GDVII and had physical properties consistent with viruses in the Picornaviridae family. The virus was propagated in cell culture, and neurologic disease was reproduced when virus was inoculated into suckling mice and suckling rats.¹² Subsequent serologic studies using crossneutralization, complement fixation, and hemagglutination inhibition assays further substantiated the antigenic relatedness between MHG virus and multiple strains of TMEV.^4,11 In addition, sera from ‘normal’ rats contained antibodies to the newly identified Theiler-like virus of rats, suggesting widespread infection of the virus in research rat colonies.¹² More recently in Japan, a Theiler-like virus was isolated after intracranial inoculation of newborn Wistar rats with intestinal homogenates from TMEV GDVII-seropositive rats.¹³ Inoculated rats did not develop clinical signs of infection, but virus was cultivated in BHK21 cells from brain homogenates of the 10-d-old Wistar rats inoculated intracranially. Physiochemical properties of the virus, designated NSG910, were consistent with those of the Cardiovirus genus. Sequence analysis also showed that NSG910 was a Cardiovirus in the family Picornaviridae that was related to, but distinct from MHG virus, and strains of TMEV. This report served to further document the existence of a unique Cardiovirus of rats closely related to, but distinct from, TMEV strains.¹³ In a recent report from Brazil, neonatal mice and rats inoculated with intestinal homogenates from rats with antibodies to TMEV strain GDVII developed neurologic signs of flaccid hindlimb paralysis and tremors. In addition, brain homogenates from the affected animals were positive by RT-PCR for cardioviral RNA.²⁰Picornavirus virions are approximately 30 nm in diameter, nonenveloped, with icosahedral symmetry and a single-stranded, positive-sense RNA genome.¹⁹ Encephalomyocarditis virus and Theilovirus are 2 species of Cardiovirus in the Picornaviridae family. Encephalomyocarditis virus species includes mengovirus, Maus Elberfeld virus, and Columbia SK virus.⁷ Strains of Theilovirus species include TMEV, Vilyuisk virus, and RTV.^13,18,22 Most often studied are the TMEV strains, which are classified according to their neurovirulence after intracerebral inoculation. Included are the highly neurovirulent GD VII and FA strains²³ and the less virulent, more persistent DA, BeAn 8386, WW, and TO (Theiler original) strains.^9,17,22 Studies have shown that the virus replicates in the alimentary tract and is shed in the feces of infected mice.^15,19 Mice rarely show clinical disease when infected under natural conditions; however, neurologic manifestations have been reported.^21,24Sentinel animals typically are used to survey rodent colonies for the presence or absence of infectious agents. Outbred stocks are frequently used as sentinels because of their vigor, relatively low cost, and ability to mount a robust humoral immune response to infectious agents.^8,14 Sprague–Dawley (SD) and CD rats are 2 stocks that are commonly used as sentinels for rat colonies. The origins of the SD rat (Rattus norvegicus) date back to the 1920s as a result of mating Wistar stock with a hybrid rat stock of unknown origin. In the 1950s, an SD breeding stock was cesarean derived in an effort to improve microbiologic status. This nucleus of cesarean-derived rats formed the foundation of the CD rat stock.²⁵ Because SD and CD rat stocks have a common ancestry, they frequently are considered to be interchangeable for the purpose of sentinel animals.In the studies reported here, we isolated and propagated a novel strain of Theilovirus, referred to as RTV1, from the feces of infected SD rats. The entire genome of RTV1 was sequenced and compared with those of isolates of TMEV and NSG910, the only other isolate of RTV to be sequenced in its entirety. In addition, we evaluated the susceptibility of SD and CD outbred rats to RTV1 after oral inoculation with the virus.     

Multiplex fluorescent immunoassay for the simultaneous detection of serum antibodies to multiple rodent pathogens

Multiplex Fluorescent Immunoassay (MFI) is a sensitive and specific serologic test that allows simultaneous detection of antibodies to multiple viral and bacterial agents in a single reaction well. MFI is a high-throughput assay that offers several advantages over other prevalent assays, and some research animal diagnostic laboratories have adopted it as their primary technique. The authors present a detailed review of MFI and its application to laboratory animal diagnostics.     

Early-Life Telomere Dynamics Differ between the Sexes and Predict Growth in the Barn Swallow (Hirundo rustica)

Telomeres are conserved DNA-protein structures at the termini of eukaryotic chromosomes which contribute to maintenance of genome integrity, and their shortening leads to cell senescence, with negative consequences for organismal functions. Because telomere erosion is influenced by extrinsic and endogenous factors, telomere dynamics may provide a mechanistic basis for evolutionary and physiological trade-offs. Yet, knowledge of fundamental aspects of telomere biology under natural selection regimes, including sex- and context-dependent variation in early-life, and the covariation between telomere dynamics and growth, is scant. In this study of barn swallows (Hirundo rustica) we investigated the sex-dependent telomere erosion during nestling period, and the covariation between relative telomere length and body and plumage growth. Finally, we tested whether any covariation between growth traits and relative telomere length depends on the social environment, as influenced by sibling sex ratio. Relative telomere length declined on average over the period of nestling maximal growth rate (between 7 and 16 days of age) and differently covaried with initial relative telomere length in either sex. The frequency distribution of changes in relative telomere length was bimodal, with most nestlings decreasing and some increasing relative telomere length, but none of the offspring traits predicted the a posteriori identified group to which individual nestlings belonged. Tail and wing length increased with relative telomere length, but more steeply in males than females, and this relationship held both at the within- and among-broods levels. Moreover, the increase in plumage phenotypic values was steeper when the sex ratio of an individual’s siblings was female-biased. Our study provides evidence for telomere shortening during early life according to subtly different dynamics in either sex. Furthermore, it shows that the positive covariation between growth and relative telomere length depends on sex as well as social environment, in terms of sibling sex ratio.     

Different strategies for multi-enzyme cascade reaction for chiral vic-1,2-diol production

Bioprocess and Biosystems Engineering - The stereoselective three-enzyme cascade for the one-pot synthesis of (1S,2S)-1-phenylpropane-1,2-diol ((1S,2S)-1-PPD) from inexpensive starting substrates,...     

Atomic force microscopy studies provide direct evidence for dimerization of the HIV restriction factor APOBEC3G

APOBEC3G (A3G) is an antiviral protein that binds RNA and single-stranded DNA (ssDNA). The oligomerization state of A3G is likely to be influenced by these nucleic acid interactions. We applied the power of nanoimaging atomic force microscopy technology to characterize the role of ssDNA in A3G oligomerization. We used recombinant human A3G prepared from HEK-293 cells and specially designed DNA substrates that enable free A3G to be distinguished unambiguously from DNA-bound protein complexes. This DNA substrate can be likened to a molecular ruler because it consists of a 235-bp double-stranded DNA visual tag spliced to a 69-nucleotide ssDNA substrate. This hybrid substrate enabled us to use volume measurements to determine A3G stoichiometry in both free and ssDNA-bound states. We observed that free A3G is primarily monomeric, whereas ssDNA-complexed A3G is mostly dimeric. A3G stoichiometry increased slightly with the addition of Mg(2+), but dimers still predominated when Mg(2+) was depleted. A His-248/His-250 Zn(2+)-mediated intermolecular bridge was observed in a catalytic domain crystal structure (Protein Data Bank code 3IR2); however, atomic force microscopy analyses showed that the stoichiometry of the A3G-ssDNA complexes changed insignificantly when these residues were mutated to Ala. We conclude that A3G exchanges between oligomeric forms in solution with monomers predominating and that this equilibrium shifts toward dimerization upon binding ssDNA.     

Etiologic Agents of Central Nervous System Infections among Febrile Hospitalized Patients in the Country of Georgia

Objectives

There is a large spectrum of viral, bacterial, fungal, and prion pathogens that cause central nervous system (CNS) infections. As such, identification of the etiological agent requires multiple laboratory tests and accurate diagnosis requires clinical and epidemiological information. This hospital-based study aimed to determine the main causes of acute meningitis and encephalitis and enhance laboratory capacity for CNS infection diagnosis.

Methods

Children and adults patients clinically diagnosed with meningitis or encephalitis were enrolled at four reference health centers. Cerebrospinal fluid (CSF) was collected for bacterial culture, and in-house and multiplex RT-PCR testing was conducted for herpes simplex virus (HSV) types 1 and 2, mumps virus, enterovirus, varicella zoster virus (VZV), Streptococcus pneumoniae, HiB and Neisseria meningitidis.

Results

Out of 140 enrolled patients, the mean age was 23.9 years, and 58% were children. Bacterial or viral etiologies were determined in 51% of patients. Five Streptococcus pneumoniae cultures were isolated from CSF. Based on in-house PCR analysis, 25 patients were positive for S. pneumoniae, 6 for N. meningitidis, and 1 for H. influenzae. Viral multiplex PCR identified infections with enterovirus (n = 26), VZV (n = 4), and HSV-1 (n = 2). No patient was positive for mumps or HSV-2.

Conclusions

Study findings indicate that S. pneumoniae and enteroviruses are the main etiologies in this patient cohort. The utility of molecular diagnostics for pathogen identification combined with the knowledge provided by the investigation may improve health outcomes of CNS infection cases in Georgia.     

Effect of dietary copper on selenium toxicity in Fischer 344 rats

The purpose of this study was to investigate the ameliorating effects of dietary copper supplementation on selenium toxicity. Nine groups (n = 6) of weanling Fischer 344 female rats were randomly assigned to treatment groups and fed diets containing nontoxic levels of copper as CuCl2 and/or selenium as selenite or selenocystamine. Weight gain, liver and spleen weights, plasma lipid peroxidation, and liver selenium and copper content were analyzed after the 6-wk treatment period. Concentrations of up to 10 times the daily lethal dose of dietary selenium were well tolerated in rats supplemented with dietary copper. As the dietary level of selenium was increased, the ratio of selenium to copper measured in the liver decreased. In the groups of rats in which dietary copper supplementation was absent and dietary selenium was supplemented, copper stores in the liver remained unchanged from control values. Copper's protective effects from dietary selenium toxicity may come from the formation of a copper-selenide complex that renders both selenium and copper metabolically unavailable and nontoxic.     

Peroxisome proliferator-activated receptor alpha agonists as therapy for autoimmune disease

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. PPAR gamma ligands, which include the naturally occurring PG metabolite 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), as well as thiazolidinediones, have been shown to have anti-inflammatory activity. The PPAR alpha agonists, gemfibrozil, ciprofibrate, and fenofibrate, have an excellent track history as oral agents used to treat hypertriglyceridemia. In the present study, we demonstrate that these PPAR alpha agonists can increase the production of the Th2 cytokine, IL-4, and suppress proliferation by TCR transgenic T cells specific for the myelin basic protein Ac1-11, as well as reduce NO production by microglia. Oral administration of gemfibrozil and fenofibrate inhibited clinical signs of experimental autoimmune encephalomyelitis. More importantly, gemfibrozil was shown to shift the cytokine secretion of human T cell lines by inhibiting IFN-gamma and promoting IL-4 secretion. These results suggest that PPAR alpha agonists such as gemfibrozil and fenofibrate, may be attractive candidates for use in human inflammatory conditions such as multiple sclerosis.