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.     

How does precursor RNA structure influence RNA processing and gene expression?

RNA is a fundamental biomolecule that has many purposes within cells. Due to its single-stranded and flexible nature, RNA naturally folds into complex and dynamic structures. Recent technological and computational advances have produced an explosion of RNA structural data. Many RNA structures have regulatory and functional properties. Studying the structure of nascent RNAs is particularly challenging due to their low abundance and long length, but their structures are important because they can influence RNA processing. Precursor RNA processing is a nexus of pathways that determines mature isoform composition and that controls gene expression. In this review, we examine what is known about human nascent RNA structure and the influence of RNA structure on processing of precursor RNAs. These known structures provide examples of how other nascent RNAs may be structured and show how novel RNA structures may influence RNA processing including splicing and polyadenylation. RNA structures can be targeted therapeutically to treat disease.     

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.     

TRAM2 protein interacts with endoplasmic reticulum Ca2+ pump Serca2b and is necessary for collagen type I synthesis

Cotranslational insertion of type I collagen chains into the lumen of the endoplasmic reticulum (ER) and their subsequent folding into a heterotrimeric helix is a complex process which requires coordinated action of the translation machinery, components of translocons, molecular chaperones, and modifying enzymes. Here we describe a role for the protein TRAM2 in collagen type I expression in hepatic stellate cells (HSCs) and fibroblasts. Activated HSCs are collagen-producing cells in the fibrotic liver. Quiescent HSCs produce trace amounts of type I collagen, while upon activation collagen synthesis increases 50- to 70-fold. Likewise, expression of TRAM2 dramatically increases in activated HSCs. TRAM2 shares 53% amino acid identity with the protein TRAM, which is a component of the translocon. However, TRAM2 has a C terminus with only a 15% identity. The C-terminal part of TRAM2 interacts with the Ca(2+) pump of the ER, SERCA2b, as demonstrated in a Saccharomyces cerevisiae two-hybrid screen and by immunoprecipitations in human cells. TRAM2 also coprecipitates with anticollagen antibody, suggesting that these two proteins interact. Deletion of the C-terminal part of TRAM2 inhibits type I collagen synthesis during activation of HSCs. The pharmacological inhibitor of SERCA2b, thapsigargin, has a similar effect. Depletion of ER Ca(2+) with thapsigargin results in inhibition of triple helical collagen folding and increased intracellular degradation. We propose that TRAM2, as a part of the translocon, is required for the biosynthesis of type I collagen by coupling the activity of SERCA2b with the activity of the translocon. This coupling may increase the local Ca(2+) concentration at the site of collagen synthesis, and a high Ca(2+) concentration may be necessary for the function of molecular chaperones involved in collagen folding.     

Improved survival of vitrified porcine embryos after partial delipation through chemically stimulated lipolysis and inhibition of apoptosis

Mechanical removal of intracellular lipids has been the most effective approach to increase the cryosurvival of porcine embryos. In this experiment, we tested the hypotheses that the cryosurvival of porcine embryos can be improved after partial delipation through chemically stimulated lipolysis and that the survival can be further improved by inhibition of apoptosis. Porcine embryos were produced in vitro using sow oocytes. On Day 5 of embryonic development, embryos were cultured in the presence of 10 microM forskolin for 24h. On Day 6 blastocysts were vitrified using an open pulled straw (OPS) method and warmed blastocysts were cultured 18 h for them to recover. A caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was used at 20 microM during vitrification and subsequent culture to inhibit apoptosis. A 2 x 2 x 2 factorial design experiment was conducted to examine the effect of chemical delipation, vitrification and apoptosis inhibition. We also measured the lipolytic activity of porcine embryos cultured with or without forskolin. Chemical delipation increased the cryosurvival of porcine embryos compared to the controls (71.2+/-2.8% versus 37.1+/-5.1%). Apoptosis inhibition increased the ability of blastocysts to fully recover (23.8+/-3.1% versus 14.6+/-4.3%). However, there was no interaction between chemical delipation and apoptosis inhibition. Lipolytic agent treatment increased the lipolytic activity of porcine blastocysts. In conclusion, cryosurvival of porcine embryos was improved by partial delipation through chemical stimulation of lipolysis or apoptosis inhibition.     

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.