Characterization of stanniocalcin 2, a novel target of the mammalian unfolded protein response with cytoprotective properties

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces a highly conserved homeostatic response in all eukaryotic cells, termed the unfolded-protein response (UPR). Here we describe the characterization of stanniocalcin 2 (STC2), a mammalian homologue of a calcium- and phosphate-regulating hormone first identified in fish, as a novel target of the UPR. Expression of STC2 gene is rapidly upregulated in cultured cells after exposure to tunicamycin and thapsigargin, by ATF4 after activation of the ER-resident kinase PERK. In addition, STC2 expression is also activated in neuronal cells by oxidative stress and hypoxia but not by several cellular stresses unrelated to the UPR. In contrast, expression of another homologue, STC1, is only upregulated by hypoxia independent of PERK or ATF4 expression. In vivo studies revealed that rat cortical neurons rapidly upregulate STC2 after transient middle cerebral artery occlusion. Finally, siRNA-mediated inhibition of STC2 expression renders N2a neuroblastoma cells and HeLa cells significantly more vulnerable to apoptotic cell death after treatment with thapsigargin, and overexpression of STC2 attenuated thapsigargin-induced cell death. Consequently, induced STC2 expression is an essential feature of survival component of the UPR.     

Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling

Mammalian organs are typically comprised of several cell populations. Some (e.g. brain) are very heterogeneous, and this cellular complexity makes it difficult, if not impossible, to interpret expression profiles obtained with microarrays. Instruments, such as those manufactured by Leica or Arcturus, that permit laser capture microdissection of specific cells or cell groups from tissues were developed to solve this problem. To take full advantage of these instruments, however, one must be able to recognize cell populations of interest and, after they are harvested, to extract intact, unmodified RNA from them. Here we describe a novel, fast and simple method to fix and immunostain tissue sections that permits this to be done.     

Fiber fractionation to understand the effect of mechanical refining on fiber structure and resulting enzymatic digestibility of biomass

Mechanical refining results in fiber deconstruction and modifications that enhance enzyme accessibility to carbohydrates. Further understanding of the morphological changes occurring to biomass during mechanical refining and the impacts of these changes on enzymatic digestibility is necessary to maximize yields and reduce energy consumption. Although the degree of fiber length reduction relative to fibrillation/delamination can be impacted by manipulating refining variables, mechanical refining of any type (PFI, disk, and valley beater) typically results in both phenomena. Separating the two is not straightforward. In this study, fiber fractionation based on particle size performed after mechanical refining of high-lignin pulp was utilized to successfully elucidate the relative impact of fibrillation/delamination and fiber cutting phenomena during mechanical refining. Compositional analysis showed that fines contain significantly more lignin than larger size fractions. Enzymatic hydrolysis results indicated that within fractions of uniform fiber length, fibrillation/delamination due to mechanical refining increased enzymatic conversion by 20–30 percentage points. Changes in fiber length had little effect on digestibility for fibers longer than ~0.5 mm. However, the digestibility of the fines fractions was high for all levels of refining even with the high-lignin content.     

Genome Sequence of Blattabacterium sp. Strain BGIGA, Endosymbiont of the Blaberus giganteus Cockroach

Cockroaches harbor the obligate flavobacterial endosymbiont Blattabacterium sp., which resides within the host's bacteriocytes and can recycle ammonia and urea nitrogenous wastes into amino acids for the host. We report the complete genome sequence of the Blattabacterium sp. associated with the giant roach Blaberus giganteus.     

A simple and rapid nonviral approach to efficiently transfect primary tissue-derived cells using polyethylenimine

This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available off-the-shelf cationic polymer, 25-kDa branched polyethylenimine (bPEI25). Transfection efficiency of cationic polymers varies among cell lines and is highly dependent on the conditions and environment in which complexes are formed. Factors requiring optimization include the salt concentration, volume, incubation time, mixing order and ratio of polymer to DNA. In this transfection protocol, complexes are prepared in 30 min, with analysis 24 h later; thus, experiments can be completed in 2 d. In this protocol, as an example, we describe the parameters we have optimized for the transfection of bone marrow stromal cells and normal human foreskin fibroblasts. By using this protocol, we have obtained transfection efficiencies comparable to lipofection. An appropriately optimized protocol enhances the utility of cationic polymers in transfecting mammalian cells, thereby providing an effective alternative to expensive commercial reagents.     

Integrating candidate gene and quantitative genetic approaches to understand variation in timing of breeding in wild tit populations

Two commonly used techniques for estimating the effect of genes on traits in wild populations are the candidate gene approach and quantitative genetic analyses. However, whether these two approaches measure the same underlying processes remains unresolved. Here, we use these two methods to test whether they are alternative or complementary approaches to understanding genetic variation in the timing of reproduction - a key trait involved in adaptation to climate change - in wild tit populations. Our analyses of the candidate gene Clock show weak correlates with timing variables in blue tits, but no association in great tits, confirming earlier results. Quantitative genetic analyses revealed very low levels of both direct (female) and indirect (male) additive genetic variation in timing traits for both species, in contrast to previous studies on these traits, and much lower than generally assumed. Hence, neither method suggests strong genetic effects on the timing of breeding in birds, and further work should seek to assess the generality of these conclusions. We discuss how differences in the genetic control of traits, species life-history and confounding environmental variables may determine how useful integrating these two techniques is to understand the phenotypic variation in wild populations.     

Development of a quantitative assay to measure expression of transforming growth factor β (TGF-β) in Lost River sucker (Deltistes luxatus) and shortnose sucker (Chasmistes brevirostris) and evaluation of potential pitfalls in use with field-collected samples

The Nature Conservancy is in the process of restoring the Williamson River Delta in an attempt to recreate important juvenile habitat for the endangered shortnose sucker Chasmistes brevirostris and the endangered Lost River sucker Deltistes luxatus. Measurement of TGF-β mRNA expression level was one of the indicators chosen to evaluate juvenile sucker health during the restoration process. TGF-β mRNA expression level has been correlated with disease status in several laboratory studies and TGF-β mRNA expression level has been used as a species-specific indicator of immune status in field-based fish health assessments. We describe here the identification of TGF-β and a possible splice variant from shortnose sucker and from Lost River sucker. The performance of a quantitative RT-PCR assay to measure TGF-β mRNA expression level was evaluated in field-collected spleen and kidney tissue samples. The quality of extracted RNA was higher in tissues harvested in September compared to July and higher in tissues harvested at lower temperature compared to higher temperature. In addition, the expression level of both TGF-β and 18S as assessed by qRT-PCR was higher in samples with higher quality RNA. TGF-β mRNA expression was lower in kidney than in spleen in both Lost River sucker and shortnose sucker.