Conformational coupling,bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP–NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop–trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β′ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.     

Fibulin-1 Is Increased in Asthma – A Novel Mediator of Airway Remodeling?

Background

The extracellular matrix is a dynamic and complex network of macromolecules responsible for maintaining and influencing cellular functions of the airway. The role of fibronectin, an extracellular matrix protein, is well documented in asthma. However, the expression and function of fibulin-1, a secreted glycoprotein which interacts with fibronectin, has not been reported. Fibulin-1 is widely expressed in basement membranes in many organs including the lung. There are four isoforms in humans (A–D) of which fibulin-1C and 1D predominate. The objective of this study was to study the expression of fibulin-1 in volunteers with and without asthma, and to examine its function in vitro.

Methodology/Principal Findings

We used immunohistochemistry and dot-blots to examine fibulin-1 levels in bronchial biopsies, bronchoalveolar lavage fluid and serum. Real-time PCR for fibulin-1C and 1D, and ELISA and western blotting for fibulin-1 were used to study the levels in airway smooth muscle cells. The function of fibulin-1C was determined by assessing its role, using an antisense oligonucleotide, in cell proliferation, migration and wound healing. A murine model of airway hyperresponsiveness (AHR) was used to explore the biological significance of fibulin-1. Levels of fibulin-1 were significantly increased in the serum and bronchoalveolar lavage fluid of 21 asthmatics compared with 11 healthy volunteers. In addition fibulin-1 was increased in asthma derived airway smooth muscle cells and fibulin-1C contributed to the enhanced proliferation and wound repair in these cells. These features were reversed when fibulin-1C was suppressed using an antisense oligomer. In a mouse model of AHR, treatment with an AO inhibited the development of AHR to methacholine.

Conclusions

Our data collectively suggest fibulin-1C may be worthy of further investigation as a target for airway remodeling in asthma.     

Changes in upland bird abundances show associations with moorland management

Capsule: Changes in abundance of six bird species showed associations with moorland management.

Aims: To assess responses of breeding birds to moorland management over a 14-year period.

Methods: Vegetation and birds were surveyed at 2–3-year intervals and changes examined in relation to sheep and cattle grazing, vegetation burning and cutting.

Results: Seven correlations between change in management and change in bird abundance were detected, and six between change in vegetation and change in bird abundance. On plots where sheep numbers declined, Golden Plover Pluvialis apricaria and Northern Wheatear Oenanthe oenanthe declined. Where a greater area was burned, Golden Plover increased in the initial post-burning period but Red Grouse Lagopus lagopus scotica declined. Eurasian Curlew Numenius arquata and Sky Lark Alauda arvensis increased where a greater area of moorland vegetation was cut. Whinchat Saxicola rubetra declined with increasing cattle numbers on a plot.

Conclusions: Bird populations respond to changes in moorland management, but these changes are not always associated with detectable changes in vegetation. These responses of moorland breeding birds to management could help refine agri-environment options and other conservation interventions on moorland. Responses differed between bird species, ideally requiring site-specific planning where managing for multiple species is a goal.     

Brassica rapa Domestication: Untangling Wild and Feral Forms and Convergence of Crop Morphotypes

The study of domestication contributes to our knowledge of evolution and crop genetic resources. Human selection has shaped wild Brassica rapa into diverse turnip, leafy, and oilseed crops. Despite its worldwide economic importance and potential as a model for understanding diversification under domestication, insights into the number of domestication events and initial crop(s) domesticated in B. rapa have been limited due to a lack of clarity about the wild or feral status of conspecific noncrop relatives. To address this gap and reconstruct the domestication history of B. rapa, we analyzed 68,468 genotyping-by-sequencing-derived single nucleotide polymorphisms for 416 samples in the largest diversity panel of domesticated and weedy B. rapa to date. To further understand the center of origin, we modeled the potential range of wild B. rapa during the mid-Holocene. Our analyses of genetic diversity across B. rapa morphotypes suggest that noncrop samples from the Caucasus, Siberia, and Italy may be truly wild, whereas those occurring in the Americas and much of Europe are feral. Clustering, tree-based analyses, and parameterized demographic inference further indicate that turnips were likely the first crop type domesticated, from which leafy types in East Asia and Europe were selected from distinct lineages. These findings clarify the domestication history and nature of wild crop genetic resources for B. rapa, which provides the first step toward investigating cases of possible parallel selection, the domestication and feralization syndrome, and novel germplasm for Brassica crop improvement.     

The essential role of centrosomal NDE1 in human cerebral cortex neurogenesis

We investigated three families whose offspring had extreme microcephaly at birth and profound mental retardation. Brain scans and postmortem data showed that affected individuals had brains less than 10% of expected size (≤10 standard deviation) and that in addition to a massive reduction in neuron production they displayed partially deficient cortical lamination (microlissencephaly). Other body systems were apparently unaffected and overall growth was normal. We found two distinct homozygous mutations of NDE1, c.83+1G>T (p.Ala29GlnfsX114) in a Turkish family and c.684_685del (p.Pro229TrpfsX85) in two families of Pakistani origin. Using patient cells, we found that c.83+1G>T led to the use of a novel splice site and to a frameshift after NDE1 exon 2. Transfection of tagged NDE1 constructs showed that the c.684_685del mutation resulted in a NDE1 that was unable to localize to the centrosome. By staining a patient-derived cell line that carried the c.83+1G>T mutation, we found that this endogeneously expressed mutated protein equally failed to localize to the centrosome. By examining human and mouse embryonic brains, we determined that NDE1 is highly expressed in neuroepithelial cells of the developing cerebral cortex, particularly at the centrosome. We show that NDE1 accumulates on the mitotic spindle of apical neural precursors in early neurogenesis. Thus, NDE1 deficiency causes both a severe failure of neurogenesis and a deficiency in cortical lamination. Our data further highlight the importance of the centrosome in multiple aspects of neurodevelopment.     

A new insight into the role of rat cytochrome P450 24A1 in metabolism of selective analogs of 1α,25-dihydroxyvitamin D3

We examined the metabolism of two synthetic analogs of 1α,25-dihydroxyvitamin D₃ (1), namely 1α,25-dihydroxy-16-ene-23-yne-vitamin D₃ (2) and 1α,25-dihydroxy-16-ene-23-yne-26,27-dimethyl-vitamin D₃ (4) using rat cytochrome P450 24A1 (CYP24A1) in a reconstituted system. We noted that 2 is metabolized into a single metabolite identified as C26-hydroxy-2 while 4 is metabolized into two metabolites, identified as C26-hydroxy-4 and C26a-hydroxy-4. The structural modification of adding methyl groups to the side chain of 1 as in 4 is also featured in another analog, 1α,25-dihydroxy-22,24-diene-24,26,27-trihomo-vitamin D₃ (6). In a previous study, 6 was shown to be metabolized exactly like 4, however, the enzyme responsible for its metabolism was found to be not CYP24A1. To gain a better insight into the structural determinants for substrate recognition of different analogs, we performed an in silico docking analysis using the crystal structure of rat CYP24A1 that had been solved for the substrate-free open form. Whereas analogs 2 and 4 docked similar to 1, 6 showed altered interactions for both the A-ring and side chain, despite prototypical recognition of the CD-ring. These findings hint that CYP24A1 metabolizes selectively different analogs of 1, based on their ability to generate discrete recognition cues required to close the enzyme and trigger the catalytic mechanism.     

Heparin binds 8 kDa gelsolin cross-β-sheet oligomers and accelerates amyloidogenesis by hastening fibril extension

Glycosaminoglycans (GAGs) are highly sulfated linear polysaccharides prevalent in the extracellular matrix, and they associate with virtually all amyloid deposits in vivo. GAGs accelerate the aggregation of many amyloidogenic peptides in vitro, but little mechanistic evidence is available to explain why. Herein, spectroscopic methods demonstrate that GAGs do not affect the secondary structure of the monomeric 8 kDa amyloidogenic fragment of human plasma gelsolin. Moreover, monomerized 8 kDa gelsolin does not bind to heparin under physiological conditions. In contrast, 8 kDa gelsolin cross-β-sheet oligomers and amyloid fibrils bind strongly to heparin, apparently because of electrostatic interactions between the negatively charged polysaccharide and a positively charged region of the 8 kDa gelsolin assemblies. Our observations are consistent with a scaffolding mechanism whereby cross-β-sheet oligomers, upon formation, bind to GAGs, accelerating the fibril extension phase of amyloidogenesis, possibly by concentrating and orienting the oligomers to more efficiently form amyloid fibrils. Notably, heparin decreases the 8 kDa gelsolin concentration necessary for amyloid fibril formation, likely a consequence of fibril stabilization through heparin binding. Because GAG overexpression, which is common in amyloidosis, may represent a strategy for minimizing cross-β-sheet oligomer toxicity by transforming them into amyloid fibrils, the mechanism described herein for GAG-mediated acceleration of 8 kDa gelsolin amyloidogenesis provides a starting point for therapeutic strategy development. The addition of GAG mimetics, small molecule sulfonates shown to reduce the amyloid load in animal models of amyloidosis, to a heparin-accelerated 8 kDa gelsolin aggregation reaction mixture neither significantly alters the rate of amyloidogenesis nor prevents oligomers from binding to GAGs, calling into question their commonly accepted mechanism.     

Whole-genome sequences of two Borrelia afzelii and two Borrelia garinii Lyme disease agent isolates

Human Lyme disease is commonly caused by several species of spirochetes in the Borrelia genus. In Eurasia these species are largely Borrelia afzelii, B. garinii, B. burgdorferi, and B. bavariensis sp. nov. Whole-genome sequencing is an excellent tool for investigating and understanding the influence of bacterial diversity on the pathogenesis and etiology of Lyme disease. We report here the whole-genome sequences of four isolates from two of the Borrelia species that cause human Lyme disease, B. afzelii isolates ACA-1 and PKo and B. garinii isolates PBr and Far04.