Isolation and characterization of microsatellite markers from the entomopathogenic fungus Metarhizium anisopliae

Fourteen polymorphic microsatellite markers were isolated from the entomopathogenic fungus, Metarhizium anisopliae, based on enriched genomic libraries. In order to assess allelic variability, the microsatellite loci were analysed in a collection of 34 isolates sampled from across Switzerland. The number of detected alleles in 14 loci ranged from two to eight and expected heterozygosity from 0.265 to 0.808. Because of the high expected heterozygosity, the 14 microsatellite loci are very useful for ecological studies and analysis of population diversity, and to identifying, monitoring, and tracking M. anisopliae strains applied as biological control agents.     

Sensitivity of respiratory metabolism and efficiency to foliar nitrogen during growth and maintenance

Scaling of respiration from the leaf to the canopy level currently depends on identification of physiological parameters that are tightly linked to respiration and that can readily be determined. Several recent studies have helped provide guides to predicting whole canopy respiration on the basis of foliar nitrogen (N). This approach is potentially powerful owing to the well‐described patterns of allocation of N that follow interception of radiation. In the present study, we investigated the sensitivity of the N–respiration correlation to environmental and developmental factors, in order to evaluate its usage for attempts to scale respiration to the organism and ecosystem level. We studied fully expanded, 1 and 2‐year‐old, and current‐year needles from canopies of Pinus radiata that had been treated (unthinned, thinned and thinned+fertilized treatments) in ways likely to induce a wide range of growth and respiratory responses. We examined respiration in detail during the growth period in spring and again at the end of summer, using calorespirometric methods (combined measurements of CO₂ and heat rates) to determine the respiration rates , instantaneous enthalpic growth rates (R_SGΔH_B, a measure of the conservation of electrons in anabolic products) and the enthalpy conversion efficiency (η_H) of needles differing in age. A general linear model revealed that was positively correlated with needle N, but this correlation was strongly dependent on the season and the needle age – indicating an important physiological difference between expanding young needles and fully expanded old needles. Furthermore, the strength of the correlation between needle N and respiration was comparatively weak for the current year, expanding foliage, indicating that factors other than foliage N significantly influenced the respiration of young needles. The analysis of instantaneous growth rates revealed two general processes. Older, nonexpanding foliage showed considerable rates of enthalpic growth (increases in enthalpy) that was mainly caused by the increment of lignin during secondary growth. Secondly, canopy development appeared dynamic and to be optimized according to environmental drivers and constraints – such as light and water availability. In late spring, needle extension slowed in the upper, but not the lower canopy, because the upper canopy appeared to be affected first by the onset of drought stress in late spring. Growth rates were reduced in the upper canopy despite greater rates of respiration, indicating higher demand of ATP for the maintenance of protein and for export of sugars. Consequently, the enthalpy conversion efficiency and enthalpic N productivity (enthalpic growth per unit N) were comparatively poor indicating advanced development of needles in the upper canopy. We suggest that the growth and maintenance paradigm of respiration is, at best, only moderately useful when applied to whole trees, and is not valid at the cellular level or that of the plant organ. A different concept, namely that of respiratory efficiency, seems a more suitable way to represent respiration in carbon (C) balance models and should help provide a better mechanistic understanding of how respiration affects the C conversion efficiency of plants, and ultimately the net primary productivity of ecosystems.     

Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK

Short-patch repair of DNA single-strand breaks and gaps (SSB) is coordinated by XRCC1, a scaffold protein that recruits the DNA polymerase and DNA ligase required for filling and sealing the damaged strand. XRCC1 can also recruit end-processing enzymes, such as PNK (polynucleotide kinase 3′-phosphatase), Aprataxin and APLF (aprataxin/PNK-like factor), which ensure the availability of a free 3′-hydroxyl on one side of the gap, and a 5′-phosphate group on the other, for the polymerase and ligase reactions respectively. PNK binds to a phosphorylated segment of XRCC1 (between its two C-terminal BRCT domains) via its Forkhead-associated (FHA) domain. We show here, contrary to previous studies, that the FHA domain of PNK binds specifically, and with high affinity to a multiply phosphorylated motif in XRCC1 containing a pSer-pThr dipeptide, and forms a 2:1 PNK:XRCC1 complex. The high-resolution crystal structure of a PNK–FHA–XRCC1 phosphopeptide complex reveals the basis for this unusual bis-phosphopeptide recognition, which is probably a common feature of the known XRCC1-associating end-processing enzymes.     

Electron microscopy of Xrcc4 and the DNA ligase IV–Xrcc4 DNA repair complex

The DNA ligase IV–Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4–Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4–Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of ～37 Å reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4–Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4–Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.     

Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications

Exploitation of potential new targets for drug and vaccine development has an absolute requirement for multimilligram quantities of soluble protein. While recombinant expression of full-length proteins is frequently problematic, high-yield soluble expression of functional subconstructs is an effective alternative, so long as appropriate termini can be identified. Bioinformatics localizes domains, but doesn't predict boundaries with sufficient accuracy, so that subconstructs are typically found by trial and error. Combinatorial Domain Hunting (CDH) is a technology for discovering soluble, highly expressed constructs of target proteins. CDH combines unbiased, finely sampled gene-fragment libraries, with a screening protocol that provides "holistic" readout of solubility and yield for thousands of protein fragments. CDH is free of the "passenger solubilization" and out-of-frame translational start artifacts of fusion-protein systems, and hits are ready for scale-up expression. As a proof of principle, we applied CDH to p85alpha, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.     

Serum vaspin concentration in elderly patients with type 2 diabetes mellitus and macrovascular complications

Background

Adipose tissue, an endocrine organ of the body, is involved in some obesity-related disease states such as insulin resistance, diabetes mellitus, and atherosclerosis. Vaspin is a novel adipocyte with insulin sensitizing effects. In this study, we planned to estimate serum vaspin concentrations as related to glycemic status and the presence of macrovascular complications among elderly patients with type-2 diabetes mellitus (T2DM).

Methods

A total of 230 elderly patients with T2DM were evaluated. These patients were divided into two groups: patients without complications (T2DM group, n?=?110), and patients with macrovascular complications (T2DM + MC group, n?=?120). In addition, 60 healthy elderly subjects were enrolled and assigned into the control group (NC group). Relevant parameters were matched for age and gender ratio. Serum vaspin concentrations were measured by Enzyme-linked immunosorbent assay (ELISA). Anthropometric measurements, plasma glucose and HbA_1C levels, insulin concentration, liver and kidney functions, and lipid profile were measured for each participant.

Results

Serum vaspin concentrations were significantly higher in the T2DM group than in the T2DM + MC group (F?=?13.122, P?<?0.01). These concentrations were also significantly higher among females, compared to males (T?=?3.567, P?<?0.05). Logistic regression analysis revealed that serum vaspin concentration, systolic blood pressure, HDL-C and T2DM duration were independent influencing factors for diabetic macrovascular complications.

Conclusion

Serum vaspin may be considered as a potential marker to assess the status of elderly patients with T2DM and the risk of developing serious macrovascular complications. Further prospective studies are warranted.

Trial registration

ChiCTR-OPC-14005698, retrospectively registered on 20 Dec. 2014.

     

The postcranial anatomy of Suminia getmanovi (Synapsida: Anomodontia), the earliest known arboreal tetrapod

The basal anomodont Suminia getmanovi Ivakhnenko, 1994 from the late Palaeozoic of Russia is highly specialized in its masticatory apparatus, and has been suggested to represent the earliest arboreal tetrapod in the fossil record. Its postcranial anatomy is described in detail for the first time, revealing a large number of autapomorphies for this small herbivore. These include a reduced number of presacral and therein dorsal vertebrae, an elongate neck, a long and possibly prehensile tail, a procoracoid with a notch at its ventromedial margin rather than a foramen, an iliac blade with a robust ridge at its anteromedial edge, a pubis with a puboischiadic fenestra and separate pubic foramen, and elongate limbs. Additional autapomorphic characters are displayed in the autopodium, which comprises about 40% of the entire limb length. These features include an enlarged, phalangiform distal carpal 1 and tarsal 1, a short and robust first metacarpal, a crescent‐shaped distal tarsal 4, and elongate penultimate phalangeal elements. The phylogenetic relationships of basal anomodonts are revisited using an expanded data set, with the addition of key taxa and several postcranial characters. Unlike dicynodonts, Suminia retained the plesiomorphic phalangeal formula for amniotes of 2‐3‐4‐5‐3 (manus) and 2‐3‐4‐5‐4 (pes). This pattern is achieved by the retention of disc‐like phalangeal elements between the proximal and penultimate phalanges in digits III, IV (manus and pes), and V (pes only). In light of the new material, Suminia can be recognized as the most complete basal anomodont, offering new insights into the early evolution of the group. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 162 , 661–698.