Detection and structural characterization of insulin prefibrilar oligomers using surface enhanced Raman spectroscopy

In vitro fibril formation typically exhibits a lag phase followed by a rapid elongation phase. Soluble prefibrilar oligomers form as multiple assembly states occur during the lag phase and, after forming a nucleus, rapidly propagate into amyloid aggregates and fibrils. The structure and morphology of amyloid fibrils have been extensively characterized over the last decades, while little is known about the structural organization of the prefibrilar oligomers or their multiple assembly states. The main difficulty in structural characterization of prefibrilar aggregates is their low concentration (pmolar) and their continual reactive conversion. Herein we overcome these difficulties by utilizing Surface‐Enhanced Raman Spectroscopy (SERS) with a model amyloid peptide, insulin. SERS is a powerful analytic tool that is able to provide detection of small molecules down to a single‐molecule level. Using SERS we found that during the 3 lag phase before the onset of insulin fibril formation, the amount of insulin oligomers increased more than twice after the first hour of incubation under fibrillation conditions (pH 1.6, 65°C) and then slowly decreased with time. The latter finding is kinetically linked to the conversion of the prefibrilar oligomers into fibril species. This study provides valuable new information about the time‐dependent structural organization of insulin oligomers and demonstrates the power and potential of SERS for detection and structural characterization of biological specimens present at low concentrations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:488–495, 2014     

Role of vascular endothelial growth factor (VEGF) and VEGF-R genotyping in guiding the metastatic process in pT4a resected gastric cancer patients

In radically resected gastric cancer the possibility to predict the site of relapse could be clinically relevant for the selection of post-surgical management. We previously showed that specific tumour integrins genotypes are independently associated with either peritoneal or hematogenous metastases (ITGA and ITGV). Recently VEGF and VEGF-R polymorphisms have been demonstrated to potentially affect tumour angiogenesis and the metastatic process in gastric cancer. We then investigated the role of VEGFs and VEGF-R genotyping in determining either peritoneal carcinosis or hematogenous metastases in radically resected gastric cancer patients. Tumour genotyping for integrins (ITGA and ITGV) was also performed according to our previous findings. Genotyping for VEGF-A, VEGF-C, VEGFR-1,2,3 and ITGA and ITGV was carried out on pT4a radically resected gastric tumours recurring with either peritoneal-only carcinosis or hematogenous metastases. 101 patients fulfilled the inclusion criteria: 57 with peritoneal carcinomatosis only and 44 with hematogenous spread only. At multivariate analysis, intestinal histology and the AC genotype of rs699947 (VEGFA) showed to independently correlate with hematogenous metastases (p = 0.0008 and 0.008 respectively), whereas diffuse histology and the AA genotype of rs2269772 (ITGA) independently correlated with peritoneal-only diffusion (p = <0.0001 and 0.03 respectively). Our results seem to indicate that combining information from genotyping of rs699947 (VEGFA, AC), rs2269772 (ITGA, AA) and tumour histology could allow clinicians to individuate gastric cancer at high risk for recurrence either with peritoneal or hematogenous metastases. The selection tool deriving from this analysis may allow an optimal use of the available treatment strategies in these patients.     

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors.     

Agropastoral activities increase fluctuating asymmetry in tadpoles of two neotropical anuran species

Agriculture and pasture activities are the main drivers for habitat reduction, directly affecting amphibian assemblages. In the Cerrado, the progress of agricultural and pasture areas negatively affects aquatic environments and their organisms, once the suppression of marginal vegetation reduces the natural protection against allochtone stressors. One possible way to measure the stress level is the Fluctuating Asymmetry (FA), which is calculated based on the deviations in the development of bilateral morphological traits of the organism. Herein, we evaluated whether environments with a higher degree of agropastoral influence and reduced marginal vegetation can increase FA levels in tadpoles of two common and widely distributed anuran species in the Cerrado (Physalaemus cuvieri and Scinax fuscomarginatus). We sampled and classified water bodies according to the percentage of agropastoral land use and marginal vegetation, and measured four morphological traits of tadpoles to evaluate the degree of FA. We found that in environments with intensive agropastoral land use, tadpoles of P. cuvieri and S. fuscomarginatus had higher FA in nostril‐snout distance (NSD). In environments with reduced marginal vegetation, tadpoles of S. fuscomarginatus had higher FA in eye width (EW), but no effect was detected for tadpoles of P. cuvieri. These morphological traits (i.e. nostrils and eyes) are associated to individual fitness of tadpoles. Thus, these developmental deviations can affect species fitness and population homeostasis over time, contributing to generate stochastic population dynamics and increasing the vulnerability of native species to local extinctions. The use of FA as a tool to measure environmental impact on species with potential to be used as bioindicators can contribute to generate and test hypothesis when integrated with long‐term studies.     

Cytogenetic studies in six species of Scinax (Anura,Hylidae) clade Scinax ruber from northern and northeastern Brazil

Scinax species are still underrepresented in cytogenetic studies, mainly with respect to populations from northeastern and northern Brazil. In this study, we provide new chromosomal information on Scinax boesemani, S. camposseabrai, S. garbei, S. pachycrus, S. trilineatus and S. x-signatus, all belonging to clade S. ruber. They were collected at two locations in the Caatinga biome (northeastern Brazil) and at one in the Amazon (northern Brazil) biomes. Chromosomes were analyzed by conventional staining, C-banding, Ag-NOR staining, and fluorochrome staining. All species shared a modal diploid value of 2n = 24 and fundamental arm number (FN) of 48. Moreover, both chromosomal size and morphology were similar to other species in this Scinaxclade. C-banding revealed centromeric heterochromatin in all species, along with terminal species-specific C-bands in some species. Active nucleolar organizer regions (Ag-NORs) were identified at 11q in most species, except for S. boesemani and S. garbei (Ag-NORs at interstitial region of 8q). Differing from most anurans, GC-rich regions were not restricted to NORs, but also coincident with some centromeric and terminal C-bands. These data contribute to the cytotaxonomy of Scinax by providing chromosomal markers and demonstrating the occurrence of microstructural rearrangements and inversions on chromosomal evolution of Scinax.     

Major diffusion leaks of clamp‐on leaf cuvettes still unaccounted: how erroneous are the estimates of Farquhar et al. model parameters?

Estimates of leaf gas-exchange characteristics using standard clamp-on leaf chambers are prone to errors because of diffusion leaks. While some consideration has been given to CO(2) diffusion leaks, potential water vapour diffusion leaks through chamber gaskets have been neglected. We estimated diffusion leaks of two clamp-on Li-Cor LI-6400 (Li-Cor, Inc., Lincoln, NE, USA) leaf chambers with polymer foam gaskets and enclosing either 2 or 6 cm(2) leaf area, and conducted a sensitivity analysis of the diffusion leak effects on Farquhar et al. photosynthesis model parameters - the maximum carboxylase activity of ribulose 1 x 5-bisphosphate carboxylase/oxygenase (Rubisco) (V(cmax)), capacity for photosynthetic electron transport (J(max)) and non-photorespiratory respiration rate in light (R(d)). In addition, net assimilation rate (A(n)) versus intercellular CO(2) (C(i)) responses were measured in leaves of Mediterranean evergreen species Quercus ilex L. enclosing the whole leaf chamber in a polyvinyl fluoride bag flushed with the exhaust air of leaf chamber, thereby effectively reducing the CO(2) and water vapour gradients between ambient air and leaf chamber. For the empty chambers, average diffusion leak for CO(2), K(CO2), (molar flow rate corresponding to unit CO(2) mole fraction difference) was ca. 0.40 micromol s(-1). K(CO2) increased ca. 50% if a dead leaf was clamped between the leaf chamber. Average diffusion leak for H(2)O was ca. 5- to 10-fold larger than the diffusion leak for CO(2). Sensitivity analyses demonstrated that the consequence of a CO(2) diffusion leak was apparent enhancement of A(n) at high CO(2) mole fraction and reduction at lower CO(2) mole fraction, and overall compression of C(i) range. As the result of these modifications, Farquhar et al. model parameters were overestimated. The degree of overestimation increased in the order of V(cmax) < J(max) < R(d), and was larger for smaller chambers and for leaves with lower photosynthetic capacity, leading to overestimation of all three parameters by 70-290% for 2 cm(2), and by 10-60% for 6 cm(2) chamber. Significant diffusion corrections (5-36%) were even required for leaves with high photosynthetic capacity measured in largest chamber. Water vapour diffusion leaks further enhanced the overestimation of model parameters. For small chambers and low photosynthetic capacities, apparent C(i) was simulated to decrease with increasing A(n) because of simultaneous CO(2) and H(2)O diffusion leaks. Measurements in low photosynthetic capacity Quercus ilex leaves enclosed in 2 cm(2) leaf chamber exhibited negative apparent C(i) values at highest A(n). For the same leaves measured with the entire leaf chamber enclosed in the polyvinyl fluoride bag, C(i) and A(n) increased monotonically. While the measurements without the bag could be corrected for diffusion leaks, the required correction in A(n) and transpiration rates was 100-500%, and there was large uncertainty in Farquhar et al. model parameters derived from 'corrected'A(n)/C(i) response curves because of uncertainties in true diffusion leaks. These data demonstrate that both CO(2) and water vapour diffusion leaks need consideration in measurements with clamp-on leaf cuvettes. As plants in natural environments are often characterized by low photosynthetic capacities, cuvette designs need to be improved for reliable measurements in such species.     

Detection and reduction of microaggregates in insulin preparations

Insulin is an important biotherapeutic protein, and it is also a model protein used to study amyloid diseases, such as Alzheimer's and Parkinson's. The preparation of the protein can lead to small amounts of aggregate in the solution, which in turn may lead to irreproducible in vitro results. Using several pre‐treatment methods, we have determined that pH cycling and diafiltration of the insulin removes microaggregates that may be present in the solution. These microaggregates were not detectable with traditional biochemical methods, but using small‐angle neutron scattering, we were able to show that pH cycling reduces the radius of gyration of the insulin. Diafiltration removes the aggregates by size and pH cycling dissolves the aggregates by adjusting the pH through the pI of the protein. Pre‐treating the insulin with either pH cycling or diafiltration allowed reproducible kinetics of fibrillation for the insulin protein. Microaggregates are a common problem in protein production, formulation, and preparation; here we show that they are the main cause for inconsistent behavior and how pH cycling and diafiltration can mitigate this problem. Biotechnol. Bioeng. 2011; 108:237–241. © 2010 Wiley Periodicals, Inc.