Beta-ketoacyl-acyl carrier protein synthase IV: a key enzyme for regulation of medium-chain fatty acid synthesis in Cuphea lanceolata seeds

With the aim of elucidating the mechanisms involved in the biosynthesis of medium-chain fatty acids in Cuphea lanceolata Ait., a crop accumulating up to 90% decanoic acid in seed triacylglycerols, cDNA clones of a beta-ketoacyl-acyl carrier protein (ACP) synthase IV (clKAS IV, EC 2.3.1.41) were isolated from C. lanceolata seed embryos. The amino acid sequence deduced from clKAS IV cDNA showed 80% identity to other plant KAS II-type enzymes, 55% identity towards plant KAS I and over 90% towards other Cuphea KAS IV-type sequences. Recombinant clKAS IV was functionally overexpressed in Escherichia coli, and substrate specificity of purified enzyme showed strong preference for elongation of short-chain and medium-chain acyl-ACPs (C4- to C10-ACP) with nearly equal activity. Further elongation steps were catalysed with distinctly less activity. Moreover, short- and medium-chain acyl-ACPs exerted a chain-length-specific and concentration-dependent substrate inhibition of clKAS IV. Based on these findings a regulatory mechanism for medium-chain fatty acid synthesis in C. lanceolata is presented.     

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.     

A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography

Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000.     

High-performance liquid chromatographic total particles quantification of retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein

A novel and rapid method for the total particles quantification of murine leukemia virus derived retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein was developed using high performance liquid chromatography. Virus particles were detected by absorbance at 260 nm and quantified using a calibration curve generated from highly purified and concentrated viral stock characterized by negative stain electron microscopy. The method requires Benzonase digestion and concentration of the supernatant prior to analysis. The virus eluted in 12.55 min at a flow rate of 1 mL/min in 20 mM Tris-Cl, pH 7.4 + 1.1 M NaCl. The limits of detection and quantification of this assay were 4.71 x 10(8) and 1.57 x 10(9) viral particles/mL, respectively. Linearity was between 3.0 x 10(9) and 1.0 x 10(11) viral particles/mL with a correlation coefficient of 0.9923 and a slope of 6 x 10(-6). The assay precision was <5% and <10% for intra- and inter-day analysis, respectively. This assay was used for the total particles quantification of a 7-day, large-scale perfusion culture production of a retroviral vector grown in 293 cells expressing the beta-galactosidase gene.     

Micro-quantitation of lipids in serum-free cell culture media: a critical aspect is the minimization of interference from medium components and chemical reagents

Lipids (fatty acids) at a concentration range of 10-100 microg/L are essential components included in most serum-free cell culture medium formulations. A gas chromatography/mass spectrometry (GC/MS) method for the micro-quantitation of lipids, determined as fatty acid methyl esters (FAMEs), in complex serum-free cell culture media was developed. The interference of derivatizing reagents, extraction solvents and medium additives in the micro-quantitation of lipids was also examined. The results show that the concentration of fatty acids such as palmitic and stearic acids detected in derivatizing reagents or extraction solvents was in the range of 10-230 microg/L. Tween-80, a surfactant and medium additive, produced nearly 20 FAMEs alone when methylated using a derivatizing agent. Moreover, the surfactant Pluronic F-68, a medium additive, interfered in the FAME recovery. Procedures, which include use of low volumetric ratio of reagent to medium and precipitation of the above surfactants, were developed to minimize background FAMEs to levels which do not significantly affect the quantitation of medium lipids and to diminish the interference caused by Pluronic F-68. Fatty acid concentrations in several complex serum-free culture media were quantitated by this method and were very close to the values indicated in their formulations.     

Upward Altitudinal Shifts in Habitat Suitability of Mountain Vipers since the Last Glacial Maximum

We determined the effects of past and future climate changes on the distribution of the Montivipera raddei species complex (MRC) that contains rare and endangered viper species limited to Iran, Turkey and Armenia. We also investigated the current distribution of MRC to locate unidentified isolated populations as well as to evaluate the effectiveness of the current network of protected areas for their conservation. Present distribution of MRC was modeled based on ecological variables and model performance was evaluated by field visits. Some individuals at the newly identified populations showed uncommon morphological characteristics. The distribution map of MRC derived through modeling was then compared with the distribution of protected areas in the region. We estimated the effectiveness of the current protected area network to be 10%, which would be sufficient for conserving this group of species, provided adequate management policies and practices are employed. We further modeled the distribution of MRC in the past (21,000 years ago) and under two scenarios in the future (to 2070). These models indicated that climatic changes probably have been responsible for an upward shift in suitable habitats of MRC since the Last Glacial Maximum, leading to isolation of allopatric populations. Distribution will probably become much more restricted in the future as a result of the current rate of global warming. We conclude that climate change most likely played a major role in determining the distribution pattern of MRC, restricting allopatric populations to mountaintops due to habitat alterations. This long-term isolation has facilitated unique local adaptations among MRC populations, which requires further investigation. The suitable habitat patches identified through modeling constitute optimized solutions for inclusion in the network of protected areas in the region.     

Association of laryngeal and nasopharyngeal tuberculosis: a case report

Introduction

To the best of our knowledge, the association of nasopharyngeal and laryngeal tuberculosis has never been described before in the literature. We report here a first observation.

Case presentation

We report the case of a 38-year-old Arab man who presented with an isolated hoarseness. Radiological and endoscopic examinations showed a thickening of the left lateral wall of his nasopharynx and the left vocal cord. Pathology revealed the diagnosis of tuberculosis of both localizations. He received a 6-month antituberculous chemotherapy with a satisfying uneventful evolution.

Conclusions

Tuberculosis should be considered in the differential diagnosis of soft tissue masses of the head and neck, particularly when the imaging findings and clinical presentation are atypical. The diagnosis of tuberculosis is mainly based on histopathological and/or bacteriological examination.     

An integrative approach to identify hexaploid wheat miRNAome associated with development and tolerance to abiotic stress

Background

Wheat is a major staple crop with broad adaptability to a wide range of environmental conditions. This adaptability involves several stress and developmentally responsive genes, in which microRNAs (miRNAs) have emerged as important regulatory factors. However, the currently used approaches to identify miRNAs in this polyploid complex system focus on conserved and highly expressed miRNAs avoiding regularly those that are often lineage-specific, condition-specific, or appeared recently in evolution. In addition, many environmental and biological factors affecting miRNA expression were not yet considered, resulting still in an incomplete repertoire of wheat miRNAs.

Results

We developed a conservation-independent technique based on an integrative approach that combines machine learning, bioinformatic tools, biological insights of known miRNA expression profiles and universal criteria of plant miRNAs to identify miRNAs with more confidence. The developed pipeline can potentially identify novel wheat miRNAs that share features common to several species or that are species specific or clade specific. It allowed the discovery of 199 miRNA candidates associated with different abiotic stresses and development stages. We also highlight from the raw data 267 miRNAs conserved with 43 miRBase families. The predicted miRNAs are highly associated with abiotic stress responses, tolerance and development. GO enrichment analysis showed that they may play biological and physiological roles associated with cold, salt and aluminum (Al) through auxin signaling pathways, regulation of gene expression, ubiquitination, transport, carbohydrates, gibberellins, lipid, glutathione and secondary metabolism, photosynthesis, as well as floral transition and flowering.

Conclusion

This approach provides a broad repertoire of hexaploid wheat miRNAs associated with abiotic stress responses, tolerance and development. These valuable resources of expressed wheat miRNAs will help in elucidating the regulatory mechanisms involved in freezing and Al responses and tolerance mechanisms as well as for development and flowering. In the long term, it may help in breeding stress tolerant plants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1490-8) contains supplementary material, which is available to authorized users.     

Study of Thermal Decomposition of Li1‐x(Ni1/3Mn1/3Co1/3)0.9O2 Using In‐Situ High‐Energy X‐Ray Diffraction

Safety has been a major technological concern hindering the deployment of lithium‐ion batteries for automobile applications. We investigated the decomposition mechanism of delithiated cathode materials at thermal abuse conditions using Li_1.1[Ni_1/3Mn_1/3Co_1/3]_0.9O₂ as a model cathode material. An in‐situ high‐energy X‐ray diffraction technique was established as an alternative to conventional thermal analysis techniques like differential scanning calorimetry and accelerating rate calorimetry. The X‐ray diffraction data revealed that the thermal decomposition pathway of delithiated Li_1‐x[Ni_1/3Mn_1/3Co_1/3]_0.9O₂ strongly depended on the exposed chemical environment, like solvents and lithium salts. A phase transformation of dry delithiated Li_1‐x[Ni_1/3Mn_1/3Co_1/3]_0.9O₂ was observed at about 278 °C, and its onset temperature was reduced to about 197°C with the presence of the electrolyte. It is suggested that the reduction in thermal stability is possibly related to proton intercalation into the delithiated material.