Exploring the interplay of stability and function in protein evolution

A new split β‐lactamase assay promises experimental testing of the interplay of protein stability and function. Proteins are sufficiently stable to act effectively within cells. However, mutations generally destabilize structure, with effects on free energy that are comparable to the free energy of folding. Assays of protein functionality and stability in vivo enable a quick study of factors that influence these properties in response to targeted mutations. These assays can help molecular engineering but can also be used to target important questions, including why most proteins are marginally stable, how mutations alter structural makeup, and how thermodynamics, function, and environment shape molecular change. Processes of self‐organization and natural selection are determinants of stability and function. Non‐equilibrium thermodynamics provides crucial concepts, e.g., cells as emergent energy‐dissipating entities that do work and build their own parts, and a framework to study the sculpting role of evolution at different scales.     

Recovery of Sugars from Ionic Liquid Biomass Liquor by Solvent Extraction

The dissolution of biomass into ionic liquids (ILs) has been shown to be a promising alternative biomass pretreatment technology, facilitating faster breakdown of cellulose through the disruption of lignin and the decrystallization of cellulose. Both biological and chemical catalysis have been employed to enhance the conversion of IL-treated biomass polysaccharides into monomeric sugars. However, biomass-dissolving ILs, sugar monomers, and smaller carbohydrate oligomers are all soluble in water. This reduces the overall sugar content in the recovered solid biomass and complicates the recovery and recycle of the IL. Near-complete recovery of the IL and the holocellulose is essential for an IL-based pretreatment technology to be economically feasible. To address this, a solvent extraction technique, based on the chemical affinity of boronates such as phenylboronic acid and naphthalene-2-boronic acid for sugars, was applied to the extraction of glucose, xylose, and cellobiose from aqueous mixtures of 1-ethyl-3-methylimidazolium acetate. It was shown that boronate complexes could extract up to 90% of mono- and disaccharides from aqueous IL solutions, 100% IL systems, and hydrolysates of corn stover containing IL. The use of boronate complexes shows significant potential as a way to recover sugars at several stages in ionic liquid biomass pretreatment processes, delivering a concentrated solution of fermentable sugars, minimizing toxic byproducts, and facilitating ionic liquid cleanup and recycle.     

The diversity of mycorrhizal fungi in Japanese Cephalanthera species

The diversity of mycorrhizal fungi among four Japanese Cephalanthera (Orchidaceae) species was examined at a total of seven sites, based on sequence variation in nuclear ribosomal DNA. This is the first report to detect mycorrhizal fungi in C. subaphylla. Two patterns of mycorrhizal associations were confirmed from our results. C. falcata has lower fungal specificity, associating with Russulaceae, Sebacinales and Thelephoraceae. By contrast, the three Cephalanthera species C. erecta, C. subaphylla, and C. longifolia were associated mainly with Thelephoraceae fungi. There is no large difference found in mycobionts between green and albino individuals of C. falcata and no distinct preference of Japanese Cephalanthera species for a specific fungal group of Thelephoraceae.     

Occurrence and Genome Analysis of Cucurbit chlorotic yellows virus in Iran

In 2011 and 2012, several cucurbit‐growing regions of Iran were surveyed and samples with symptoms similar to those induced by Cucurbit chlorotic yellows virus (CCYV) were collected. The pathogen was transmitted to cucumber and melon under greenhouse conditions by whiteflies (Bemisia tabaci). RT‐PCR using designed CCYV‐specific primer pair (CCYV‐F/CCYV‐R) resulted in amplification of the predicted size DNA fragment (870 bp) for the coat protein (CP) gene in samples collected from Boushehr, Eyvanakay and Varamin. Nucleotide sequences of the CP of the three Iranian CCYV isolates were compared with five CCYV isolates obtained from GenBank and analysed. Phylogenetically, all CCYV isolates clustered in two groups; Group I is composed of five non‐Iranian isolates from China, Lebanon, Japan, Sudan and Taiwan, and the three Iranian isolates formed Group 2. Among Iranian isolates, the Eyvanakay isolate clustered in a distinct clade with the Boushehr and Varamin isolates. A phylogenetic tree based on amino acid identity of CP showed that CCYV was closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). This is the first report of CCYV in Iran.     

Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization

Mutations in cryopyrin and pyrin proteins are responsible for several autoinflammatory disorders in humans, suggesting that these proteins play important roles in regulating inflammation. Using a HEK293 cell-based reconstitution system that stably expresses ASC and procaspase-1 we demonstrated that neither cryopyrin nor pyrin or their corresponding disease-associated mutants could significantly activate NF-kappaB in this system. However, both cryopyrin and two disease-associated cryopyrin mutants induced ASC oligomerization and ASC-dependent caspase-1 activation, with the disease-associated mutants being more potent than the wild-type (WT) cryopyrin, because of increased self-oligomerization. Contrary to the proposed anti-inflammatory activity of WT pyrin, our results demonstrated that pyrin, like cryopyrin, can also assemble an inflammasome complex with ASC and procaspase-1 leading to ASC oligomerization, caspase-1 activation and interleukin-1beta processing. Thus, we propose that pyrin could function as a proinflammatory molecule.     

Genetic and molecular characterization of the human Osteosarcoma 3AB‐OS cancer stem cell line: A possible model for studying osteosarcoma origin and stemness

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second‐line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB‐OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB‐OS cells have hypertriploid karyotype with 71–82 chromosomes. By comparing 3AB‐OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan® Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let‐7/98 and miR‐29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets. J. Cell. Physiol. 228: 1189–1201, 2013. © 2012 Wiley Periodicals, Inc.