Prolonged treatment with 3-isobutyl-1-methylxanthine improves the efficiency of differentiating 3T3-L1 cells into adipocytes

Until now, the low efficiency of current protocols or kits for the differentiation of 3T3-L1 preadipocytes makes it difficult to continue the studies of the cellular and molecular mechanisms in adipocytes. Here we present a productive and highly efficient protocol for the differentiation of 3T3-L1 cells that uses a prolonged treatment with 3-isobutyl-1-methylxanthine (IBMX) during the differentiated process. 3T3-L1 cells of unknown passage +3 and unknown passage +7 treated with a prolonged exposure to IBMX showed significantly increased differentiation efficiency by day 15, in contrast to low levels of differentiation seen with protocols that lacked additional IBMX.     

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein.     

Bacteriophage immobilized graphene electrodes for impedimetric sensing of bacteria (Staphylococcus arlettae)

Bacteriophages are a class of viruses that specifically infect and replicate within a bacterium. They possess inherent affinity and specificity to the particular bacterial cells. This property of bacteriophages makes them an attractive biorecognition element in the field of biosensor development. In this work, we report the use of an immobilized bacteriophage for the development of a highly sensitive electrochemical sensor for Staphylococcus arlettae, bacteria from the pathogenic family of coagulase-negative staphylococci (CNS). The specific bacteriophages were covalently immobilized on the screen-printed graphene electrodes. Thus, the fabricated bacteriophage biosensor displayed quantitative response for the target bacteria (S. arlettae) for a broad detection range (2.0–2.0 × 10⁶ cfu). A fast response time (2 min), low limit of detection (2 cfu), specificity, and stability over a prolonged period (3 months) are some of the important highlights of the proposed sensor. The practical utility of the developed sensor has been demonstrated by the analysis of S. arlettae in spiked water and apple juice samples.     

Integrative insect taxonomy based on morphology,mitochondrial DNA,and hyperspectral reflectance profiling

Integrative taxonomy is considered a reliable taxonomic approach of closely related and cryptic species by integrating different sources of taxonomic data (genetic, ecological, and morphological characters). In order to infer the boundaries of seven species of the evacanthine leafhopper genus Bundera Distant, 1908 (Hemiptera: Cicadellidae), an integrated analysis based on morphology, mitochondrial DNA, and hyperspectral reflectance profiling (37 spectral bands from 411–870 nm) was conducted. Despite their morphological similarities, the genetic distances of the cytochrome c oxidase subunit I (COI) gene among the tested species are relatively large (5.8–17.3%). The species‐specific divergence of five morphologically similar species (Bundera pellucida and Bundera spp. 1–4) was revealed in mitochondrial DNA data and reflectance profiling. A key to identifying males is provided, and their morphological characters are described. Average reflectance profiles from the dorsal side of specimens were classified based on linear discriminant analysis. Cross‐validation of reflectance‐based classification revealed that the seven species could be distinguished with 91.3% classification accuracy. This study verified the feasibility of using hyperspectral imaging data in insect classification, and our work provides a good example of using integrative taxonomy in studies of closely related and cryptic species. © 2015 The Linnean Society of London     

Synthesis of pyrimidine-2,4,6-trione derivatives: Anti-oxidant,anti-cancer, α-glucosidase, β-glucuronidase inhibition and their molecular docking studies

This paper describes a facile protocol, efficient, and environmentally benign for the synthesis a series of barbiturate acid substituted at C5 position 3a–o. The desired compounds subjected in vitro for different set of bioassays including against anti-oxidant (DPPH and super oxide scavenger assays), anti-cancer, α-glucosidase and β-glucuronidase inhibitions. Compound 3m (IC₅₀ = 22.9 ± 0.5 μM) found to be potent α-glucosidase enzyme inhibitors and showed more activity than standard acarbose (IC₅₀ = 841 ± 1.73 μM). Compound 3f (IC₅₀ = 86.9 ± 4.33 μM) found to be moderate β-Glucuronidase enzyme inhibitors and showed activity comparatively less than the standard d-saccharic acid 1,4-lactone (IC₅₀ = 45.75 ± 2.16 μM). Furthermore, in sillico investigation was carried out to investigate bonding mode of barbiturate acid derivatives.     

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.     

Microbial Communities Associated with Zones of Elevated Magnetic Susceptibility in Hydrocarbon-Contaminated Sediments

Recent studies suggest that magnetic susceptibility (MS) measurements can play an important role in identifying zones where microbial-mediated iron mineral transformations are occurring. Here we investigated the microbial community variations within zones of elevated MS in a petroleum hydrocarbon-contaminated aquifer near Bemidji, Minnesota, USA. Our main objective was to 1) identify the key microbial populations that may play a role in hydrocarbon degradation, 2) analyze which microbial populations could be connected to the elevated MS and 3) explore the use of non-destructive geophysical techniques as a tool to guide microbial sampling. Clone libraries based on the 16S rRNA gene revealed the presence of iron-reducing β-Proteobacteria in the vadose zone, whereas the free petroleum phase on the water table was characterized by a methanogenic consortium, in which the syntrophic δ-proteobacterium Smithella and the hydrogenotrophic Methanoregula predominated. Nonmetric multidimensional scaling (NMDS) found a close relationship between elevated MS values and the methanogenic hydrocarbon-degrading consortium. Our results suggest that magnetic susceptibility measurements can guide microbiologists to zones of active microbial biodegradation in aged petroleum spills.     

Firmicutes is an Important Component of Microbial Communities in Water-Injected and Pristine Oil Reservoirs,Western Siberia,Russia

The dominant microbial components of fluids from wells in pristine and water-injected, high-temperature, Western Siberian oil fields, were analyzed by PCR-DGGE. Particular emphasis was placed on sulphate-reducing organisms, due to their ecological and industrial importance. Bacterial phylotypes obtained from the non-water-injected Stolbovoye oil field were more diverse than those from the Samotlor field, which is subject to secondary oil recovery by reinjection of recycled production water. The majority of phylotypes from both sites were related to Firmicutes. The low similarity to their closest relatives indicates unique bacterial communities in deep underground production waters and crude oil. Archaeal phylotypes detected only in the Samotlor samples were represented by Methanosarcinales and Methanobacteriales.