The label free picomolar detection of insulin in blood serum

Insulin, a polypeptide hormone secreted by pancreatic cells, is a key regulator in glucose homeostasis. Its deficiency leads to insulin-dependent (type I) diabetes whereas resistance to insulin is common in type II diabetes, obesity and a range of endocrine disorders. Its determination is of considerable value, particularly in the clinical diagnosis of diabetes mellitus and the doping control of athletes. It has, additionally, been noted as a potential breast cancer marker (serum insulin levels being found to be raised in comparison to control patients). Electrochemical assays are potentially very cheap, highly sensitive, and very readily transposed to a point of care. Though there exist numerous examples of label free impedimetric or capacitative assaying of biomolecules, these are rarely demonstrated to be effective in complex biological mixtures or to be applicable to low molecular weight targets (since they operate through the interfacial displacement of water/ions and/or the steric blocking of a redox probe). We report herein an ultrasensitive electrochemical and label-free biosensor for insulin in blood serum with a clinically relevant linear range and detection limit of 1.2pM. The transducing surfaces, based on readily prepared, antibody modified, polyethylene glycol monolayer modified polycrystalline gold surfaces, respond in a highly specific and re-useable manner to the target in up to 50% blood serum.     

Porcine skeletal muscle differentially expressed gene ATP5B: molecular characterization, expression patterns, and association analysis with meat quality traits

The 2-DE/MS-based proteomics approach was used to investigate the differences of porcine skeletal muscle, and ATP5B was identified as one differential expression protein. In the present study, ATP5B gene was further cloned by RT-PCR, the sequence was analyzed using the bioinformatics method, and the mRNA expression was detected by qRT-PCR. Sequence analysis showed that the porcine ATP5B gene contains an ORF encoding 528-amino-acid residues with 49 and 166 nucleotides in the 5′ and 3′ UTRs, respectively. The mRNA of ATP5B was widely expressed in all 14 tissues tested, but especially highly expressed in parorchis and fat. The expression pattern of ATP5B was similar in Large White and Meishan breeds, showing that the expression was upregulated by 3 days after birth and downregulated during postnatal development of skeletal muscle. Comparing the two breeds, the mRNA abundance of ATP5B in Large White was more highly expressed than in Meishan at all developmental stages (P < 0.05). Moreover, a synonymous mutation, G75A in exon 8, was identified and association analysis with the traits of meat quality showed that it was significantly associated with the RLF, FMP, IFR, IMF, and IMW (P < 0.05). These results suggested that ATP5B probably plays a key role in porcine skeletal muscle development and may provide further insight into the molecular mechanisms responsible for breed-specific differences in meat quality.     

Morphology of three new marine Frontonia species (Ciliophora; Peniculida) with note on the phylogeny of this genus

Members of the ciliate genus Frontonia are common colonizers of periphytic communities in aquatic biotopes. Recent studies indicate that their species diversity is higher than previously supposed. In this study the morphology and infraciliature of three new species, Frontonia sinica spec. nov., F. pusilla spec. nov., and F. elegans spec. nov., isolated from coastal waters of China, were investigated using live observation and silver impregnation methods. Frontonia sinica differs from its congeners by the following combination of characters: ellipsoidal body, about 116 somatic and five or six vestibular kineties, peniculi 1 and 2 four-rowed, peniculus 3 two-rowed, and a single contractile vacuole. Frontonia pusilla has about 72 somatic kineties, four-rowed peniculi 1 and 2, a two-rowed peniculus 3, and two contractile vacuoles. Frontonia elegans has 73 somatic kineties, four-rowed peniculi 1 and 2, a three-rowed peniculus 3, and two contractile vacuoles. In the present work, six new small-subunit rRNA gene sequences of six Frontonia species are used to construct the phylogenetic trees. Our phylogenetic analysis supports that the genus Frontonia may be paraphyletic. Meanwhile, no pattern of correlation could be found between the structures of peniculi and the phylogenetic relationships of Frontonia species in the present study.     

Two dimensional electrophoresis of the exo-proteome produced from community acquired methicillin resistant Staphylococcus aureus belonging to clonal complex 80

Two-dimensional electrophoresis (2DE) combined with mass spectrometry was used to characterize the exo-proteome secreted by two strains (ER13 and ER21) representing community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) belonging to clonal complex 80 (CC80). Common spots were detected between the 2 gels using the Progenesis SameSpots software. Two hundred and fifty-one and 312 spots from the exo-proteome of ER13 and ER21 were resolved, respectively. 2DE overlap comparison showed that 59 spots were shared. LC–MS/MS analysis identified 57 proteins from these spots comprising about 21% extracellular, 48% cytoplasmic, 2% cytoplasmic membrane, 2% cell wall, and 26% with unknown localization. The identified proteins were classified with respect to their Gene Ontology (GO) annotation as ～24% virulence determinants and toxins, ～17% involved in carbohydrate metabolism, ～14% involved in environmental stress, and ～12% associated with cell division. The identification of the enterotoxin B from the exo-products of both strains used in our study, as belonging to CC80 was interesting.     

Genetic analysis of Agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motile‐to‐sessile switch

Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a u nip olar p olysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c‐di‐GMP) lead to surface‐contact‐independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP‐negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c‐di‐GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive c‐di‐GMP phosphodiesterase also elevate UPP production and attachment, consistent with c‐di‐GMP activation of surface‐dependent adhesin deployment.     

Chiral discrimination and interaction mechanism between enantiomers and serum albumins

The chiral discrimination studies of biological system are theoretically and practically significant for the development of chiral drugs and life science. Our work has embarked upon the interaction between serum albumin (SA) (including human SA and bovine SA), R,S‐1‐(4‐methoxyphenyl)ethylamine, and R,S‐1‐(3‐methoxyphenyl)ethylamine. The formation of intermediate transition state, binding sites, and chiral discrimination ability can be investigated by ultraviolet‐visible spectra and fluorescence spectra. Moreover, both the changes of hydrophobic microenvironment and energy transfer can be detected by synchronous fluorescence spectra and fluorescence lifetime. Copyright © 2013 John Wiley & Sons, Ltd.     

Biomass production, relative competitive ability and water use efficiency of two dominant species in semiarid Loess Plateau under different water supply and fertilization treatments

Water stress and nutrient deficiency are considered to be the main environmental factors limiting plant growth and species interaction in semiarid regions. However, less is known about the interactive effects of soil water, nitrogen and phosphorus on native species growth and relative competitive ability. A replacement series design method was used with 12 mixed plants of Bothriochloa ischaemum and Lespedeza davurica grown in a pot experiment under three water regimes and four fertility treatments. Intercropping systems were assessed on the basis of indices such as biomass production and allocation, relative competitive ability, aggressivity, relative yield total and water use efficiency (WUE). Water stress decreased significantly the total biomass production for each species, either in monoculture or in mixtures. N, P, or NP application can significantly improve biomass production of the two species in their mixtures. There was no obvious change trend in root/shoot ratio of B. ischaemum or L. davurica in different mixture proportions. Relative yield total (RYT) values ranged from 0.98 to 1.39. Aggressivity values of B. ischaemum to L. davurica were positive in all water regimes and fertilizations, implying that B. ischaemum was the dominant species. Relative competition intensity values of B. ischaemum (i.e., RCI_B) were less than zero, while greater than zero for L. davurica (i.e., RCI_L), indicating that the effects of intraspecific competition with L. davurica were stronger for B. ischaemum, and the opposite for L. davurica. WUE increased gradually as the proportion of B. ischaemum increased in mixtures, and a 10:2 B. ischaemum:L. davurica mixture proportion had significantly higher WUE. Results suggest that it is advantageous to grow the two species together to maximize biomass production and the recommended mixture ratio was 10:2 of B. ischaemum to L. davurica because it gave higher RYT and significantly higher WUE under conditions of water deficit.     

Response of Pyropia haitanensis to agaro-oligosaccharides evidenced mainly by the activation of the eicosanoid pathway

The marine red alga Pyropia haitanensis (Protoflorideophyceae, Bangiaceae) has a nonvascular and multicellular structure and emerged earlier in evolution than other cultivatable red algae. It has been reported that lipid mediators from both the eicosanoid and octadecanoid pathways are involved in the innate immunity of other marine algae. But the defense strategies of P. haitanensis are not clearly understood. Here, we investigated the lipid defense of P. haitanensis elicited by agaro-oligosaccharides. The results indicate that the resistance of P. haitanensis was elicited and hydrogen peroxide was released by agaro-oligosaccharides. In P. haitanensis, C20 fatty acids are the essential fatty acids. Phospholipase A₂ was activated, and the free fatty acids decreased 3 h after treatment with agaro-oligosaccharides. Gas chromatography–mass spectrometry analyses revealed that the contents of volatile organic compounds increased after treatment for 3 h, which indicated that these free fatty acids were metabolized to volatile organic compounds. In conclusion, the lipid metabolic defense pathway of P. haitanensis was mainly via the C20 metabolism pathway. The C20 fatty acid was rapidly metabolized to volatile organic compounds, but not oxidized to oxylipins in response to agaro-oligosaccharides.