Evaluation of a novel, integrated approach using functionalized magnetic beads, bench-top MALDI-TOF-MS with prestructured sample supports, and pattern recognition software for profiling potential biomarkers in human plasma.

The advantage of using proteins and peptides as biomarkers is that they can be found readily in blood, urine, and other biological fluids. Such sample types are easily obtained and represent a potentially rich palette of biologically informative molecules. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) represents a key tool for rapidly interrogating such sample types. The goal of clinical proteomics is to harness the power of this tool for identifying novel, condition-specific protein fingerprints that may, in turn, lead to the elucidation and use of diseasespecific biomarkers that may be used to diagnose disease as well as to evaluate disease severity, disease progression, and intervention efficacy. Here we have evaluated a simple, affordable bench-top MALDI-TOF mass spectrometer to generate protein profiles from human plasma samples of asthma patients and healthy individuals. We achieve this profiling by using C8-functionalized magnetic beads that enrich a specific subset of plasma proteins based on their absorption by this resin. This step is followed by elution, transfer onto a prestructured sample support (AnchorChip technology), and analysis in a bench-top MALDI-TOF mass spectrometer (OmniFLEX) with AutoXecute acquisition control which enables automated operation with reproducible results. Resulting spectra are compiled and analyzed through the pattern recognition component of ClinProTools software. This approach in combination with ClinProTools software permits the investigator to rapidly scan for potential biomarker peptides/proteins in human plasma. The reproducibility of plasma profiles within and between days has been evaluated. The results show that the novel and facile approach with manual magnetic-bead sample preparation and a low-cost bench-top MALDI-TOF mass spectrometer is suitable for preliminary biomarker discovery studies.     

Characterization of N-glycans from mouse brain neural cell adhesion molecule.

The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.     

Suppression of GLUT1; A new strategy to prevent diabetic complications

High blood glucose results in high glucose levels in retina, because GLUT1, the sole glucose transporter between blood and retina, transports more glucose when blood glucose is high. This is the ultimate cause of diabetic retinopathy. Knockdown of GLUT1 by intraocular injections of a pool of siRNAs directed against SLC2A1 mRNA which codes for GLUT1 significantly reduced mean retinal glucose levels in diabetic mice. Systemic treatment of diabetic mice with forskolin or genistein, which bind GLUT1 and inhibit glucose transport, significantly reduced retinal glucose to the same levels seen in non‐diabetics. 1,9‐Dideoxyforskolin, which binds GLUT1 but does not stimulate adenylate cyclase had an equivalent effect to that of forskolin regarding lowering retinal glucose in diabetics indicating that cyclic AMP is noncontributory. GLUT1 inhibitors also reduced glucose and glycohemoglobin levels in red blood cells providing a peripheral biomarker for the effect. In contrast, brain glucose levels were not increased in diabetics and not reduced by forskolin. Treatment of diabetics with forskolin prevented early biomarkers of diabetic retinopathy, including elevation of superoxide radicals, increased expression of the chaperone protein β2 crystallin, and increased expression of vascular endothelial growth factor (VEGF). These data identify GLUT1 as a promising therapeutic target for prevention of diabetic retinopathy. J. Cell. Physiol. 228: 251–257, 2013. © 2012 Wiley Periodicals, Inc.     

Tyrosinase inhibitory effects and inhibition mechanisms of nobiletin and hesperidin from citrus peel crude extracts

The inhibitory effects of nobiletin and hesperidin from citrus peel crude extracts on tyrosinase diphenolase activity are evaluated. IC₅₀ of nobiletin and hesperidin is 1.49 mM and 16.08 mM, respectively and their inhibition mechanism is competitive type with K_i = 2.82 mM and noncompetitive with K_i = 9.16 mM, respectively. Crude extracts from citrus peel (C. unshiu Marc.) were extracted with 95% ethanol and fractionated by petroleum ether (PCPE). The ethanol phase (ECPE) was further desorbed from macroporous adsorption resin (FGRE). Their IC₅₀ values were 8.09 mg/mL, 7.53 mg/mL and 4.80 mg/mL, respectively. Their inhibition on melanogenesis in B16 mouse melanoma cells was also evaluated. FGRE showed a significant inhibition (42.5% at 31.25 μg/mL, p < 0.01) while hesperidin showed almost no inhibition. Nobiletin and PCPE give efficacious antiproliferation effects on B16 mouse melanoma cell with IC₅₀ values 88.6 μM and 62.96 μg/mL, respectively, by the MTT test. Hesperidin and other crude extracts showed very low cytotoxity to the B16 cell.     

Plastome phylogeny and lineage diversification of Salicaceae with focus on poplars and willows

Phylogenetic relationships and lineage diversification of the family Salicaceae sensu lato (s.l.) remain poorly understood. In this study, we examined phylogenetic relationships between 42 species from six genera based on the complete plastomes. Phylogenetic analyses of 77 protein coding genes of the plastomes produced good resolution of the interrelationships among most sampled species and the recovered clades. Of the sampled genera from the family, Flacourtia was identified as the most basal and the successive clades comprised both Itoa and Poliothyrsis, Idesia, two genera of the Salicaceae sensu stricto (s.s.) (Populus and Salix). Five major subclades were recovered within the Populus clade. These subclades and their interrelationships are largely inconsistent with morphological classifications and molecular phylogeny based on nuclear internal transcribed spacer sequence variations. Two major subclades were identified for the Salix clade. Molecular dating suggested that species diversification of the major subclades in the Populus and Salix clades occurred mainly within the recent Pliocene. In addition, we found that the rpl32 gene was lost and the rps7 gene evolved into a pseudogene multiple times in the sampled genera of the Salicaceae s.l. Compared with previous studies, our results provide a well‐resolved phylogeny from the perspective of the plastomes.     

Programming living sensors for environment,health and biomanufacturing

Synthetic biology offers new tools and capabilities of engineering cells with desired functions for example as new biosensing platforms leveraging engineered microbes. In the last two decades, bacterial cells have been programmed to sense and respond to various input cues for versatile purposes including environmental monitoring, disease diagnosis and adaptive biomanufacturing. Despite demonstrated proof-of-concept success in the laboratory, the real-world applications of microbial sensors have been restricted due to certain technical and societal limitations. Yet, most limitations can be addressed by new technological developments in synthetic biology such as circuit design, biocontainment and machine learning. Here, we summarize the latest advances in synthetic biology and discuss how they could accelerate the development, enhance the performance and address the present limitations of microbial sensors to facilitate their use in the field. We view that programmable living sensors are promising sensing platforms to achieve sustainable, affordable and easy-to-use on-site detection in diverse settings.     

Environmental factors and hygiene behaviors associated with facial cleanliness and trachoma in Kongwa,Tanzania

BackgroundHaving a clean face is protective against trachoma. In the past, long distances to water were associated with unclean faces and increased trachoma. Other environmental factors have not been extensively explored. We need improved clarity on the environmental factors associated with facial cleanliness and trachoma prevalence, especially when the disease burden is low.Methodology/Principle findingsA cross-sectional survey focusing on household environments was conducted in all 92 villages in Kongwa, Tanzania, in a random selection of 1798 households. Children aged 0–5 years in these households were examined for facial cleanliness. In each of the 50 randomly-selected villages, 50 children aged 1–9 years were randomly selected and examined for trachoma. In a multivariate model adjusting for child age, we found that children were more likely to have clean faces if the house had a clean yard (OR 1.62, 95% CI 1.37–1.91), an improved latrine (OR 1.11, 95% CI 1.01–1.22), and greater water storage capacity (OR 1.02, 95% CI 1.00–1.04), and if there were clothes washed and drying around the house (OR 1.30, 95% CI 1.09–1.54). However, measures of crowding, wealth, time spent on obtaining water, or the availability of piped water was not associated with clean faces. Using a cleanliness index (clean yard, improved latrine, washing clothes, ≥1 child in the household having a clean face), the community prevalence of trachoma decreased with an increase in the average value of the index (OR 2.28, 95% CI 1.17–4.80).Conclusions/SignificanceAccess to water is no longer a significant limiting factor in children’s facial cleanliness in Kongwa. Instead, water storage capacity and the way that water is utilized are more important in facial cleanliness. A household cleanliness index with a holistic measure of household environment is associated with reduced community prevalence of trachoma.