Technical note: Morphometric maps of long bone shafts and dental roots for imaging topographic thickness variation

Qualitative and quantitative characterization through functional imaging of mineralized tissues is of potential value in the study of the odontoskeletal remains. This technique, widely developed in the medical field, allows the bi‐dimensional, planar representation of some local morphometric properties, i.e., topographic thickness variation, of a three‐dimensional object, such as a long bone shaft. Nonetheless, the use of morphometric maps is still limited in (paleo)anthropology, and their feasibility has not been adequately tested on fossil specimens. Using high‐resolution microtomographic images, here we apply bi‐dimensional virtual “unrolling” and synthetic thickness mapping techniques to compare cortical bone topographic variation across the shaft in a modern and a fossil human adult femur (the Magdalenian from Chancelade). We also test, for the first time, the possibility to virtually unroll and assess for dentine thickness variation in modern and fossil (the Neanderthal child from Roc de Marsal) human deciduous tooth roots. The analyses demonstrate the feasibility of using two‐dimensional morphometric maps for the synthetic functional imaging and comparative biomechanical interpretation of cortical bone thickness variation in extant and fossil specimens and show the interest of using this technique also for the subtle characterization of root architecture and dentine topography. More specifically, our preliminary results support the use of virtual cartography as a tool for assessing to what extent internal root morphology is capable of responding to loading and directional stresses and strains in a predictable way. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Pattern‐mapped multiple detection of 11 pathogenic bacteria using a 16s rDNA‐based oligonucleotide microarray

Pathogen detection is an important issue in human health due to the threats posed by severe communicable diseases. In the present study, to achieve efficient and accurate multiple detection of 11 selected pathogenic bacteria, we constructed a 16S rDNA oligonucleotide microarray containing doubly specific capture probes. Many target pathogens were specifically detected by the microarray with the aid of traditional perfect match‐based analysis using our previously proposed two‐dimensional visualization plot tool. However, some target species or subtypes were difficult to discriminate by perfect match analysis due to nonspecific binding of conserved 16S rDNA‐derived capture probes with high sequence similarity. We noticed that the patterns of specific spots for each strain were somewhat different in the two‐dimensional gradation plot. Therefore, to discriminate subtle differences between phylogenetically related pathogens, a pattern‐mapping statistical model was established using an artificial neural network algorithm trained by experimental repeats. The oligonucleotide microarray system harboring doubly specific capture probes combined with the pattern‐mapping analysis tool resulted in successful detection of all target pathogens including even subtypes of two closely related species showing strong nonspecific binding. Collectively, the results indicate that our novel combined system of a 16S rDNA‐based DNA microarray and a pattern‐mapping statistical analysis tool is a simple and effective method for detecting multiple pathogens. Biotechnol. Bioeng. 2010;106: 183–192. © 2010 Wiley Periodicals, Inc.     

Compartmental culture of embryonic stem cell-derived neurons in microfluidic devices for use in axonal biology

Axonal pathology has been clearly implicated in neurodegenerative diseases making the compartmental culture of neurons a useful research tool. Primary neurons have already been cultured in compartmental microfluidic devices but their derivation from an animal is a time-consuming and difficult work and has a limit in their sources. Embryonic stem cell (ESC)-derived neurons (ESC_Ns) overcome this limit, since ESCs can be renewed without limit and can be differentiated into ESC_Ns by robust and reproducible protocols. In this research, ESC_Ns were derived from mouse ESCs in compartmental microfluidic devices, and their axons were isolated from the somal cell bodies. Once embryoid bodies (EBs) were localized in the microfluidic culture chamber, ESC_Ns spread out from the EBs and occupied the cell culture chamber. Their axons traversed the microchannels and finally were isolated from the somata, providing an arrangement comparable to dissociated primary neurons. This ESC_N compartmental microfluidic culture system not only offers a substitute for the primary neuron counterpart system but also makes it possible to make comparisons between the two systems.     

Soluble pig lymphocyte activation gene-3 (LAG-3; CD223) inhibits human-to-pig xenogeneic mixed lymphocyte reaction

Lymphocyte activation gene-3 (LAG-3; CD223) is a transmembrane protein that is structurally similar to CD4. Since LAG-3 has a much higher binding affinity to MHC class II than that of CD4, several approaches using soluble LAG-3 were used to modulate immune responses by activation or inhibition of MHC class II expressing antigen presenting cells. In this study, we constructed soluble pig LAG-3 containing a critical binding site (D1 and D2 region) to MHC class II molecules, combined with a constant region of an immunoglobulin (Ig) heavy chain. Flow cytometry analyses indicated that soluble pig LAG-3 binds to both pig and human MHC class II molecules. Moreover, soluble pig LAG-3 can inhibit human lymphocyte proliferation in the human–pig xenogeneic mixed lymphocyte reaction in a dose-dependent manner. These results indicate that soluble pig LAG-3 may be useful for controlling the xenogeneic T cell immune responses between the human and pig.     

Metabolic engineering of <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> for malonyl-CoA biosynthesis to enhance heterologous production of polyketides

Using metabolic engineering, we developed Streptomyces venezuelae YJ028 as an efficient heterologous host to increase the malonyl-CoA pool to be directed towards enhanced production of various polyketides. To probe the applicability of newly developed hosts in the heterologous production of polyketides, we expressed type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase, in these hosts. Flaviolin production was doubled by expression of acetyl-CoA carboxylase (ACCase) and 4-fold by combined expression of ACCase, metK1-sp and afsR-sp. Thus, the newly developed Streptomyces venezuelae YJ028 hosts produce heterologous polyketides more efficiently than the parent strain.     

Antitumor activity of 3,4-dihydroquinazoline dihydrochloride in A549 xenograft nude mice

In the previous article we have reported that 3,4-dihydroquinazoline 1 is a potent and selective T-type calcium channel blocker that exhibited strong anti-cancer activity in vitro. Compound 1·2HCl was further in vivo evaluated against A549 xenograft in BALB/c nude mice, which exhibited 49% tumor-weight inhibition through intravenous administration of 2 mg/kg of body weight and was more potent than doxorubicin. Moreover, compound 1·2HCl has an oral bioavailability of 98% with LD(50) values of 693 mg/kg (p.o. route) and 40.0 mg/kg (i.v. route) of body weight. In addition, its efficient scale-up synthetic method was developed.     

Synthesis and PGE2 production inhibition of 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione derivatives

3,4-Diphenyl-substituted 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione derivatives were synthesized and evaluated for the inhibitory activities on LPS-induced PGE₂ production in RAW 264.7 macrophage cells. Both 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione rings as main scaffolds were easily obtained using one of three synthetic methods. Among the compounds investigated, 1H-3-(4-sulfamoylphenyl)-4-phenyl-pyrrole-2,5-dione (6l) showed a strong inhibitory activity (IC₅₀ = 0.61 μM) of PGE₂ production.     

Synthesis and evaluation of stilbene derivatives as a potential imaging agent of amyloid plaques

Fluorescence probes that can detect Aβ (β-amyloid peptide) plaque are important tools for diagnosis of Alzheimer’s disease (AD), and 4-N-methylamino-4′-hydroxystilbene (SB-13) is one of the promising candidate molecules. We report here the synthesis of SB-13 derivatives that consist of various electron donating/withdrawing moieties and distinct size of N-substituents. The synthesized compounds were screened for detection of Aβ40 fibrils in vitro. Four compounds exhibited more than sixfold intensity increase, and they were further analyzed for detail bindings and Aβ plaque imaging. Among these molecules, compound 42 meets two critical requirements for imaging agent; high fluorescence responsiveness and strong binding affinity. This compound showed more than 25-fold increase with the dissociation constant of 1.13 ± 0.37 μM. In AD mouse brain tissue, 42 selectively stained Aβ plaque, more specifically peripheral regions of Aβ plaque. This finding demonstrated its potential use as brain-imaging agents for AD studies.     

Mining of serum glycoproteins by an indirect approach using cell line secretome

Glycosylation is the most important and abundant post-translational modification in serum proteome. Several specific types of glycan epitopes have been shown to be associated with various types of disease. Direct analysis of serum glycoproteins is challenging due to its wide dynamic range. Alternatively, glycoproteins can be discovered in the secretome of model cell lines and then confirmed in blood. However, there has been little experi-mental evidence showing cell line secretome as a tractable target for the study of serum glycoproteins. We used a hydrazine-based glycocapture method to selectively enrich glycoproteins from the secretome of the breast cancer cell line Hs578T. A total of 132 glycoproteins were identified by nanoLC-MS/MS analysis. Among the identified proteins, we selected 13 proteins that had one or more N-glycosylation motifs in the matched peptides, which were included in the Secreted Protein Database but not yet in the Plasma Proteome Database (PPD), and whose antibodies were commercially available. Nine out of the 13 selected proteins were detected from human blood plasma by western analysis. Furthermore, eight proteins were also detected from the plasma by targeted LC-MS/MS, which had never been previously identified by data-dependent LC-MS/MS. Our results provide novel proteins that should be enrolled in PPD and suggest that analysis of cell line secretome with subfractionation is an efficient strategy for discovering disease-relevant serum proteins.     

Acute stress responsive RGS proteins in the mouse brain

Regulator of G-protein signaling (RGS) proteins play an important role in G-protein coupled receptor (GPCR) signaling and the activity of some GPCRs is modulated via RGS protein levels during stress response. The aim of this study was to investigate changes in RGS protein mRNA expressions in the mouse brain after 2h restraint stress. The mRNA level of 19 RGS proteins was analyzed using real-time PCR in six brain regions, which included the prefrontal cortex, amygdala, hippocampus, hypothalamus, striatum, and pituitary gland, from control and stressed mouse. We found that the level of mRNA of each RGS varied according to brain region and that two to eight RGS proteins exhibited changes in mRNA levels in each brain region by restraint stress. It was also revealed that RGS4 protein amount was consistent with mRNA level, indicating RGS4 protein may have regulatory roles in the acute stress response.