The early topography of thalamocortical projections is shifted in Ebf1 and Dlx1/2 mutant mice

The prevailing model to explain the formation of topographic projections in the nervous system stipulates that this process is governed by information located within the projecting and targeted structures. In mammals, different thalamic nuclei establish highly ordered projections with specific neocortical domains and the mechanisms controlling the initial topography of these projections remain to be characterized. To address this issue, we examined Ebf1(-/-) embryos in which a subset of thalamic axons does not reach the neocortex. We show that the projections that do form between thalamic nuclei and neocortical domains have a shifted topography, in the absence of regionalization defects in the thalamus or neocortex. This shift is first detected inside the basal ganglia, a structure on the path of thalamic axons, and which develops abnormally in Ebf1(-/-) embryos. A similar shift in the topography of thalamocortical axons inside the basal ganglia and neocortex was observed in Dlx1/2(-/-) embryos, which also have an abnormal basal ganglia development. Furthermore, Dlx1 and Dlx2 are not expressed in the dorsal thalamus or in cortical projections neurons. Thus, our study shows that: (1) different thalamic nuclei do not establish projections independently of each other; (2) a shift in thalamocortical topography can occur in the absence of major regionalization defects in the dorsal thalamus and neocortex; and (3) the basal ganglia may contain decision points for thalamic axons' pathfinding and topographic organization. These observations suggest that the topography of thalamocortical projections is not strictly determined by cues located within the neocortex and may be regulated by the relative positioning of thalamic axons inside the basal ganglia.     

Long-term operation of slurry bioreactor for decomposition of food wastes

A pilot scale slurry bioreactor was used for the treatment of food wastes. Food wastes were continuously added (750 g wet weight per day) into the reactor and successfully decomposed to inorganic carbon without intermittent removal of suspended solids. During operation for 90 days, 91% reduction of food wastes was achieved. Microorganisms actively grew during the initial 20 days of operation, and reached a stationary phase with a cell concentration of around 5 x 10(10) cells ml(-1), which indicated that food waste was utilized as a respiratory substrate during this phase. Using data for time variation of dissolved oxygen, the oxygen requirement for decomposition of food wastes was estimated to be 5.0 g O2 g(-1) dry weight of food wastes.     

Sensitive liquid chromatographic--mass spectrometric assay for norfloxacin in poultry tissue

A highly sensitive and specific method for the determination of norfloxacin in poultry tissues by LC-MS was developed and validated. An extract of the sample was separated on a C(18) reversed-phase column and analyzed by LC-MS. The mobile phase was gradiently flowed with 2% acetic acid and acetonitrile. The limit of detection and limit of quantitation were 1 and 5 ng/g, respectively. Mean recoveries from various spiked tissues were 87.2% (ranging from 82.5 to 92.7%) for norfloxacin. The method has been successfully applied to determine norfloxacin in poultry muscle.     

A direct repeat of N-type Ca2+ channel alpha1B gene functions as a negative regulatory element in HeLa cells

The expression of the N-type voltage-gated calcium channel alpha1B gene is restricted to neurons by a 5'-upstream region (-3992 to -1788) that contains negative regulatory element(s) that are active in non-neuronal cells. A 39 bp DNA element, which is repeated nine times in a head-to-tail fashion, was found within the same region. To examine whether this direct repeat (DR) may function as a negatively acting cis-regulatory element, several fusion plasmids, DR-110alpha1BLUC (1X), DR-SV40LUC (IX, 2X), in which one or two copies of the DR fragment were subcloned upstream of the homologous and heterologous promoters, were transiently transfected into HeLa and NS20Y cells. The promoter activity of DR-110alpha1BLUC (1X) decreased to approximately 17% of the 110alph(a1B)LUC construct in HeLa cells. The expression of the DR-SV40LUC (1X) and DR-SV40LUC (2X) plasmids was also reduced to 50 to 23% of the levels that were observed in the pGL2-Promoter in the same cells. However, no repression of the DR constructs was observed in NS20Y cells. An electrophoretic mobility shift assay showed that two DR-specific complexes were detected in HeLa cells, but not in NS20Y cells. In addition, Southwestern blotting revealed the presence of approximately 33 and 43 kDa proteins in HeLa cells. Overall, these results suggest that a 39 bp DNA element might act as repressor in non-neuron cells through the specific interactions of the DNA-proteins.     

Activity enhancement of Cel5Z from Pectobacterium chrysanthemi PY35 by removing C-terminal region

The phytopathogenic bacterium Pectobacteium chrysanthemi PY35 secretes Cel5Z endoglucanase belonging to the glycoside hydrolase family 5 of EC 3.2.1.4. The mutation of cel5Z::Omega gene was constructed by cloning the 2.0-kb SmaI fragment containing the streptomycin/spectinomycin-resistance gene of pHP45(Omega) into the BalI site of pPY100. The insertion of Omega fragment generated a new stop codon, removing the Ser/Thr-rich linker region and the cellulose binding domain (CBD) in the C-terminal region of cel5Z gene. By subsequent subcloning from this 4.9-kb fragment (pPY1001), a 1.0-kb (pPY1002) fragment was obtained and designated as cel5Z::Omega. The cel5Z::Omega gene had an open reading frame (ORF) of 1011 bp, encoding 336 amino acids, starting with an ATG codon and ending with a new TGA stop codon. The molecular mass of the Cel5Z::Omega protein in E. coli transformant appeared to be 32 kDa by SDS-PAGE analysis in the presence of carboxymethyl-cellulose (CMC). The Cel5Z::Omega protein hydrolyzed CMC with 1.7-fold higher activity than the intact Cel5Z cellulase.     

Functional integration of serial dilution and capillary electrophoresis on a PDMS microchip

For the quantitative analysis of an unknown sample a calibration curve should be obtained, as analytical instruments give relative, rather than absolute measurements. Therefore, researchers should make standard samples with various known concentrations, measure each standard and the unknown sample, and then determine the concentration of the unknown by comparing the measured value to those of the standards. These procedures are tedious and time-consuming. Therefore, we developed a polymer based microfluidic device from polydimethylsiloxane, which integrates serial dilution and capillary electrophoresis functions in a single device. The integrated microchip can provide a one-step analytical tool, and thus replace the complex experimental procedures. Two plastic syringes, one containing a buffer solution and the other a standard solution, were connected to two inlet holes on a microchip, and pushed by a hydrodynamic force. The standard sample is serially diluted to various concentrations through the microfluidic networks. The diluted samples are sequentially introduced through microchannels by electro-osmotic force, and their laser-induced fluorescence signals measured by capillary electrophoresis. We demonstrate the integrated microchip performance by measuring the fluorescence signals of fluorescein at various concentrations. The calibration curve obtained from the electropherograms showed the expected linearity.     

A karyometric approach to the characterization of atypical endometrial hyperplasia with and without co-occurring adenocarcinoma

OBJECTIVE: To develop a karyometric image analysis approach to distinguishing atypical endometrial hyperplasia with and without co-occurring adenocarcinoma. STUDY DESIGN: Six cases of atypical hyperplasia without and 6 cases with co-occurring adenocarcinoma, 4 cases of normal endometrium and 3 cases of adenocarcinoma were identified. From each case 100 nuclei were measured in representative diagnostic areas identified by an experienced pathologist. Discriminant analyses were performed. An unsupervised learning algorithm was applied to define and characterize different nuclear phenotypes, and those data were used to identify cases with co-occurring adenocarcinoma. RESULTS: Discriminant analysis showed that nuclei from atypical hyperplasia and atypical hyperplasia with co-occurring adenocarcinoma are statistically different. The unsupervised learning algorithm revealed differences in nuclear subpopulations that can be used to correctly identify an estimated 85% of individual cases. CONCLUSION: Nuclei from atypical hyperplasia without and with co-occurring adenocarcinoma have statistically different karyometric characteristics that may facilitate case classification.     

TIP,a T-cell factor identified using high-throughput screening increases survival in a graft-versus-host disease model

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.