The role of Lin28b in myeloid and mast cell differentiation and mast cell malignancy.

Mast cells (MCs) are critical components of the innate immune system and important for host defense, allergy, autoimmunity, tissue regeneration and tumor progression. Dysregulated MC development leads to systemic mastocytosis (SM), a clinically variable but often devastating family of hematologic disorders. Here we report that induced expression of Lin28, a heterochronic gene and pluripotency factor implicated in driving a fetal hematopoietic program, caused MC accumulation in adult mice in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in LIN28B-expressing hematopoietic progenitors, with increased levels of LIN28B in common myeloid and basophil-MC progenitors altering gene expression patterns to favor cell fate choices that enhanced MC specification. In addition, LIN28B-induced MCs appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of MC terminal differentiation in the context of LIN28B upregulation. Finally, interrogation of human MC leukemia samples revealed upregulation of LIN28B in abnormal MCs from patients with SM. This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in MC disease.

Fabrication of visible-light-driven N-doped ordered mesoporous TiO2 photocatalysts and their photocatalytic applications.

Herein we report a facile method for the synthesis of N-doped crystalline mesoporous titanium dioxide (NMT) with ordered structure. Structural characterization and HR-TEM studies revealed that NMT exhibits pure anatase phase with highly crystalline ordered mesoporous structure in NMT. The N2 isotherms are of type IV with an H1 hysteresis loop and a pronounced capillary condensation step at high relative pressure for NMT, suggesting the presence of well-ordered mesoporous structure. The reflectance spectrum of NMT shows stronger absorption in the visible region above 400 nm, owing to the substitution of the lattice oxygen by nitrogen. XPS results proved the doping of nitrogen in to oxygen in TiO2 lattice, which confirmed by the presence of peak at 401 eV for N1s. The efficiency of photocatalyst was evaluated by the degradation of Rhodamine-B and antibacterial activity against E. coli under visible-light irradiation. N-doped mesoporous TiO2 shows superior photocatalytic and anti-bacterial activity compared to pure TiO2 under visible-light irradiation. The enhanced photocatalytic activity of NMT is attributed to synergistic effect of NMT that is N-doping and well ordered crystalline mesoporous structure with high surface area of NMT. These findings suggest that N-doped mesoporous TiO2 has potential application in many areas such as degradation of hazardous pollutants, anti-bacterial agents, fuel cells, battery electrode, sensors, opto electronic devices, photo active self-cleaning surfaces.

Doped nanocrystalline ZnO powders for non-linear resistor applications by spray pyrolysis method.

Homogeneous and doped nanocrystalline ZnO powders (30-200 nm) were synthesized by spray pyrolysis technique. The spray pyrolysed powders were calcined in the temperature range of 500-750 degrees C. Formation of insulating pyrochlore phase started from 700 degrees C during the calcination itself. The calcined powders were compacted and sintered at different temperatures ranging from 900-1200 degrees C for 0.5-4 h. The densification behavior was found to be dependent on calcination temperature of the nanopowder. The resulting discs were found to have density (5.34-5.62 g/cc) in the range of 96-99% of theoretical density. The breakdown voltage value obtained for the nanopowder based non-linear resistor is 10.3 kV/cm with low leakage current density of 0.7 microA/cm2 and coefficient of nonlinearity as high as 193. The activation energy for grain growth of the doped ZnO nanopowder powders is 449.4 +/- 15 kJ/mol.

Prognostic significance of the radiographic pattern of disease in patients with severe acute respiratory syndrome.

OBJECTIVE: This study was performed to evaluate the prognostic significance of the radiographic pattern of disease in probable cases of severe acute respiratory syndrome (SARS). MATERIALS AND METHODS: A retrospective review of 439 radiographs was performed for 51 patients with a final diagnosis of probable SARS. Forty-nine patients were followed up for a mean interval of 23 days (range, 2-63 days). RESULTS: Abnormal findings on a chest radiograph were noted at presentation in 80.4% (41/51) of patients. Four radiographic patterns were seen: normal (group 1) in 19.6% (10/51), focal opacity (group 2) in 39.2% (20/51), multifocal opacities (group 3) in 27.5% (14/51), and diffuse air-space opacification (group 4) in 13.7% (7/51). Radiographic progression of disease occurred in 38.8% (19/49) of the patients in groups 1-4. There were no deaths in groups 1 and 2. In group 3, one (7.7%) of the 13 patients died. Five (71.4%) of the seven patients in group 4 died. Overall, 12.2% (6/49) of the patients died, all of whom had diffuse air-space opacification on the last chest radiograph. In these patients, medical comorbidity was present in 66.7% (4/6), and the exposure history was known in 83.3% (5/6). Death occurred at a mean interval of 18.2 days (range, 9-36 days) from the initial exposure. CONCLUSION: Patients presenting with normal findings or focal air-space opacity on chest radiographs had a good clinical outcome. Patients with multifocal opacities that progressed to diffuse air-space opacification and patients presenting with diffuse air-space opacification had a high fatality rate, but patients in this group were also older and more likely to have comorbid conditions. Patients with SARS present with recognizable patterns of disease that have prognostic significance.

Electrochemical oxidation of underivatized-nucleic acids at highly boron-doped diamond electrodes.

Boron-doped diamond (BDD) electrodes have been examined for the electrochemical oxidation of underivatized-nucleic acids in terms of single stranded and double stranded DNA. Cyclic voltammetry and square wave voltammetry have been used to study the oxidation reactions and to detect DNA without derivatization or hydrolysis steps. At the diamond electrode, at least two well-defined voltammetric peaks were observed for both single stranded and double stranded DNA. Diamond electrode is the first material to show a well-defined voltammetric peaks for adenine group oxidation directly in the helix structure of nucleic acid due to its wide potential window. For single stranded DNA, a third peak, related to the pyrimidine group oxidation was also observed. As-deposited diamond film with predominantly hydrogen-terminated surface exhibited superior performance over oxygen-terminated diamond in terms of sensitivity. However, by optimizing the ionic strength, sensitivity of O-terminated films could be improved. Linear calibration results have shown linearity of current with concentration in the range 0.1-8 microg mL(-1) for both guanine and adenine residues at as-deposited BDD. Detection limits (S/N = 3) of 3.7 and 10 ng mL(-1) for adenine and guanine residue in single stranded DNA, respectively, and 5.2 and 10 ng mL(-1) for adenine and guanine residue in double stranded DNA, respectively, were observed. This work shows the promising use of diamond as an electrochemical detector for direct detection of nucleic acids. The results also show the possibility of using the oxidation peak current of adenine group that is more sensitive for the direct detection of nucleicacids.

Gradient liquid chromatography of leucine-enkephalin peptide and its metabolites with electrochemical detection using highly boron-doped diamond electrode.

Boron-doped diamond thin film (BDD) electrodes have been used to study the oxidation reactions and to detect leucine-enkephalinamide (LEA) and its metabolites, tyrosine (T), tyrosyl-alanine (TA), tyrosyl-alanine-glycine (TAG) and leucine-enkephalin (LE) using cyclic voltammetry (CV), flow-injection analysis (FIA), and gradient liquid chromatography (LC) with amperometric detection. At diamond electrodes, well-defined and highly reproducible cyclic voltammograms were obtained with signal-to-background (S/B) ratios 5-10 times higher than those observed for glassy carbon (GC) electrodes. The analytical peaks of LC for LEA and its metabolites were well resolved. No deactivation of BDD electrodes was found after several experiments with standard as well as plasma samples, indicating high stability of the electrode. Calibration curves were linear over a wide range from 0.06 to 30 microM with regression coefficients of 0.999 for all compounds. The limits of detection obtained based on a signal-to-noise ratio of 3:1 were 3, 2.2, 2.7, 20 and 11 nM for T, TA, TAG, LE and LEA, respectively. These values were at least one order lower than those obtained at GC electrodes, which has given limits of detection of 22.88, 20.64, 89.57, 116.04 and 75.67 for T, TA, TAG, LE and LEA, respectively. Application of this method to real samples was demonstrated and validated using rabbit serum samples. This work shows the promising use of conducting diamond as an amperometric detector in gradient LC, especially for the analysis of enkephalinamide and its metabolites.

Selective amperometric detection of dopamine using OPPy-modified diamond microsensor system.

Highly boron-doped diamond microfiber electrodes (BDDMF) were fabricated and characterized by the use of Scanning Electron Microscopy (SEM), Raman spectroscopy, and cyclic voltammetry. Amperometric detection of dopamine (DA), a neurotransmitter was achieved at pH 7.0, using BDDMF electrodes. The interferences from ascorbic acid (AA) and DOPAC were efficiently eliminated by using overoxidized polypyrrole-modified BDDMF electrodes, which also increased the sensitivity for the detection of dopamine. The limit of detection (S/N = 3) for dopamine was 0.1 nM, which is one order lower than that observed for carbon microfiber electrodes (CMFE), and the linear dynamic range was obtained from 0.5 nM to 100 microM (r2 = 0.997). The amperometric response for 0.5 nM dopamine has shown high stability with an RSD of 5.4% (n = 5). Highly reproducible results were obtained with an RSD of 6.2% for 10 measurements of 1 nM DA taken during 10 h and also remained the same, during measurements for 7 days, with no variation in efficiency for rejection of AA and DOPAC.

Inheritance of flower color in chickpea.

Flower color is a useful morphological marker in chickpea (Cicer arietinum L.). Inheritance of this trait was studied using two white-flowered chickpea genotypes, P 9623 and RS 11, and one blue-flowered genotype, T 39-1. The genetic constitutions of the white flower colors of P 9623 and RS 11 were different, for in an earlier study their F1 produced pink flowers. The two F1s of the crosses P 9623 x T 39-1 and RS 11 x T 39-1 also produced pink flowers. Each of the two F2 populations segregated in 9 pink:3 blue:4 white-flowered plants. These results can be explained by a three-gene model. These three independently segregating genes are probably the same as C, B, and P reported in the literature earlier. Allelic tests could not be undertaken, as the genetic stocks used in the earlier studies are not available. The genetic constitutions of the three parents and their F1s are proposed. These accessions should be useful for conducting allelic tests for determining flower color loci in chickpea and for comparative studies with field pea. The seeds of these genetic stocks are maintained at the Genetic Resources and Enhancement Program at ICRISAT and are available for research purposes on request.

Electrochemical oxidation of histamine and serotonin at highly boron-doped diamond electrodes.

The electrochemistry of histamine and serotonin in neutral aqueous media (pH 7.2) was investigated using polycrystalline, boron-doped diamond thin-film electrodes. Cyclic voltammetry, hydrodynamic voltammetry, and flow injection analysis (FIA) with amperometric detection were used to study the oxidation reactions. Comparison experiments were carried out using polished glassy carbon (GC) electrodes. At diamond electrodes, highly reproducible and well-defined cyclic voltammograms were obtained for histamine with a peak potential at 1.40 V vs SCE. The voltammetric signal-to-background ratios obtained at diamond were 1 order of magnitude higher than those obtained for GC electrodes at and above 100 microM analyte concentrations. A linear dynamic range of 3-4 orders of magnitude and a detection limit of 1 microM were observed in the voltammetric measurements. Well-defined sweep rate-dependent voltammograms were also obtained for 5-hydroxytryptamine (5-HT). The characteristics of the voltammogram indicated lack of adsorption of its oxidation products on the surface. No fouling or deactivation of the electrode was observed within the experimental time of several hours. A detection limit of 0.5 microM (signal-to-noise ratio 13.8) for histamine was obtained by use of the FIA technique with a diamond electrode. A remarkably low detection limit (10 nM) was obtained for 5-HT on diamond by the same method. Diamond electrodes exhibited a linear dynamic range from 10 nM to 100 microM for 5-HT determination and a range of 0.5-100 microM for histamine determination. The FIA response was very reproducible from film to film, and the response variability was below 7% at the actual detection limits.

Microbiologic spectrum and susceptibility of isolates: part I. Postoperative endophthalmitis. Endophthalmitis Research Group.

PURPOSE: To present the microbial spectrum and susceptibilities of isolates in postoperative endophthalmitis. METHOD: Isolates from 206 eyes of 206 patients who underwent vitrectomy for postoperative endophthalmitis were examined. RESULTS: One-hundred twelve (54.4%) of 206 vitreous samples were culture positive and 14 (12.5%) of 112 culture-positive cases were polymicrobial, yielding a total of 126 isolates. Isolates included 59 (46.8%) gram-positive cocci, eight (6.3%) gram-positive bacilli, 33 (26.2%) gram-negative organisms, five (4.0%) Actino-mycetes-related organisms, and 21 (16.7%) fungi. Susceptibilities to amikacin, ceftazidime, chloramphenicol, cefazolin, ciprofloxacin, gentamicin, and vancomycin are reported. CONCLUSIONS: This is the largest, single-center, prospective series on microbial susceptibilities in postoperative endophthalmitis. We report a high prevalence of gram-negative species and fungi, suggesting that empiric therapy should include coverage for gram-negative pathogens and for fungal pathogens in appropriate settings.

Microbiologic spectrum and susceptibility of isolates: part II. Posttraumatic endophthalmitis. Endophthalmitis Research Group.

PURPOSE: To present the microbial spectrum and susceptibilities of isolates in posttraumatic endophthalmitis. METHOD: Isolates from 182 eyes of 182 patients who underwent vitrectomy for posttraumatic endophthalmitis were examined. RESULTS: One hundred thirteen (62.1%) of 182 vitreous samples were culture-positive, and 23 (20.4%) of 113 culture-positive cases were polymicrobial, including three (2.7%) trimicrobial cases, yielding a total of 139 isolates. Isolates included 63 (45.3%) gram-positive cocci, 24 (17.3%) gram-positive bacilli, 25 (18.0%) gram-negative organisms, seven (5.0%) Actinomycetes-related organisms, and 20 (14.4%) fungi. Susceptibilities to amikacin, ceftazidime, chloramphenicol, cefazolin, ciprofloxacin, gentamicin, and vancomycin are reported. CONCLUSIONS: This study represents a large series on microbial spectrum and susceptibilities in posttraumatic endophthalmitis. We report a high prevalence of gram-positive bacilli species and polymicrobial infections containing gram-negative species, underscoring the importance of broad-spectrum, combination antibiotics in the empiric treatment of posttraumatic endophthalmitis.

CSF orbitorrhoea with tension pneumocephalus.

A seventy eight year old man sustained penetrating injury to right orbit about 15 years ago. Later he developed right orbital infection leading to phthisis bulbi. Two months before admission he developed CSF leak from the right orbit, tension pneumocephalous and meningitis. A rare case of CSF orbitorrhoea is reported here along with the discussion on mechanisms and management.

Electrochemical Oxidation of NADH at Highly Boron-Doped Diamond Electrodes.

Conductive boron-doped chemical vapor-deposited diamond thin films, already known to have superior properties for general electroanalysis, including low background current and a wide potential window, are here shown to have additional advantages with respect to electrochemical oxidation of nicotinamide adenine dinucleotide (NADH), including high resistance to deactivation and insensitivity to dissolved oxygen. Cyclic voltammetry, amperometry, and the rotating disk electrode technique were used to study the reaction in neutral pH solution. Highly reproducible cyclic voltammograms for NADH oxidation were obtained at as-deposited diamond electrodes. The response was stable over several months of storage in ambient air, in contrast to glassy carbon electrodes, which deactivated within 1 h. The diamond electrode exhibited very high sensitivity for NADH, with an amperometric detection limit of 10 nM (S/N = 7). The response remained stable, even in the very low concentration range, for several months. In addition, interference effects due to ascorbic acid were minimal when the concentrations of NADH and ascorbic acid were comparable. An NADH-mediated dehydrogenese-based ethanol biosensor incorporating an unmodified diamond electrode is demonstrated. The present results indicate that diamond is a useful electrode material for the analytical detection of NADH, making it attractive for use in sensors based on enzyme-catalyzed reactions involving NADH as a cofactor.

Simplified preformed chelate protein radiolabeling with technetium-99m mercaptoacetamidoadipoylglycylglycine (N3S-adipate).

A simplified kiet has been developed for 99mTc protein radiolabeling using an N3S triamide mercaptide bifunctional chelating agent and the preformed chelate approach. The process combined N3S chelating agent, gluconate intermediate transfer agent, stannous reducing agent, and gentisic acid stabilizer into a lyophilized formulation. With sulfur donor atom hemithioacetal protection of the ligand, delta-2,3,5,6-tetrafluorothiophenyl alpha-S-(1-ethoxyethyl)mercaptoacetamido-L-adipoylglycylglycine , optimum 99mTc chelation was achieved in a single step. Subsequent reaction with NR-LU-10 antibody Fab fragment followed by purification via QAE Sephadex anion exchange resin filter afforded 99mTc-N3S-NR-LU-10 Fab conjugate with retained immunoreactivity and effective tumor targeting properties.

Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells.

Thyroid peroxidase (TPO) is an essential enzyme for thyroid hormone biosynthesis and is an autoantigen against which antibodies are found in a number of autoimmune thyroid disorders. Large quantities of pure TPO are essential for understanding its structure and role in normal thyroid function and thyroid diseases. In this study, we describe the production of human TPO (hTPO) using a baculovirus expression vector in insect cells. TPO was sequentially extracted from insect cells using various buffers and the protein was purified to homogeneity on a C4 reversed-phase semipreparative column using high-performance liquid chromatography. The purified protein was identified as hTPO by enzyme-linked immunosorbent assay, Western blot, and amino acid sequence analyses. Carbohydrate analysis of the recombinant hTPO showed that the protein is glycosylated and mannose is the major oligosaccharide. We have extended the carbohydrate analysis by establishing the occurrence of N-acetyl galactosamine which suggested that the recombinant hTPO might contain O-glycosyl moieties. Purified hTPO reacted specifically with sera from patients with Hashimoto's thyroiditis. Crude as well as purified hTPO did not show any enzymatic activity when produced in Sf9 insect cells grown in serum free medium. In contrast, hTPO produced in the presence of 10% fetal bovine serum containing 1 microgram/ml of haematin was enzymatically active. However, the enzymatic activity of the recombinant hTPO was lower than that often found with hTPO purified from thyroid tissue. Availability of purified hTPO in relatively large quantities should allow further structural and immunological studies.

Development and biologic evaluation of a kit for preformed chelate technetium-99m radiolabeling of an antibody Fab fragment using a diamide dimercaptide chelating agent.

A kit has been developed for 99mTc antibody radiolabeling via defined chemistry using an N2S2 diamide dimercaptide bifunctional chelating agent and the performed chelate method. The process involved efficient transchelation of 99mTc from gluconate to 2,3,5,6-tetrafluorophenyl 4,5-bis-S-(1-ethoxyethyl) mercaptoacetamidopentanoate as an active ester ligand and subsequent conjugation to antibody lysine amine functional groups. The use of the ethoxyethyl group for sulfur protection allowed optimum yields of 99mTc N2S2 chelate formation with complete retention of the active ester. Subsequent addition of antibody Fab fragment gave 99mTc chelate conjugates indistinguishable from the stepwise in situ esterification and purification of the 99mTc N2S2 complex followed by conjugation as previously shown to give stable 99mTc antibody fragments with retained immunoreactivity and tumor-targeting properties.