DNA demethylation and tri-methylation of H3K4 at the TACSTD2 promoter are complementary players for TROP2 regulation in colorectal cancer cells.

TROP2 is a powerful cancer driver in colorectal cancer cells. Divergent epigenetic regulation mechanisms for the corresponding TACSTD2 gene exist such as miRNAs or DNA methylation. However, the role of TACSTD2 promoter hypermethylation in colorectal cancer has not been investigated yet. In this study, TROP2 expression strongly correlated with promoter methylation in different colorectal tumor cell lines. Treatment with 5-Azacytidine, a DNMT1 inhibitor, led to demethylation of the TACSTD2 promoter accompanied by an increase in TROP2 protein expression. TROP2 expression correlated with promoter methylation in vivo in human colon tumor tissue, thereby verifying promoter methylation as an important factor in the regulation of TROP2 expression in colorectal cancer. When performing a ChIP-Seq analysis in HCT116 and HT29 cells, we found that TACSTD2 promoter demethylation was accompanied by tri-methylation of H3K4. In silico analysis of GSE156613 data set confirmed that a higher binding of histone mark H3K4me3 around the TACSTD2 promoter was found in TACSTD2 high expressing tumors of colon cancer patients compared to the corresponding adjacent tumor tissue. Moreover, the link between TROP2 and the H3K4me3 code was even evident in tumors showing high intratumoral heterogeneity for TROP2 staining. Our data provide novel evidence for promoter demethylation and simultaneous gains of the active histone mark H3K4me3 across CpG-rich sequences, both being complementary mechanisms in the transcriptional regulation of TACSTD2 in colon cancer. The functional consequences of TROP2 loss in colorectal cancer needs to be further investigated.

Interaction-induced partitioning and magnetization jumps in the mixed-spin oxide FeTiO3-Fe2O3.

In this study we report on jumps in the magnetic moment of the hemo-ilmenite solid solution (x)FeTiO(3)-(1-x)Fe(2)O(3) above Fe(III) percolation at low temperature (T<3 K). The first jumps appear at 2.5 K, one at each side of the magnetization loop, and their number increases with decreasing temperature and reaches 5 at T=0.5 K. The jumps occur after field reversal from a saturated state and are symmetrical in the trigger field and intensity with respect to the field axis. Moreover, an increase of the sample temperature by 2.8% at T=2.0 K indicates the energy released after the ignition of the magnetization jump, as the spin-currents generated by the event are dissipated in the lattice. The magnetization jumps are further investigated by Monte Carlo simulations, which show that these effects are a result of magnetic interaction-induced partitioning on a sublattice level.

Spectral characterization of flash and high flux x-ray radiographic sources with a magnetic Compton spectrometer.

In this work, we present a new analysis method applied to revitalize permanent magnet Compton spectrometers used to measure photon energy spectra in the MeV range. The inversion of the measured electron distribution to determine the original photon distribution is achieved via a method of consistent coupled radiation transport and magnetic field mapping of the input photon spectra to the measured electron distribution. The method of linear least squares was used to perform the unfolding of the electron distribution to the initial photon spectra, without any assumptions made regarding the electron distribution. We present an application of this method to data from a nominal 19.4 MeV flash radiographic source (the first axis of the Dual Axis Radiographic Hydro-Test Facility) capable of generating 500 R @ 1 m in ∼60 ns and a medical therapy source (a Scanditronix M22, Microtron) capable of variable energies with nominal endpoints of 6, 10, 15, and 20 MeV and an output of ∼1000-2000 R/min @ 1 m. The results provide agreement between the modeled and unfolded experimentally measured photon spectra as quantified by statistical tests, from 1.5 to 20 MeV. Experimental results are presented as well as a discussion of the novel MCNP6-based simulations and methods for reconstruction of the spectra.

Alterations in the mechanical, chemical and biocompatibility properties of low-cost polyethylene and polyester meshes after steam sterilization.

INTRODUCTION: In Africa and other Low Resource Settings (LRS), the guideline-based and thus in most cases mesh-based treatment of inguinal hernias is only feasible to a very limited extent. This has led to an increased use of low cost meshes (LCMs, mostly mosquito meshes) for patients in LRS. Most of the LCMs used are made of polyethylene or polyester, which must be sterilized before use. The aim of our investigations was to determine changes in the biocompatibility of fibroblasts as well as mechanical and chemical properties of LCMs after steam sterilization. MATERIAL AND METHODS: Two large-pored LCMs made of polyester and polyethylene in a size of 11 x 6 cm were cut and steam sterilized at 100, 121 and 134 °C. These probes and non-sterile meshes were then subjected to mechanical tensile tests in vertical and horizontal tension, chemical analyses and biocompatibility tests with human fibroblasts. All meshes were examined by stereomicroscopy, scanning electron microscopy (SEM), LDH (cytotoxicity) measurement, viability testing, pH, lactate and glycolysis determination. RESULTS: Even macroscopically, polyethylene LCMs showed massive shrinkage after steam sterilization, especially at 121 and 134 °C. While polyester meshes showed no significant changes after sterilization with regard to deformation and damage as well as tensile force and stiffness, only the unsterile polyethylene mesh and the mesh sterilized at 100 °C could be tested mechanically due to the shrinkage of the other specimen. For these meshes the tensile forces were about four times higher than for polyester LCMs. Chemical analysis showed that the typical melting point of polyester LCMs was between 254 and 269 °C. Contrary to the specifications, the polyethylene LCM did not consist of low-density polyethylene, but rather high-density polyethylene and therefore had a melting point of 137 °C, so that the marked shrinkage described above occurred. Stereomicroscopy confirmed the shrinkage of polyethylene LCMs already after sterilization at 100 °C in contrast to polyester LCMs. Surprisingly, cytotoxicity (LDH measurement) was lowest for both non-sterile LCMs, while polyethylene LCMs sterilized at 100 and 121 °C in particular showed a significant increase in cytotoxicity 48 hours after incubation with fibroblasts. Glucose metabolism showed no significant changes between sterile and non-sterile polyethylene and polyester LCMs. CONCLUSION: The process of steam sterilization significantly alters mechanical and structural properties of synthetic hernia mesh implants. Our findings do not support a use of low-cost meshes because of their unpredictable properties after steam sterilization.

An in vitro study on the biocompatibility of fibroblasts in sterile and non-sterile low-cost and commercial meshes.

INTRODUCTION: Despite several successful studies with low-cost meshes (LCM) for the treatment of inguinal hernias in India and Africa, a nationwide application has not been possible for a variety of reasons. One problem is the special preparation and sterilization of these meshes-naturally, they should comply with international standards and demands, which is often difficult to achieve in Africa. Our primary approach was to determine whether there are differences in the biocompatibility of fibroblasts between non-sterile and sterile LCMs and commercial meshes (CM). MATERIALS AND METHODS: Two polyester CMs with different pore size and a polyester LCM were examined as both sterile and non-sterile. LCM was plasma sterilized at 60 °C and steam sterilized at 134 °C. Sterile and non-sterile meshes were soaked with an antibiotic (penicillin/streptomycin) and antimycotic solution (amphotericin B). Human fibroblasts from healthy subcutaneous tissue were used. Various tests for evaluating the growth behavior and cell morphology of human fibroblasts were conducted. Semiquantitative (light microscopy) and qualitative (scanning electron microscopy) analyses were performed after 1 week and again after 12 weeks. The metabolism of fibroblasts was checked by pH measurements and glucose analyses. Biocompatibility of fibroblasts on sterile and non-sterile meshes was carried out by luminescence methods (cell viability and apoptosis) as well as calorimetric methods for proliferation determination (BrDU assay) and cytotoxicity (LDH assay). RESULTS: Light and electron microscopy revealed a moderate growth of fibroblasts on all investigated mesh types. The results of glycolysis and the pH value were within the normal range for all sterile and non-sterile meshes. In biocompatibility studies, no elevated level of apoptosis was detected. The viability measurement of mitochondrial activity of fibroblasts showed a 50% inhibition of mitochondria in all nets, with the exception of non-sterile CM, whereas mitochondrial activity was increased in the non-sterile CM. A proliferation measurement (BrdU test) revealed different growth inhibition in the sterile and non-sterile meshes. This growth inhibition was significantly stronger, particularly for non-sterile CM light meshes, than it was for the non-sterile LCM. CONCLUSION: Again, our studies show no significant differences in biocompatibility of fibroblasts between expensive and low-cost meshes. In addition, we detected fibroblast growth even in sterile meshes, independent of the mesh group. To our knowledge, the present study is the first of its kind in terms of qualitative equivalence of sterile and non-sterile in vitro mesh samples. We do not wish to create future patient studies with non-sterilized meshes saturated with antibiotics/antimycotics. However, perhaps we can prove in future studies that under semi-sterile conditions with certain LCMs, wound infection rates can be acceptable.

Peripheral blood dendritic cells are phenotypically and functionally intact in chronic hepatitis B virus (HBV) infection.

Persistence of hepatitis B virus (HBV) infection is associated with reduced anti-viral T cell responses. Impaired dendritic cell (DC) function was suggested as the cause of reduced T cell stimulation in chronic HBV carriers. Thus, we compared myeloid (mDC) and plasmacytoid DC (pDC) from chronic HBV carriers and controls. Frequency and phenotype of isolated DC were analysed by fluorescence activated cell sorter staining, DC function by mixed lymphocyte reaction, cytokine bead array, intracellular cytokine staining, enzyme-linked immunosorbent assay and enzyme-linked immunospot. Expression of HBV DNA and mRNA was studied by polymerase chain reaction (PCR). Circulating total DC, mDC or pDC were not reduced in chronic HBV carriers. Isolated mDC and pDC from chronic HBV carriers exhibited similar expression of co-stimulatory molecules and alloreactive T helper cell stimulation as control DC, whether tested directly ex vivo or after in vitro maturation. Secretion of pro- and anti-inflammatory cytokines by CD40 or Toll-like receptor ligand-stimulated patient DC was intact, as was human leucocyte antigen A2-restricted HBV-specific cytotoxic lymphocyte stimulation. Although both DC populations contained viral DNA, viral mRNA was undetectable by reverse transcription-PCR, arguing against viral replication in DC. We found no quantitative, phenotypic or functional impairment of mDC or pDC in chronic hepatitis B, whether studied ex vivo or after in vitro maturation.

The expression and function of NKG2D molecule on intrahepatic CD8+ T cells in chronic viral hepatitis.

The natural killer (NK) cell receptor, NKG2D is a member of the c-type lectin-activating receptor family. It is expressed by all NK cells and by a sub-population of CD8+ T cells. NKG2D engagement with its ligands directly activates NK cells and acts as a co-stimulator on CD8+ T cells. Recent reports, however, have demonstrated a role for NKG2D in direct T-cell activation in chronic inflammation. The aim of this study was to investigate the pattern of expression and the functional role of NKG2D on circulating and intrahepatic CD8+ T cells in chronic viral hepatitis. Peripheral blood lymphocytes and intrahepatic lymphocytes from 45 patients with chronic viral hepatitis (HBV and HCV) were studied. Phenotypic NKG2D expression and its functional ability to activate intrahepatic and circulating lymphocytes were analysed. Intrahepatic CD8+ T cells display increased NKG2D expression in chronic viral hepatitis in comparison with circulating CD8+ T cells. NKG2D co-stimulates intrahepatic CD8+ T cells and hepatitis B virus-specific CD8+ T cells. However, we could not demonstrate an ability to directly activate CD8+ T cells through the NKG2D signalling pathway alone. NKG2D is up-regulated on intrahepatic CD8+ T cells in type B and C chronic viral hepatitis; however, its function appears to be restricted to that of a co-stimulatory molecule.

The relation between local structural distortion and the low-temperature magnetic anomaly in Fe7S8.

Structural defects on an atomic level can crucially impact the magnetic properties of a material. We study this phenomenon by means of magnetometry and powder neutron diffraction on a stoichiometric, monoclinic pyrrhotite (Fe7S8), which is a classic omission structure with a magnetic anomaly at about 30 K. The initial structural distortion of the pyrrhotite at 300 K caused by the vacancy arrangement decreases upon cooling, and simultaneous to the magnetic anomaly the anisotropic contraction of the unit cell homogenizes the covalency of the Fe-Fe bonds with lengths less than 3.0 Å and the Fe-S-Fe bond angles. These changes on the atomic level affect the spin-orbit coupling and the super-exchange interactions in Fe7S8, and trigger the low-temperature magnetic anomaly within a crystallographically stable system.

Post-translational modification of polyketide and nonribosomal peptide synthases.

The past year has witnessed a major advance in the study of polyketide and nonribosomal peptide biosynthesis with the identification of the phosphopantetheinyl transferase enzyme family, enzymes required to produce active, post-translationally modified polyketide and peptide synthases. Phosphopantetheinyl transferases required for fatty acid, peptide and siderophore biosynthesis have been characterized and a consensus sequence noted in order to facilitate future identification of additional proteins catalyzing phosphopantetheinyl transfer.

Ability of Streptomyces spp. acyl carrier proteins and coenzyme A analogs to serve as substrates in vitro for E. coli holo-ACP synthase.

INTRODUCTION: The polyketide natural products are assembled by a series of decarboxylation/condensation reactions of simple carboxylic acids catalyzed by polyketide synthase (PKS) complexes. The growing chain is assembled on acyl carrier protein (ACP), an essential component of the PKS. ACP requires posttranslational modification on a conserved serine residue by covalent attachment of a 4'-phosphopantetheine (P-pant) cofactor to yield active holo-ACP. When ACPs of Streptomyces type II aromatic PKS are overproduced in E. coli, however, typically little or no active holo-ACP is produced, and the ACP remains in the inactive apo-form. RESULTS: We demonstrate that E. coli holo-ACP synthase (ACPS), a fatty acid biosynthesis enzyme, can catalyze P-pant transfer in vitro to the Streptomyces PKS ACPs required for the biosynthesis of the polyketide antibiotics granaticin, frenolicin, oxytetracycline and tetracenomycin. The catalytic efficiency of this P-pant transfer reaction correlates with the overall negative charge of the ACP substrate. Several coenzyme A analogs, modified in the P-pant portion of the molecule, are likewise able to serve as substrates in vitro for ACPS. CONCLUSIONS: E coli ACPS can serve as a useful reagent for the preparation of holo-forms of Streptomyces ACPs as well as holo-ACPs with altered phosphopantetheine moieties. Such modified ACPs should prove useful for studying the role of particular ACPs and the phosphopantetheine cofactor in the subsequent reactions of polyketide and fatty acid biosynthesis.

Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E. coli EntF catalyzes both elongation and cyclolactonization.

BACKGROUND: EntF is a 142 kDa four domain (condensation-adenylation-peptidyl carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzyme that assembles the Escherichia coli N-acyl-serine trilactone siderophore enterobactin from serine, dihydroxybenzoate (DHB) and ATP with three other enzymes (EntB, EntD and EntE). To assess how EntF forms three ester linkages and cyclotrimerizes the covalent acyl enzyme DHB-Ser-S-PCP (peptidyl carrier protein) intermediate, we mutated residues of the proposed catalytic Ser-His-Asp triad of the thioesterase (TE) domain. RESULTS: The Ser1138-->Cys mutant (kcat decreased 1000-fold compared with wild-type EntF) releases both enterobactin (75%) and linear (DHB-Ser)2 dimer (25%) as products. The His 1271-->Ala mutant (kcat decreased 10,000-fold compared with wild-type EntF) releases only enterobactin, but accumulates both DHB-Ser-O-TE and (DHB-Ser)2-O-TE acyl enzyme intermediates. Electrospray ionization and Fourier transform mass spectrometry of proteolytic digests were used to analyze the intermediates. CONCLUSIONS: These results establish that the TE domain of EntF is both a cyclotrimerizing lactone synthetase and an elongation catalyst for ester-bond formation between covalently tethered DHB-Ser moieties, a new function for chain-termination TE domains found at the carboxyl termini of multimodular NRPSs and polyketide synthases.

A new enzyme superfamily - the phosphopantetheinyl transferases.

BACKGROUND: All polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases require posttranslational modification of their constituent acyl carrier protein domain(s) to become catalytically active. The inactive apoproteins are converted to their active holo-forms by posttranslational transfer of the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a conserved serine residue in each acyl carrier protein domain. The first P-pant transferase to be cloned and characterized was the recently reported Escherichia coli enzyme ACPS, responsible for apo to holo conversion of fatty acid synthase. Surprisingly, initial searches of sequence databases did not reveal any proteins with significant peptide sequence similarity with ACPS. RESULTS: Through refinement of sequence alignments that indicated low level similarity with the ACPS peptide sequence, we identified two consensus motifs shared among several potential ACPS homologs. This has led to the identification of a large family of proteins having 12-22 % similarity with ACPS, which are putative P-pant transferases. Three of these proteins, E. coli EntD and o195, and B. subtilis Sfp, have been overproduced, purified and found to have P-pant transferase activity, confirming that the observed low level of sequence homology correctly predicted catalytic function. Three P-pant transferases are now known to be present in E. coli (ACPS, EntD and o195); ACPS and EntD are specific for the activation of fatty acid synthase and enterobactin synthetase, respectively. The apo-protein substrate for o195 has not yet been identified. Sfp is responsible for the activation of the surfactin synthetase. CONCLUSIONS: The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains. This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.

Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis.

BACKGROUND: Virulence in the pathogenic bacterium Yersinia pestis, causative agent of bubonic plague, has been correlated with the biosynthesis and transport of an iron-chelating siderophore, yersiniabactin, which is induced under iron-starvation conditions. Initial DNA sequencing suggested that this system is highly conserved among the pathogenic Yersinia. Yersiniabactin contains a phenolic group and three five-membered thiazole heterocycles that serve as iron ligands. RESULTS: The entire Y. pestis yersiniabactin region has been sequenced. Sequence analysis of yersiniabactin biosynthetic regions (irp2-ybtE and ybtS) reveals a strategy for siderophore production using a mixed polyketide synthase/nonribosomal peptide synthetase complex formed between HMWP1 and HMWP2 (encoded by irp1 and irp2). The complex contains 16 domains, five of them variants of phosphopantetheine-modified peptidyl carrier protein or acyl carrier protein domains. HMWP1 and HMWP2 also contain methyltransferase and heterocyclization domains. Mutating ybtS revealed that this gene encodes a protein essential for yersiniabactin synthesis. CONCLUSIONS: The HMWP1 and HMWP2 domain organization suggests that the yersiniabactin siderophore is assembled in a modular fashion, in which a series of covalent intermediates are passed from the amino terminus of HMWP2 to the carboxyl terminus of HMWP1. Biosynthetic labeling studies indicate that the three yersiniabactin methyl moieties are donated by S-adenosylmethionine and that the linker between the thiazoline and thiazolidine rings is derived from malonyl-CoA. The salicylate moiety is probably synthesized using the aromatic amino-acid biosynthetic pathway, the final step of which converts chorismate to salicylate. YbtS might be necessary for converting chorismate to salicylate.

Enzyme-linked immunomagnetic electrochemical detection of Salmonella typhimurium.

There is a need for rapid methods to detect pathogenic bacteria in food products as alternatives to the current laborious and time-consuming culture procedures. We report a microbial detection technique that combines the selectivity of antibody-coated superparamagnetic beads with the rapidity and sensitivity of electrochemical detection in a format termed enzyme-linked immunomagnetic electrochemistry. In it, Salmonella typhimurium were sandwiched between antibody-coated magnetic beads and an enzyme-conjugated antibody. With the aid of a magnet, the beads (with or without bound bacteria) were localized onto the surface of disposable graphite ink electrodes in a multi-well plate format. Enzyme substrate was added and conversion of substrate to an electroactive product was measured using electrochemical detection. The electrochemical response was directly proportional to the number of captured bacteria. Using this technique, a minimum detectable level of 8 x 10(3) cells/ml of Salmonella typhimurium in buffer was achieved in ca. 80 min.

Large degree of racemization observed in the amide bond forming reaction on silica gel.

A systematic study of racemization from the coupling of DNB-L-Leu and 3-aminopropyl silica gel with several amide coupling reagents was investigated. Significant amounts of racemization were observed from all except one coupling reagent. In comparison, similar reactions completed in homogeneous solution can be accomplished with much lower racemization and in much higher yields.

Ultraviolet difference spectral studies in the gamma radiolysis of DNA and model compounds. I. Aqueous solutions of DNA bases.

Ultraviolet difference spectra of gamma-irradiated, air-saturated aqueous solutions of DNA bases vs. unirradiated solutions of the same bases are shown to be a very sensitive supplemental tool with which to investigate the yields, postirradiation kinetics, and general nature of DNA base radiation products. Irradiated pyrimidines yield difference spectra which are approximately negative mirror-images of the base absorption spectra in the near-UV, indicating loss of ring conjugation. Difference spectra of irradiated purines yield a more complex pattern containing a positive long-wavelength peak, interpreted as radiation-induced extension of conjugation of the pi electron system beyond that of the unirradiated purine. On the basis of the spectroscopic evidence from these studies, 8-hydroxyguanine appears to be the dominant UV-absorbing radiation product in air-saturated guanine solutions with a G-value of 0.3 molec (100 eV)-1. Difference spectral studies provide isosbestic points which can be used in testing proposed radiation products and their yields. Such spectral studies are a rapid, non-invasive, supplemental tool which can be employed in conjunction with other analytical techniques in radiation-chemical studies, and which is one of the few tools able to detect short-lived chemical intermediates observed in oxygenated solutions of irradiated purines.

The nanophase iron mineral(s) in Mars soil.

A series of surface-modified clays containing nanophase (np) iron oxide/oxyhydroxides of extremely small particle sizes, with total iron contents as high as found in Mars soil, were prepared by iron deposition on the clay surface from ferrous chloride solution. Comprehensive studies of the iron mineralogy in these "Mars-soil analogs" were conducted using chemical extractions, solubility analyses, pH and redox, x ray and electron diffractometry, electron microscopic imaging, specific surface area and particle size determinations, differential thermal analyses, magnetic properties characterization, spectral reflectance, and Viking biology simulation experiments. The clay matrix and the procedure used for synthesis produced nanophase iron oxides containing a certain proportion of divalent iron, which slowly converts to more stable, fully oxidized iron minerals. The clay acted as an effective matrix, both chemically and sterically, preventing the major part of the synthesized iron oxides from ripening, i.e., growing and developing larger crystals. The precipitated iron oxides appear as isodiametric or slightly elongated particles in the size range 1-10 nm, having large specific surface area. The noncrystalline nature of the iron compounds precipitated on the surface of the clay was verified by their complete extractability in oxalate. Lepidocrocite (gamma-FeOOH) was detected by selected area electron diffraction. It is formed from a double iron Fe(II)/Fe(III) hydroxy mineral such as "green rust," or ferrosic hydroxide. Magnetic measurements suggested that lepidocrocite converted to the more stable maghemite (gamma-Fe2O3) by mild heat treatment and then to nanophase hematite (alpha-Fe2O3) by extensive heat treatment. After mild heating, the iron-enriched clay became slightly magnetic, to the extent that it adheres to a hand-held magnet, as was observed with Mars soil. The chemical reactivity of the iron-enriched clays strongly resembles, and offers a plausible mechanism for, the somewhat puzzling observations of the Viking biology experiments. Their unique chemical reactivities are attributed to the combined catalytic effects of the iron oxide/oxyhydroxides and silicate phase surfaces. The reflectance spectrum of the clay-iron preparations in the visible range is generally similar to the reflectance curves of bright regions on Mars. This strengthens the evidence for the predominance of nanophase iron oxides/oxyhydroxides in Mars soil. The mode of formation of these nanophase iron oxides on Mars is still unknown. It is puzzling that despite the long period of time since aqueous weathering took place on Mars, they have not developed from their transitory stage to well-crystallized end-members. The possibility is suggested that these phases represent a continuously on-going, extremely slow weathering process.

Minimum detectable level of Salmonellae using a binomial-based bacterial ice nucleation detection assay (BIND).

A modified bacterial ice nucleation detection (BIND) assay was used for rapid and sensitive detection of several Salmonella species. For the BIND assay, Salmonella cells are infected with bacteriophage genetically modified to contain DNA encoding an ice nucleation protein (INP). After infection, de novo protein synthesis occurs and INPs are incorporated into the outer membrane of the organism. After supercooling (-9.3 degrees C), only buffer solutions containing transfected salmonellae freeze, causing a phase-sensitive dye to change color. This technique, and a probability-based protocol modification, provided quantitative detection with a minimum detectable level (MDL) of 2.0 +/- 0.3 S. enteritidis cells/mL in buffer (about 3 h). The MDLs for S. typhimurium DT104 and S. abaetetuba were 4.2 +/- 0.2 and 11.1 +/- 0.4 cells/mL, respectively. Using salmonellae-specific immunomagnetic bead separation technology in conjunction with the modified BIND protocol, we achieved an MDL of about 4.5 S. enteritidis cells/mL with an apparent capture efficiency of 56%.

Identification of tetrahydrocarbazoles as novel multifactorial drug candidates for treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder and the most frequent cause of dementia. To date, there are only a few approved drugs for AD, which show little or no effect on disease progression. Impaired intracellular calcium homeostasis is believed to occur early in the cascade of events leading to AD. Here, we examined the possibility of normalizing the disrupted calcium homeostasis in the endoplasmic reticulum (ER) store as an innovative approach for AD drug discovery. High-throughput screening of a small-molecule compound library led to the identification of tetrahydrocarbazoles, a novel multifactorial class of compounds that can normalize the impaired ER calcium homeostasis. We found that the tetrahydrocarbazole lead structure, first, dampens the enhanced calcium release from ER in HEK293 cells expressing familial Alzheimer's disease (FAD)-linked presenilin 1 mutations. Second, the lead structure also improves mitochondrial function, measured by increased mitochondrial membrane potential. Third, the same lead structure also attenuates the production of amyloid-beta (Aß) peptides by decreasing the cleavage of amyloid precursor protein (APP) by ß-secretase, without notably affecting α- and γ-secretase cleavage activities. Considering the beneficial effects of tetrahydrocarbazoles addressing three key pathological aspects of AD, these compounds hold promise for the development of potentially effective AD drug candidates.