Superquenching as a detector for microsphere-based flow cytometric assays.

BACKGROUND: Fluorescent conjugated polymers display high fluorescence quantum yields and enhanced sensitivity to quenching (superquenching) by oppositely charged quenchers through energy or electron transfer. Fluorescent polymers and their quenchers are used in bead-based biosensor applications where the polymers are coated on particles. In this work, we investigate a detection method that utilizes superquenching on microspheres, which can be used for flow cytometric assays. METHODS: Microspheres were coated with the fluorescent cationic polyelectrolyte poly(p-phenylene-ethynylene) (PPE), and its superquenching by 9,10-anthraquinone-2,6-disulfonic acid (AQS) was examined by fluorometric methods in presence and in absence of a barrier to superquenching in the form of an anionic lipid bilayer. RESULTS: Flow cytometry detected superquenching of PPE on microspheres (MS-PPE) by AQS where high levels of reduction in fluorescence were observed. Adding different concentrations of AQS to MS-PPE yielded a Stern-Volmer quenching constant of 0.8x10(6) M-1. While forming an anionic lipid bilayer around the MS-PPE acted as a barrier to superquenching by AQS, disrupting the lipid bilayer allowed superquenching to take place. CONCLUSIONS: The sensitivity of flow cytometry in detecting fluorescence of microspheres and the amplified quenching sensitivity of fluorescent conjugated polymers both offer advantages over other fluorometric methods and conventional quenching detection. This study used superquenching of fluorescent polymers as a new tool in flow cytometry, thus combining the advantages offered by both method and detector. In addition, we employed the formation and the disruption of a supported lipid bilayer in mediating superquenching to offer new biosensing applications.     

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

Altered placental function as a consequence of aberrant imprinted gene expression may be one mechanism mediating the association between low birth weight and increased cardiometabolic disease risk. Imprinted gene expression is regulated by epigenetic mechanisms, particularly DNA methylation (5mC) at differentially methylated regions (DMRs). While 5-hydroxymethylcytosine (5hmC) is also present at DMRs, many techniques do not distinguish between 5mC and 5hmC. Using human placental samples, we show that the expression of the imprinted gene CDKN1C associates with birth weight. Using specific techniques to map 5mC and 5hmC at DMRs controlling the expression of CDKN1C and the imprinted gene IGF2, we show that 5mC enrichment at KvDMR and DMR0, and 5hmC enrichment within the H19 gene body, associate positively with birth weight. Importantly, the presence of 5hmC at imprinted DMRs may complicate the interpretation of DNA methylation studies in placenta; future studies should consider using techniques that distinguish between, and permit quantification of, both modifications.     

The synthesis and structure-activity relationships of 3-amino-4-benzylquinolin-2-ones; discovery of novel KCNQ2 channel openers

3-amino-4-benzylquinolin-2-ones have been identified as a novel class of KCNQ2 channel openers. Synthesis and SAR is described along with their electrophysiological evaluation as activators of the cloned mKCNQ2 channel expressed in Xenopus laevis oocytes. The preliminary SAR data suggest the importance of both the trifluoromethylsulfonamido group and electron-withdrawing substituents on the quinolone nucleus for expression of KCNQ2 channel opening properties.     

The synthesis and structure-activity relationships of 4-aryl-3-aminoquinolin-2-ones: a new class of calcium-dependent,large conductance,potassium (maxi-K) channel openers targeted for post-stroke neuroprotection

A series of 4-aryl-3-aminoquinoline-2-one derivatives was synthesized and evaluated as activators of the cloned maxi-K channel mSlo (hSlo) expressed in Xenopus laevis oocytes using electrophysiological methods. A brain penetrable activator of maxi-K channels was identified and shown to be significantly active in the MCAO model of stroke.     

Synthesis of water-soluble prodrugs of BMS-191011: a maxi-K channel opener targeted for post-stroke neuroprotection

A variety of water-soluble prodrugs of BMS-191011 was synthesized and evaluated for solution state stability and rate of conversion to BMS-191011 in rat and human plasma. The deoxycarnitine ester prodrug (11c) was selected for clinical evaluation based on its superior chemical stability, crystallinity and cleavage to BMS-191011 in human plasma.     

Antibacterial activity of α-mangostin against vancomycin resistant Enterococci (VRE) and synergism with antibiotics

alpha-Mangostin, isolated from the stem bark of Garcinia mangostana L., was found to be active against vancomycin resistant Enterococci (VRE) and methicillin resistant Staphylococcus aureus (MRSA), with MIC values of 6.25 and 6.25 to 12.5 microg/ml, respectively. Our studies showed synergism between alpha-mangostin and gentamicin (GM) against VRE, and alpha-mangostin and vancomycin hydrochloride (VCM) against MRSA. Further studies showed partial synergism between alpha-mangostin and commercially available antibiotics such as ampicillin and minocycline. These findings suggested that alpha-mangostin alone or in combination with GM against VRE and in combination with VCM against MRSA might be useful in controlling VRE and MRSA infections.     

One-dimensional acoustic standing waves in rectangular channels for flow cytometry

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry.     

Association of tumor necrosis factor alpha gene polymorphisms with Helicobacter pylori infection in dyspeptic patients in Sri Lanka

Single nucleotide polymorphisms present on the promoter sequence of the TNF‐α gene may affect production of TNF‐α, a pro‐inflammatory cytokine, during immune responses. The presence of TNF‐α polymorphisms is also reportedly associated with more severe manifestations of Helicobacter pylori infection. However, the frequency of TNF‐α polymorphisms and the associated disease severity vary between different patient groups. In this study, gastric biopsies and blood specimens were collected from 138 patients with dyspepsia undergoing routine upper gastrointestinal endoscopy. Our institution's Ethics Review Committee approved the study and written informed consent was obtained from all participants. The presence of H. pylori was confirmed histologically in all patients. The frequency of TNF‐α polymorphisms in the study cohort was investigated using PCR–restriction fragment length polymorphism and expression of serum TNF‐α quantitated using a commercial ELISA assay. The proportions of selected TNF‐α polymorphisms (TNF‐α ‐238, ‐308 and ‐863) were similar in H. pylori‐positive and ‐negative patients. Homozygous mutations of TNF‐α polymorphisms were rarely detected in the study group. There was a significant difference in TNF‐α concentrations between patients with mild chronic gastritis and TNF‐α ‐308 GG genotype and patients with moderate to severe chronic gastritis (P = 0.008). It was not possible to identify an association between these genotypes and disease severity because of the low frequency of heterozygous and homozygous mutated genes in Sri Lankan patients with dyspepsia.

     

3-Thio-quinolinone maxi-K openers for the treatment of erectile dysfunction

A series of Maxi-K openers for the treatment of erectile dysfunction based on the 3-thio-quinolinone core is described. Significant levels of channel opening (up to 550% of control) are seen in transfected oocytes. Functional activity in rabbit corpus cavernosum tissue strips confirms the potential to effect therapy for ED, the effect being maximal for the 3-amino-2-hydroxy thiol side chain.     

4-Aryl-3-(mercapto)quinolin-2-ones: novel maxi-K channel opening relaxants of corporal smooth muscle

A novel series of 4-aryl-3-(mercapto)quinolin-2-one derivatives was prepared and evaluated as openers of the cloned maxi-K channel hSlo expressed in Xenopus laevis oocytes by utilizing electrophysiological methods. The effect of these maxi-K openers on corporal smooth muscle was studied in vitro using isolated rabbit corpus cavernosum. In vivo efficacy has been demonstrated with a selective maxi-K opening relaxant in a rat model of erectile function.