Application of metasurface-enhanced infra-red spectroscopy to distinguish between normal and cancerous cell types.

Fourier transform infrared (FTIR) spectra of biological cells can reveal clinically important information about cells' composition, including their normal or cancerous status. The recently emerged diagnostic technique of spectral cytopathology (SCP) combines FTIR with multivariate statistical analysis to detect cell abnormalities, differentiate between cell types, and monitor disease progression. We demonstrate a new variant of SCP, a metasurface-enhanced infrared reflection spectroscopic cytopathology (MEIRSC) that utilises judiciously designed plasmonic metasurfaces to localize and enhance the evanescent field near the cell's membrane, and to carry out spectroscopic interrogations of the cells attached to the metasurface using reflected infrared light. Our findings indicate that the MEIRSC approach enables us to differentiate between normal and cancerous human colon cells. The sensitivity of MEIRSC is such that a very small (about 50 nm deep) portion of the cell can yield valuable diagnostic information.

Effectiveness of mebeverine in patients with post-cholecystectomy gastrointestinal spasm: results of prospective observational program "odyssey".

AIM: To assess the effectiveness of mebeverine 200 mg BID in patients with post-cholecystectomy gastrointestinal spasm not requiring surgical treatment. MATERIALS AND METHODS: 218 patients were included in 16 clinical centers in 14 cities in Russia. All patients had post-cholecystectomy gastrointestinal spasms, not requiring surgical treatment and received mebeverine (Duspatalin®) 200 mg BID. The observational assessment period lasted from the moment of their inclusion into the study up to 6 weeks post inlusion. The therapy results were evaluated using visual analog scales (GPA and 11-point numeric rating scale) by patient self-assessment of the dynamics of spasm/discomfort and other post-cholecystectomic gastrointestinal symptoms after 2 and 6 weeks of treatment. Gastrointestinal Quality of Life Index (GIQLI) was used to assess patient quality of life. RESULTS: All 218 patients completed the 2-week mebeverine treatment course, 101 of them finished the 6-week course ("prolonged population"). Significant positive changes in the relief of abdominal pain and dyspepsia were noted as well as normalization of stool frequency and consistency. A more marked change in values was observed during prolonged (up to 6 weeks) therapy. Both 2-week and 6-week mebeverine courses led to a normalization of patient quality of life. After 6 week therapy, an effect of mebeverine on the quality of life 91% of patients was observed comparable to cholecystectomy itself, speficially related to the quality of life subscore 'symptoms'. CONCLUSION: The results of our study demonstrate that mebeverine (Duspatalin®) therapy leads to an effective elimination of clinical symptoms associated with post-cholecystectomy GI-spasm disorders, like abdominal pain, symptoms of dyspepsia and stooldisorders. A more marked change in values was observed during prolonged (up to 6 weeks) therapy.

Rapid real-time recirculating PCR using localized surface plasmon resonance (LSPR) and piezo-electric pumping.

Rapid detection and characterization of pathogens in patients with bloodstream infections (BSIs) is a persistent problem for modern medicine, as current techniques are slow or provide incomplete diagnostic information. Real-time polymerase chain reaction (qPCR) allows specific detection of a wide range of targets and quantification of pathogenic burdens to aid in treatment planning. However, new technological advances are required for a rapid and multiplex implementation of qPCR in clinical applications. In this paper, the feasibility of a novel microfluidic platform for qPCR is presented, integrating highly sensitive, label-free localized surface plasmon resonance (LSPR) imaging of DNA hybridization into a recirculating chip design for real-time analysis. Single target and multiplex detection of DNA target amplification are demonstrated, with a limit of detection of 5 fg µL-1 of E. coli DNA for single target PCR, correlating with approximately 300 bacteria per mL. The results of this study demonstrate the potential of this platform for simultaneous real-time detection of multiple target genes within 15 minutes that could provide live saving benefits in patients with BSIs.

Growth of textured thin Au coatings on iron oxide nanoparticles with near infrared absorbance.

A homologous series of Au coated iron oxide nanoparticles with hydrodynamic diameters smaller than 60 nm was synthesized with very low Au-to-iron mass ratios, as low as 0.15. The hydrodynamic diameter was determined by dynamic light scattering and the composition by atomic absorption spectroscopy and energy dispersive x-ray spectroscopy. Unusually low Au precursor supersaturation levels were utilized to nucleate and grow Au coatings on iron oxide relative to the formation of pure Au nanoparticles. This approach produced unusually thin coatings by lowering autocatalytic growth of Au on Au, as shown by transmission electron microscopy. Nearly all of the nanoparticles were attracted by a magnet, indicating a minimal number of pure Au particles. The coatings were sufficiently thin to shift the surface plasmon resonance to the near infrared with large extinction coefficients, despite the small particle hydrodynamic diameters observed from dynamic light scattering to be less than 60 nm.

Telomerase from yeast Saccharomyces cerevisiae is active in vitro as a monomer.

A system for isolation of yeast telomerase via RNA affinity tag in TLC1 RNA was developed. Streptavidin aptamer was inserted at two different positions in TLC1 RNA. Telomerase with TLC1 RNA with one of these inserts is functional in vivo and can be isolated by affinity chromatography in vitro. A telomerase preparation isolated using this technique from a strain producing two distinguishable TLC1 RNA molecules (with and without aptameric insertion) resulted in isolation of active telomerase containing only TLC1 RNA with the aptamer. Our results indicate that yeast telomerase is active in vitro as a monomer.

Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties.

Molecular optical imaging has shown promise in visualizing molecular biomarkers with subcellular resolution both noninvasively and in real-time. Here, we use gold nanoparticles as optical probes to provide meaningful signal in the presence of targeted biomarkers. We present a novel conjugation technique to control the binding orientation of antibodies on the surface of gold nanoparticles to maximize antibody functionality. Briefly, a heterobifunctional linker, hydrazide-polyethylene glycol-dithiol, is used to directionally attach the Fc, or nonbinding region of the antibody, to the gold nanoparticle surface. The conjugation strategy allows for multiplexing various glycosylated antibodies on a single nanoparticle. We present a method to prepare multifunctional nanoparticles by incorporating targeting and delivery moieties on the same nanoparticle that addresses the challenge of imaging intracellular biomarkers. The time estimate for the entire protocol is approximately 6 h.

Molecular imaging of carcinogenesis with immuno-targeted nanoparticles.

Molecular characterization of cancer could have important clinical benefits such as earlier cancer detection based on molecular characterization, the ability to predict the risk of cancer progression, real time margin detection, the ability to rationally select molecular therapy and to monitor response to the therapy. We present a new class of molecular specific contrast agents for optical imaging of carcinogenesis in vivo - gold nanoparticles conjugated with monoclonal antibodies specific for cancer biomarkers.

Optical systems for in vivo molecular imaging of cancer.

Progress toward a molecular characterization of cancer would have important clinical benefits; thus, there is an important need to image the molecular features of cancer in vivo. In this paper, we describe a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer. We describe initial results obtained using optically active contrast agents to image the expression of three well known molecular signatures of neoplasia: including over expression of the epidermal growth factor receptor (EGFR), matrix metallo-proteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection. At the same time, we are developing inexpensive, portable optical systems to image the morphologic and molecular signatures of neoplasia noninvasively in real time. These real-time, portable, inexpensive systems can provide tools to characterize the molecular features of cancer in vivo.

Understanding the contributions of NADH and collagen to cervical tissue fluorescence spectra: modeling, measurements, and implications.

OBJECTIVE: At 380 nm excitation, cervical tissue fluorescence spectra demonstrate characteristic changes with both patient age and the presence of dysplasia. A Monte Carlo model was developed in order to quantitatively examine how intrinsic NADH and collagen fluorescence, in combination with tissue scattering and absorption properties, yield measured tissue spectra. METHODS: Excitation-emission matrices were measured for live cervical cells and collagen gel phantoms. Fluorescence microscopy of fresh tissue sections was performed to obtain the location and density of fluorophores as a function of patient age and the presence of dysplasia. A Monte Carlo model was developed which incorporated measurements of fluorophore line shapes and spatial distributions. RESULTS: Modeled spectra were consistent with clinical measurements and indicate that an increase in NADH fluorescence and decrease in collagen fluorescence create clinically observed differences between normal and dysplastic tissue spectra. Model predictions were most sensitive to patient age and epithelial thickness. CONCLUSIONS: Monte Carlo techniques provide an important means to investigate the combined contributions of multiple fluorophores to measured emission spectra. The approach will prove increasingly valuable as a more sophisticated understanding of in vivo optical properties is developed.

A specific DNA-dependent DNA polymerase is associated with Saccharomyces cerevisiae telomerase.

The telomere DNA of most eucaryotes consists of tandem DNA repeats and a number of associated proteins. The synthesis of the G-rich DNA strand is performed by the telomerase complex. The complementary C-strand is synthesized by DNA-dependent DNA polymerases. Using telomerase reverse transcriptase tagging followed by immunoprecipitation coupled with two-step purification, we have found a specific DNA-dependent DNA polymerase activity associated with telomerase of Saccharomyces cerevisiae. Investigation of the biochemical properties of this DNA polymerase activity confirms the hypothesis of tight interaction of DNA polymerase delta with telomerase.

Reflectance spectroscopy with polarized light: is it sensitive to cellular and nuclear morphology.

We present a method for selective detection of size-dependent scattering characteristics of epithelial cells in vivo based on polarized illumination and polarization sensitive detection of scattered light. We illustrate the method using phantoms designed to simulate squamous epithelial tissue and progressing to epithelial tissue in vitro and in vivo. Elastic light scattering spectroscopy with polarized illumination/detection dramatically reduces background signals due to both diffuse stromal scattering and hemoglobin absorption. Resulting spectra can be described as a linear combination of forward and backscattering components determined from Mie theory. Nuclear sizes and refractive indices extracted by fitting experimental spectra to this model agree well with previous measurements. Reflectance spectroscopy with polarized light can provide quantitative morphological information which could potentially be used for non-invasive detection of neoplastic changes.

Telomerase from Saccharomyces cerevisiae contains several protein subunits and may have different activities depending on the protein content.

Telomerase is a ribonucleoprotein responsible for maintaining telomeres during the cell cycle [1,2]. Here we describe a two-step purification procedure for the Saccharomyces cerevisiae telomerase complex. We have found that the properties (processivity, nuclease activity) of telomerase depend on the isolation procedure. Using a cross-linking approach, we have revealed several proteins that could be components of the telomerase complex. Furthermore, spectra of cross-linked proteins differ in processive and non-processive telomerase complexes.

Enhancement of molecular fluorescence near the surface of colloidal metal films.

Fluorescence enhancement was studied on silver colloidal metal films (CMFs) using two systems: (1) Langmuir--Blodgett monolayers of fluorescein-labeled phospholipids separated from the surface of the films by spacer layers of octadecanoic acid and (2) biotin--fluorescein conjugates captured by avidin molecules adsorbed on top of a multilayer structure formed by alternating layers of bovine serum albumin--biotin conjugate (BSA--biotin) and avidin. The dependence of fluorescence intensity on the number of lipid or protein spacer layers deposited on the surface of the CMF was investigated. The results demonstrate the requirement for adsorbate location within the region between Ag particles for maximal enhancement. The density of avidin molecules on the surface of the BSA--biotin/avidin multilayers adsorbed on the CMF was also determined. A procedure for forming a rigid, uniform silica layer around the Ag particles on the CMF is described. The layer protects the particles from undesirable chemical reactions such as etching by halide ions, for example, and provides the requisite stability for bioanalytical applications. Colloidal films composed of Ag particles covered by approximately 10-nm-thick silica layers were tested for fluorescence enhancement using goat immunoglobulin and a conjugate of rabbit anti-goat immunoglobulin with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)hexanoate. An enhancement factor of approximately 20 was obtained.

Scanning Force Microscopy Study of Methanol-Induced Changes in the Distribution of Silver Particles in Colloidal Metal Films

Colloidal silver particles were attached to a solid surface for use as a substrate for surface-enhanced Raman scattering and surface enhanced fluorescence spectroscopies. The enhancement of Raman scattering is dramatically increased after exposure of the substrates to methanol solution. Scanning force microscopy was used to visualize the substrate before and after the methanol treatment, and a statistical evaluation of the particle distribution revealed some differences. The nearest-neighbor distance was decreased after the exposure to methanol. This effect suggests that an aggregation of the particles occurred. This aggregation is probably based on increased attractive interactions and lateral mobility of the particles in methanol.

[Tactile sensitivity as a part of the kinesthetic system (a comparative clinico-physiological approach)]. / Taktil'naia chuvstvitel'nost' kak chast' kinesteticheskoi sistemy (sravnitel'nyi kliniko-fiziologicheskii podkhod).

In experiments on healthy children as well as on children with inborn and postamputation stumps of the forearm, studies have been made of the relationship between the level of tactile sensitivity in the skin of the forearm and the level of its muscular motor activity; these studies were performed using focused ultrasound in children at the age of 7, 10 and 14 years. It was found that with the increase in motor activity of the forearm, irrespectively of the age, tactile thresholds decrease. The success of prosthetic appliance depends on the ratio between skin tactile sensitivity and motor activity of the forearm. The lowest thresholds were found in 10-year children and in a zone innervated by the median skin nerve of the forearm.