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Biomedical subjects

Rumiana Bakalova

Publications and source records attributed to Rumiana Bakalova.

At least 19 recordsLinked to original sources
PubMed2006

Ultra-fast biosensors and multi-photon microscopy in the future of brain studies.

The direct, highly selective and sensitive real-time imaging of neuro- and biochemical mediators is the only way to clarify precisely the chemistry of the brain and to discover the key molecular targets involved in regulation of brain homeostasis. To realize that, we need: high-speed deep-tissue imaging techniques with high spatial and temporal resolution; and ultra-fast and highly selective molecular sensors, giving a possibility to monitor target molecules directly in their physiological environment; in addition, these molecular sensors have to be comparatively small and permeable for blood-brain barrier, to be applicable in brain studies. The present view accents on the perspectives for development of direct approach for investigation of function/flow coupling phenomenon in the brain, based on the current progress in development of ultra-fast molecular sensors for direct visualization of biochemical mediators (e.g., nitric oxide, Ca ions), and high-speed two-photon/multi-photon deep-tissue imaging.

Animals↗
PubMed2006

Dual-labeled telomere sensing probes for quantification of telomerase activity assay.

The present study describes an empirically discovered phenomenon that might be useful for development of a sensitive and rapid methodology for quantification of telomerase activity assay with simple data acquisition and possibility for calculation of telomerase product in absolute units. The method is based on the design and application of two single-stranded telomere sensing probes consisting of dual-labeled 16-mer oligonucleotides (fluorescent Cy3/Cy3-labeled and non-fluorescent IowaBlack/BHQ-labeled) that can simultaneously hybridize on the primary product of the telomerase reaction.

Base Sequence↗
PubMed2006

Silica-shelled single quantum dot micelles as imaging probes with dual or multimodality.

The present study describes a stabilization of single quantum dot (QD) micelles by a "hydrophobic" silica precursor and an extension of a silica layer to form a silica shell around the micelle using "amphiphilic" and "hydrophilic" silica precursors. The obtained product consists of approximately 92% single nanocrystals (CdSe, CdSe/ZnS, or CdSe/ZnSe/ZnS QDs) into the silica micelles, coated with a silica shell. The thickness of the silica shell varies, starting from 3-4 nm. Increasing the shell thickness increases the photoluminescence characteristics of QDs in an aqueous solution. The silica-shelled single CdSe/ZnS QD micelles possess a comparatively high quantum yield in an aqueous solution, a controlled small size, sharp photoluminescence spectra (fwhm approximately 30 nm), an absence of aggregation, and a high transparency. The surface of the nanoparticles is amino-functionalized and ready for conjugation. A comparatively good biocompatibility is demonstrated. The nanoparticles show ability for intracellular delivery and are noncytotoxic during long-term incubation with viable cells in the absence of light exposure, which makes them appropriate for cell tracing and drug delivery. The presence of the hydrophobic layer between the QD and silica-shell ensures an incorporation of other hydrophobic molecules with interesting properties (e.g., hydrophobic paramagnetic substances, hydrophobic photosensitizers, membrane stabilizers, lipid-soluble antioxidants or prooxidants, other hydrophobic organic dyes, etc.) in the close proximity of the nanocrystal. Thus, it is possible to combine the characteristics of hybrid materials with the priority of small size. The silica-shelled single QD micelles are considered as a basis for fabrication of novel hybrid nanomaterials for industrial and life science applications, for example, nanobioprobes with dual modality for simultaneous application in different imaging techniques (e.g., fluorescent imaging and functional magnetic resonance imaging).

Contrast Media↗
PubMed2006

Single quantum dot-micelles coated with silica shell as potentially non-cytotoxic fluorescent cell tracers.

The present study describes a stabilization of single quantum dot (QD) micelles by hydrophobic silica precursors and an extension of the silica layer to form a silica shell around the micelle. The obtained product consists of up to 92% of single nanocrystals (CdSe, CdSe/ZnS, or CdSe/ZnSe/ZnS quantum dots) in the silica micelles, coated with silica shell. The thickness of silica shell could vary, starting from 3 to 4 nm. Increasing the shell thickness increases the photoluminescent characteristics of QDs in aqueous solution. The silica-shelled single CdSe/ZnS QD micelles possess a high quantum yield in aqueous solution, a controlled small size, sharp photoluminescence spectra (fwhm approximately 30 nm), an absence of aggregation, and a high transparency. The presence of a hydrophobic layer between the QD and silica shell ensures an incorporation of other hydrophobic molecules (with interesting properties) in the close proximity of nanocrystal. Thus, it is possible to combine the characteristics of hybrid material with the priority of small size. The nanoparticles are amino functionalized and ready for conjugation. A comparatively good biocompatibility is demonstrated. The nanoparticles show ability for intracellular delivery and are noncytotoxic during long-term incubation with viable cells in the absence of light exposure, which makes them appropriate for cell tracing and drug delivery.

Biocompatible Materials↗
PubMed2006

Uncoated, broad fluorescent, and size-homogeneous CdSe quantum dots for bioanalyses.

In the present study, we describe the synthesis of highly luminescent uncoated water-soluble CdSe quantum dots (QDs) possessing the following characteristics: approximately 2 nm in diameter, with very good size distribution (in 95% homodispersed) accompanied by a broad-band photoluminescent spectrum. The synthetic procedure is simple, is conducted at room temperature, in the absence of the most popular coordinating ligands (as TOPO or HDA), and is highly reproducible. The obtained CdSe core QDs possessed a comparatively long fluorescence half-life (approximately 30-90 ns, depending on the emission wavelength) detected by time-resolved spectroscopy. These QDs were further conjugated with antibodies and applied in several biochemical analyses.

Antigen-Antibody Complex↗
PubMed2006

Self-surface passivation of CdX (X = Se, Te) quantum dots.

A small portion of a reaction mixture including unpurified CdX (X = Se or Te) quantum dots (QDs), in which unreacted Cd and Se ions were left together with coordinating solvents, was dropped into an organic solvent. The CdX QDs in this organic solution showed enhancement of photoluminescence (PL) efficiency, growth of particles, and focusing of size distribution for more than 10 h at room temperature (RT, -23 degrees C). These effects were attributed to passivation of QDs' surface by Cd and X ions present in the solution. No external energy source was used for these achievements; therefore, the process is termed as self-surface passivation. The self-surface passivation was reproduced using purified CdX QDs with additional Cd and X ions in an organic solvent. The self-surface passivation method was applied to RT-synthesized CdSe QDs, which is characterized by a broad PL spectrum (fwhm - 150 nm) for monodispersed QDs, to modify their emission characteristics. On self-surface passivation, the broad PL spectrum was narrowed (fwhm - 35 nm) and the QDs were grown. The X-ray diffraction measurements of RT-synthesized CdSe QDs and that subsequently aged in 1-butanol showed that crystallinity of the samples was improved on aging.

Cadmium Compounds↗
PubMed2005

The NK-lysin derived peptide NK-2 preferentially kills cancer cells with increased surface levels of negatively charged phosphatidylserine.

The NK-lysin derived peptide NK-2 is a potent antibacterial, but non-toxic to a human keratinocyte cell line and of low hemolytic activity. Its target selectivity is based upon a strong binding preference to membranes containing anionic phospholipids, which are normally not found on the surface of human cells. Here, we analyzed the interaction of NK-2 with normal human lymphocytes and seven different human cancer cell lines and demonstrate that some of these cells expose negatively charged surface phosphatidylserine (PS), which presumably facilitates killing of the cells by NK-2. This is underlined by the specific intercalation of the peptide into PS-containing liposomes analyzed by fluorescence-resonance energy transfer spectroscopy.

Antineoplastic Agents↗
PubMed2005

Quantum dot-conjugated hybridization probes for preliminary screening of siRNA sequences.

In the present study, we describe the design and fabrication of quantum dot-conjugated hybridization probes and their application to the development of a comparatively simple and rapid procedure for the selection of highly effective small-interfering RNA (siRNA) sequences for RNA interference (RNAi) in mammalian cells, for example, siRNAs with high accessibility and affinity to the respective mRNA target. A single-stranded siRNA was conjugated with a quantum dot and used as a hybridization probe. The target mRNA was amplified in the presence of Cy5-labeled nucleotides, and Cy5-mRNA served as a hybridization sample. The formation of siRNA/mRNA duplexes during a comparatively short hybridization time (1 h) was used as a criterion for the selection of highly effective, target-specific siRNA sequences. The accessibility and affinity of the siRNA sequence for the target mRNA site were determined by fluorescence resonance energy transfer (FRET) between a quantum dot (donor) and a fluorescent dye molecule (Cy5, acceptor) localized at an appropriate distance from each other when hybridization occurred. The FRET signal was observed only when there was high accessibility between an antisense siRNA and a sense mRNA and did not appear in the case of mismatch siRNAs. Moreover, the amplitude of the FRET signal significantly correlated with the specific effect of siRNA on the expression of the target mRNA and protein, determined in native cells by RT-PCR and immunoblot analysis, respectively.

Base Sequence↗
PubMed2005

Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation.

Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by solid phase fragment condensation. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugate-RNA hybrids could activate RNase H as effectively as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into the cell nucleus, (f) the conjugates with NES could be localized in the cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.

Amino Acid Sequence↗
PubMed2005

Efficient cleavage of RNA, enhanced cellular uptake, and controlled intracellular localization of conjugate DNAzymes.

Conjugate DNAzymes with polyamines and peptides were successfully prepared by solid phase fragment condensation (SPFC) and showed up to 4.2 times higher catalytic efficiency (k(cat)/K(m)) and enhanced tolerance against DNase 1digestion. To be pointed out, intracellular localization of DNAzymes could be controlled by conjugated with naturally occurring signal peptides responsible for nuclear cytoplasmic transport of proteins.

Base Sequence↗
PubMed2005

Nonradioactive telomerase activity assay by microchip electrophoresis: privileges to the classical gel electrophoresis assay.

The present study accents on the privileges of microchip-based electrophoresis to the conventional gel electrophoresis in separation of telomerase repeat amplification protocol/polymerase chain reaction (PCR) ladder products obtained in telomerase-catalyzed reaction in cancer cells. We try to clarify the interpretation of the results obtained by both electrophoretic procedures and to avoid misinterpretation as a result of PCR-dependent artefacts.

Electrophoresis, Microchip↗
PubMed2005

Highly sensitive inhibition of hTERT mRNA expression and telomerase activity by DNA-signal-peptide conjugates.

In the present study, we investigated the antisense properties of conjugate oligonucleotides (ODNs) inhibiting human telomerase activity. Conjugate oligonucleotides assembled with signal peptides, artificially designed peptides, amines and sugars were synthesized by solid phase fragment condensation (SPFC) in sufficient yields. Conjugate ODNs showed a high resistance to nuclease degradation and sufficient binding affinity to target RNA, comparatively rapid and sufficient intracellular delivery and specific localization controlled by signal peptides (nuclear localization signals, NLS; nuclear export signals, NES). ODN-NLS conjugates demonstrated high antisense inhibitory effects against human telomerase activity into the nucleus (e. g, phosphorothioate conjugate inhibited the telomerase activity over 95%), whereas ODN-NES conjugates inhibited target mRNA expression into the cytoplasm.

Biological Transport↗
PubMed2005

Role of free cadmium and selenium ions in the potential mechanism for the enhancement of photoluminescence of CdSe quantum dots under ultraviolet irradiation.

The present study describes an enhancement of the photoluminescence of CdSe quantum dots under long-term ultraviolet irradiation in organic solvents. The photoenhancement effect followed multiexponential kinetics and was found to depend on several factors: intensity of ultraviolet light, polarity of the solvent, presence of capping agents on the nanocrystal surface, and presence of free Cd and Se ions in the solution. High intensity ultraviolet irradiation provoked a rapid enhancement of the photoluminescence of CdSe nanocrystals, reaching the maximum with subsequent photoluminescence decay. Low-intensity ultraviolet irradiation provoked a comparatively slow enhancement of the photoluminescence of CdSe nanocrystals, reaching saturation after 5-6 hours of irradiation in organic solvents (butanol and chloroform). The photoenhancement effect was reversible or irreversible depending on the additional ingredients. The role of free Cd and Se in these processes was clarified. The results are discussed in the context of ultraviolet induced liberation of free Cd and Se ions from the nanocrystal surface and their hypothetical reversible deposition with trapping of the surface holes and influencing the efficiency of radiative versus nonradiative exciton decay during the enhancement of photoluminescence.

Cadmium↗
PubMed2005

A history of microarrays in biomedicine.

The fundamental strategy of the current postgenomic era or the era of functional genomics is to expand the scale of biologic research from studying single genes or proteins to studying all genes or proteins simultaneously using a systematic approach. As recently developed methods for obtaining genome-wide mRNA expression data, oligonucleotide and DNA microarrays are particularly powerful in the context of knowing the entire genome sequence and can provide a global view of changes in gene expression patterns in response to physiologic alterations or manipulation of transcriptional regulators. In biomedical research, such an approach will ultimately determine biologic behavior of both normal and diseased tissues, which may provide insights into disease mechanisms and identify novel markers and candidates for diagnostic, prognostic and therapeutic intervention. However, microarray technology is still in a continuous state of evolution and development, and it may take time to implement microarrays as a routine medical device. Many limitations exist and many challenges remain to be achieved to help inclusion of microarrays in clinical medicine. In this review, a brief history of microarrays in biomedical research is provided, including experimental overview, limitations, challenges and future developments.

Animals↗
PubMed2004

Inhibition of bcr-abl and/or c-abl gene expression by small interfering, double-stranded RNAs: cross-talk with cell proliferation factors and other oncogenes.

BACKGROUND: Short, 21-mer, double-stranded/small interfering RNAs (ds/siRNAs) were designed to target bcr-abl mRNA in chronic myelogenous leukemia (CML) with a potential also to target c-abl mRNA. METHODS: ds/siRNAs were transfected into bcr-abl-positive K-562 cells (derived from blast-crisis) or bcr-abl-negative/c-abl-positive Jurkat cells (derived from acute lymphoblastic leukemia) using lipofectamine. ds/siRNAs intracellular uptake was detected by fluorescent confocal microscopy using fluorescein-labeled ds/siRNAs. The treatment was performed over 6 days with repetitive siRNA transfections. Efficiency of the siRNAs was determined 24 hours after single siRNA transfection and 6 days after repetitive siRNA transfections. RESULTS: Two of the designed ds/siRNAs decreased the target mRNA levels markedly (determined by reverse transcriptase-polymerase chain reaction analysis) and bcr-abl/c-abl oncoproteins (determined by flow cytometry using Fluor-488-labeled, anti-c-abl antibody as well as by Western blot analysis). These sequences also inhibited protein tyrosine kinase activity significantly and suppressed cell proliferation. One of the three selected ds/siRNAs expressed only slight effects on the bcr-abl/c-abl mRNA in K-562 cells (but not on the oncoprotein level), on protein tyrosine kinase activity, and on cell proliferation. The combination of the three ds/siRNA constructs provoked stronger decreases in bcr-abl/c-abl mRNAs and their respective oncoproteins and produced the strongest suppression of cell proliferation. CONCLUSIONS: The cross-talk between siRNA interference of bcr-abl oncogene and the expression of several apoptotic/antiapoptotic factors, cell proliferation factors, and other oncogenes exists and it was determined by microarray analysis in K-562 cells that were treated over 6 days.

Base Sequence↗
PubMed2004

Enhancement of the photoluminescence of CdSe quantum dots during long-term UV-irradiation: privilege or fault in life science research?

The present study describes an impressive enhancement of the photoluminescence (PL) intensity of low-temperature synthesized CdSe nanocrystals (75 degrees C) during long-term UV-irradiation. The integrated PL-intensity of CdSe core and CdSe/ZnS core/shell nanocrystals, dispersed in chloroform, enhanced about 3 and 6 times, respectively, during 9 h exposure to UV-light, without any significant changes in the characteristic absorbance spectra and shifting of PL-spectra. After termination of the irradiation a comparatively slow photobleaching was detected with tau(1/2) = 6 h for CdSe core and tau(1/2) = 14 h for CdSe/ZnS core/shell nanocrystals. The most impressive was the effect of UV-irradiation on the photoluminescence of water-soluble CdSe nanocrystals. The integrated PL-intensity enhanced about 10 times during 11 h exposure to UV-light and the improved PL-intensity was preserved during 3 days after termination of the irradiation without any significant photobleaching. The results are discussed in the context of application of CdSe nanocrystals as novel fluorophores in life science experiments.

Biological Science Disciplines↗