Alexandra Alimova

Bacteria size determination by elastic light scattering

Light extinction and angular scattering measurements were performed on three species of bacteria with different sizes and shapes ( Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis). The Gaussian ray approximation of anomalous diffraction theory was used to determine the average bacteria size from transmission measurements. A rescaled spectra combining multiple angular data was analyzed in the framework of the Rayleigh-Gans theory of light scattering. Particle shape and size distribution is then obtained from the rescale spectra. Particle characteristics (size and/or shape) retrieved from both methods are in good agreement with size and shape measured under scanning electron microscopy. These results demonstrate that light scattering may be able to detect and identify microbial contamination in the environment.

Native fluorescence and excitation spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria subjected to conditions of starvation

Fluorescence emission and excitation spectra were measured over a 7-day period for Bacillus subtilis (Bs), a spore-forming, and Staphylococcus aureus (Sa), a nonspore-forming bacteria subjected to conditions of starvation. Initially, the Bs fluorescence was predominantly due to the amino acid tryptophan. Later, a fluorescence band with an emission peak at 410 nm and excitation peak at 345 m, from dipicolinic acid, appeared. Dipicolinic acid is produced during spore formation and serves as a spectral signature for detection of spores. The intensity of the 410-nm band continued to increase over the next 3 days. The Sa fluorescence was predominantly from tryptophan and did not change over time. In 6 of the 17 Bs specimens studied, an additional band appeared with a weak emission peak at 460 cm and excitation peaks at 250, 270, and 400 nm. The addition of beta-hydroxybutyric acid to the Bs or the Sa cultures resulted in a two-order of magnitude increase in the 460-nm emission. The addition of Fe2+ quenched the 460 emission, indicating that a source of the 460-nm emission was a siderophore produced by the bacteria. We demonstrate that optical spectroscopy-based instrumentation can detect bacterial spores in real time.

Zika virus-like particle (VLP) based vaccine

The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development.

In vivo molecular evaluation of guinea pig skin incisions healing after surgical suture and laser tissue welding using Raman spectroscopy.

The healing process in guinea pig skin following surgical incisions was evaluated at the molecular level, in vivo, by the use of Raman spectroscopy. After the incisions were closed either by suturing or by laser tissue welding (LTW), differences in the respective Raman spectra were identified. The study determined that the ratio of the Raman peaks of the amide III (1247 cm(-1)) band to a peak at 1326 cm(-1) (the superposition of elastin and keratin bands) can be used to evaluate the progression of wound healing. Conformational changes in the amide I band (1633-1682 cm(-1)) and spectrum changes in the range of 1450-1520 cm(-1) were observed in LTW and sutured skin. The stages of the healing process of the guinea pig skin following LTW and suturing were evaluated by Raman spectroscopy, using histopathology as the gold standard. LTW skin demonstrated better healing than sutured skin, exhibiting minimal hyperkeratosis, minimal collagen deposition, near-normal surface contour, and minimal loss of dermal appendages. A wavelet decomposition-reconstruction baseline correction algorithm was employed to remove the fluorescence wing from the Raman spectra.

BACTERIA-CLAY INTERACTION: STRUCTURAL CHANGES IN SMECTITE INDUCED DURING BIOFILM FORMATION

Bacteria play an important role in determining the properties and behavior of clay minerals in natural environments and such interactions have great potential for creating stable biofilms and carbon storage sites in soils, but our knowledge of these interactions are far from complete. The purpose of this study was to understand better the effects of bacteria-generated biofilms on clay interlayer expansion. Mixtures of a colloidal, 2-water hectorite clay and Pseudomonas syringae in a minimal media suspension evolve into a polysaccharide-rich biofilm aggregate in time-series experiments lasting up to 1 week. X-ray diffraction analysis reveals that upon aggregation, the clay undergoes an initial interlayer contraction. Short-duration experiments, up to 72 h, result in a decrease in the d001 value from 1.50 to 1.26 nm. The initial interlayer contraction is followed in long-duration (up to 1 week) experiments by an expansion of the d001 value of 1.84 nm. The expansion is probably a result of large, biofilm-produced, polymeric molecules being emplaced in the interlayer site. The resultant organo-clay could provide a possible storage medium for carbon in a microbial colony setting.

In situ determination of refractive index and size of Bacillus spores by light transmission

Light-extinction measurements in the wavelength range of 400 to 1000 nm are performed in situ on Bacillus subtilis endospores during heat-shock-induced activation. Simultaneous information on particle size and refractive indices during activation is calculated from the transmission spectra by use of the Gaussian ray approximation of anomalous diffraction theory. During activation the refractive index of the core decreases from 1.51 to 1.39, and the size increases from 0.38 to 0.6 microm.

Hybrid phosphorescence and fluorescence native spectroscopy for breast cancer detection

Fluorescence and phosphorescence measurements are performed on normal and malignant ex vivo human breast tissues using UV LED and xenon lamp excitation. Tryptophan (trp) phosphorescence intensity is higher in both normal glandular and adipose tissue when compared to malignant tissue. An algorithm based on the ratio of trp fluorescence intensity at 345 nm to phosphorescence intensity at 500 nm is successfully used to separate normal from malignant tissue types. Normal specimens consistently exhibited a low I(345)I(500) ratio (<10), while for malignant specimens, the I(345)I(500) ratio is consistently high (>15). The ratio analysis correlates well with histopathology. Intensity ratio maps with a spatial resolution of 0.5 mm are generated in which local regions of malignancy could be identified.

Proteins and dipicolinic acid released during heat shock activation of Bacillus subtilis spores probed by optical spectroscopy

UV fluorescence and absorption spectroscopy from Bacillus subtilis spores detected proteins and dipicolinic acid (DPA) released into the supernatant after heat treatments ranging from 20 degrees to 90 degrees C. The protein and DPA concentration in the supernatant was greater with higher heat treatment temperatures, undergoing a substantial increase for T > or = 60 degrees C, and supporting the theory that spores undergo a phase transition from a glassylike to a rubberylike state at 56 degrees C. Gel electrophoresis detected several small proteins with molecular weights between 6 and 11 kDa. These proteins may be small acid-soluble spore proteins that are present in spores but break down during germination. A 30 kDa protein extracted above 60 degrees C is related to the rubber-glass phase transition.

Effects of smectite clay on biofilm formation by microorganisms

We have investigated the role of smectite clay particles in biofilm formation by several different species of bacteria ( Pseudomonas syringae, Escherichia coli , Staphylococcus aureus and Bacillus subtilis ). We observed that the presence of clay particles enhances the formation of biofilms and, after 24 h, the bacterial populations in the clay mixtures were greater than the respective populations in media without clay. Smectite-bearing clay slurries uniformly develop bacteria–clay aggregates with a substantial biofilm component within 24 h, while the exclusively bacterial suspensions do not develop any observable biofilm component. The biofilm–clay aggregates vary in size from tens of micrometers to several millimeters. Biofilm formation was evaluated by phase contrast microscopy and fluorescence staining. Biofilm promotion by smectite clays may indicate the importance of transport of bacteria by aerosol dust particles.

Morphology of Influenza B/Lee/40 Determined by Cryo-Electron Microscopy

Cryo-electron microscopy projection image analysis and tomography is used to describe the overall architecture of influenza B/Lee/40. Algebraic reconstruction techniques with utilization of volume elements (blobs) are employed to reconstruct tomograms of this pleomorphic virus and distinguish viral surface spikes. The purpose of this research is to examine the architecture of influenza type B virions by cryo-electron tomography and projection image analysis. The aims are to explore the degree of ribonucleoprotein disorder in irregular shaped virions; and to quantify the number and distribution of glycoprotein surface spikes (hemagglutinin and neuraminidase) on influenza B. Projection image analysis of virion morphology shows that the majority (∼83%) of virions are spherical with an average diameter of 134±19 nm. The aspherical virions are larger (average diameter = 155±47 nm), exhibit disruption of the ribonucleoproteins, and show a partial loss of surface protein spikes. A count of glycoprotein spikes indicates that a typical 130 nm diameter type B virion contains ∼460 surface spikes. Configuration of the ribonucleoproteins and surface glycoprotein spikes are visualized in tomogram reconstructions and EM densities visualize extensions of the spikes into the matrix. The importance of the viral matrix in organization of virus structure through interaction with the ribonucleoproteins and the anchoring of the glycoprotein spikes to the matrix is demonstrated.