Preliminary investigation into implication of a single cell organism in fish feed bouyancy and flotation

Two Isocaloric Isoproteic 30% crude protein diets were formulated for Clariid catfish and Tilapia with wheat grain starch (WGS) and cassava tuber starch (CTS) incorporated at 10 percent as binding agents. Saccharomyces cerevisiae was included at 2% as floating additive. The water stability, nutrient retention and flotation of pelleted feeds were observed for 60 minutes. There were generally decreasing trends in stability and retention at increasing time of immersion in water. The lipid retention was higher (P>0.05) than proteins in both diets. WGS diet was better (P>0.05) than CTS diet in flotation, which has attributed to the presence of gluten protein in wheat products. It is envisaged that a break through in floating feed development in Nigeria aquaculture would save the Nigeria economy from extruded (floating) feed importation

Sensitivity map of laser tweezers Raman spectroscopy for single-cell analysis of colorectal cancer

Raman spectroscopy on single, living epithelial cells captured in a laser trap is shown to have diagnostic power over colorectal cancer. This new single-cell technology comprises three major components: primary culture processing of human tissue samples to produce single-cell suspensions, Raman detection on singly trapped cells, and diagnoses of the cells by artificial neural network classifications. it is compared with DNA flow cytometry for similarities and differences. Its advantages over tissue Raman spectroscopy are also discussed. In the actual construction of a diagnostic model for colorectal cancer, real patient data were taken to generate a training set of 320 Raman spectra and, a test set of 80. By incorporating outlier corrections to a conventional binary neural classifier, our network accomplished significantly better predictions than logistic regressions, with sensitivity improved from 77.5% to 86.3% and specificity improved from 81.3% to 86.3% for the training set and moderate improvements for the test set. Most important, the network approach enables a sensitivity map analysis to quantitate the relevance of each Raman band to the normal-to-cancer transform at the cell level. Our technique has direct clinic applications for diagnosing cancers and basic science potential in the study of cell dynamics of carcinogenesis. (C) 2007 Society of Photo-Optical Instrumentation Engineers.

Sensitivity map of laser tweezers Raman spectroscopy for single-cell analysis of colorectal cancer

Raman spectroscopy on single, living epithelial cells captured in a laser trap is shown to have diagnostic power over colorectal cancer. This new single-cell technology comprises three major components: primary culture processing of human tissue samples to produce single-cell suspensions, Raman detection on singly trapped cells, and diagnoses of the cells by artificial neural network classifications. it is compared with DNA flow cytometry for similarities and differences. Its advantages over tissue Raman spectroscopy are also discussed. In the actual construction of a diagnostic model for colorectal cancer, real patient data were taken to generate a training set of 320 Raman spectra and, a test set of 80. By incorporating outlier corrections to a conventional binary neural classifier, our network accomplished significantly better predictions than logistic regressions, with sensitivity improved from 77.5% to 86.3% and specificity improved from 81.3% to 86.3% for the training set and moderate improvements for the test set. Most important, the network approach enables a sensitivity map analysis to quantitate the relevance of each Raman band to the normal-to-cancer transform at the cell level. Our technique has direct clinic applications for diagnosing cancers and basic science potential in the study of cell dynamics of carcinogenesis. (C) 2007 Society of Photo-Optical Instrumentation Engineers.

Development of Microfluidic Devices with the Use of Nanotechnology to Aid in the Analysis of Biological Systems Including Membrane Protein Separation, Single Cell Analysis, and Genetic Markers

Computation technology has dramatically changed the world around us; you can hardly find an area where cell phones have not saturated the market, yet there is a significant lack of breakthroughs in the development to integrate the computer with biological environments. This is largely the result of the incompatibility of the materials used in both environments; biological environments and experiments tend to need aqueous environments. To help aid in these development chemists, engineers, physicists and biologists have begun to develop microfluidics to help bridge this divide. Unfortunately, the microfluidic devices required large external support equipment to run the device. This thesis presents a series of several microfluidic methods that can help integrate engineering and biology by exploiting nanotechnology to help push the field of microfluidics back to its intended purpose, small integrated biological and electrical devices. I demonstrate this goal by developing different methods and devices to (1) separate membrane bound proteins with the use of microfluidics, (2) use optical technology to make fiber optic cables into protein sensors, (3) generate new fluidic devices using semiconductor material to manipulate single cells, and (4) develop a new genetic microfluidic based diagnostic assay that works with current PCR methodology to provide faster and cheaper results. All of these methods and systems can be used as components to build a self-contained biomedical device.

Technology for Single Cell Protein Analysis in Immunology and Cancer Prognostics

The first chapter of this thesis deals with automating data gathering for single cell microfluidic tests. The programs developed saved significant amounts of time with no loss in accuracy. The technology from this chapter was applied to experiments in both Chapters 4 and 5.

The second chapter describes the use of statistical learning to prognose if an anti-angiogenic drug (Bevacizumab) would successfully treat a glioblastoma multiforme tumor. This was conducted by first measuring protein levels from 92 blood samples using the DNA-encoded antibody library platform. This allowed the measure of 35 different proteins per sample, with comparable sensitivity to ELISA. Two statistical learning models were developed in order to predict whether the treatment would succeed. The first, logistic regression, predicted with 85% accuracy and an AUC of 0.901 using a five protein panel. These five proteins were statistically significant predictors and gave insight into the mechanism behind anti-angiogenic success/failure. The second model, an ensemble model of logistic regression, kNN, and random forest, predicted with a slightly higher accuracy of 87%.

The third chapter details the development of a photocleavable conjugate that multiplexed cell surface detection in microfluidic devices. The method successfully detected streptavidin on coated beads with 92% positive predictive rate. Furthermore, chambers with 0, 1, 2, and 3+ beads were statistically distinguishable. The method was then used to detect CD3 on Jurkat T cells, yielding a positive predictive rate of 49% and false positive rate of 0%.

The fourth chapter talks about the use of measuring T cell polyfunctionality in order to predict whether a patient will succeed an adoptive T cells transfer therapy. In 15 patients, we measured 10 proteins from individual T cells (~300 cells per patient). The polyfunctional strength index was calculated, which was then correlated with the patient's progress free survival (PFS) time. 52 other parameters measured in the single cell test were correlated with the PFS. No statistical correlator has been determined, however, and more data is necessary to reach a conclusion.

Finally, the fifth chapter talks about the interactions between T cells and how that affects their protein secretion. It was observed that T cells in direct contact selectively enhance their protein secretion, in some cases by over 5 fold. This occurred for Granzyme B, Perforin, CCL4, TNFa, and IFNg. IL- 10 was shown to decrease slightly upon contact. This phenomenon held true for T cells from all patients tested (n=8). Using single cell data, the theoretical protein secretion frequency was calculated for two cells and then compared to the observed rate of secretion for both two cells not in contact, and two cells in contact. In over 90% of cases, the theoretical protein secretion rate matched that of two cells not in contact.

Towards in situ Single Cell Systems Biology

Systems-level studies of biological systems rely on observations taken at a resolution lower than the essential unit of biology, the cell. Recent technical advances in DNA sequencing have enabled measurements of the transcriptomes in single cells excised from their environment, but it remains a daunting technical problem to reconstruct in situ gene expression patterns from sequencing data. In this thesis I develop methods for the routine, quantitative in situ measurement of gene expression using fluorescence microscopy.

The number of molecular species that can be measured simultaneously by fluorescence microscopy is limited by the pallet of spectrally distinct fluorophores. Thus, fluorescence microscopy is traditionally limited to the simultaneous measurement of only five labeled biomolecules at a time. The two methods described in this thesis, super-resolution barcoding and temporal barcoding, represent strategies for overcoming this limitation to monitor expression of many genes in a single cell. Super-resolution barcoding employs optical super-resolution microscopy (SRM) and combinatorial labeling via-smFISH (single molecule fluorescence in situ hybridization) to uniquely label individual mRNA species with distinct barcodes resolvable at nanometer resolution. This method dramatically increases the optical space in a cell, allowing a large numbers of barcodes to be visualized simultaneously. As a proof of principle this technology was used to study the S. cerevisiae calcium stress response. The second method, sequential barcoding, reads out a temporal barcode through multiple rounds of oligonucleotide hybridization to the same mRNA. The multiplexing capacity of sequential barcoding increases exponentially with the number of rounds of hybridization, allowing over a hundred genes to be profiled in only a few rounds of hybridization.

The utility of sequential barcoding was further demonstrated by adapting this method to study gene expression in mammalian tissues. Mammalian tissues suffer both from a large amount of auto-fluorescence and light scattering, making detection of smFISH probes on mRNA difficult. An amplified single molecule detection technology, smHCR (single molecule hairpin chain reaction), was developed to allow for the quantification of mRNA in tissue. This technology is demonstrated in combination with light sheet microscopy and background reducing tissue clearing technology, enabling whole-organ sequential barcoding to monitor in situ gene expression directly in intact mammalian tissue.

The methods presented in this thesis, specifically sequential barcoding and smHCR, enable multiplexed transcriptional observations in any tissue of interest. These technologies will serve as a general platform for future transcriptomic studies of complex tissues.

An ATP-dependent mechanism mediates intercellular calcium signaling in bone cell network under single cell nanoindentation

To investigate the roles of intercellular gap junctions and extracellular ATP diffusion in bone cell calcium signaling propagation in bone tissue, in vitro bone cell networks were constructed by using microcontact printing and self-assembled monolayer technologies. In the network, neighboring cells were interconnected through functional gap junctions. A single cell at the center of the network was mechanically stimulated by using an AFM nanoindenter. Intracellular calcium ([Ca2+](i)) responses of the bone cell network were recorded and analyzed. In the untreated groups, calcium propagation from the stimulated cell to neighboring cells was observed in 40% of the tests. No significant difference was observed in this percentage when the intercellular gap junctions were blocked. This number, however, decreased to 10% in the extracellular ATP-pathway-blocked group. When both the gap junction and ATP pathways were blocked, intercellular calcium waves were abolished. When the intracellular calcium store in ER was depleted, the indented cell can generate calcium transients, but no [Ca2+](i) signal can be propagated to the neighboring cells. No [Ca2+](i) response was detected in the cell network when the extracellular calcium source was removed. These findings identified the biochemical pathways involved in the calcium signaling propagation in bone cell networks. Published by Elsevier Ltd.

Pulsating tandem microbubble for localized and directional single-cell membrane poration.

The interaction of laser-generated tandem microbubble (maximum diameter of about 50  μm) with single (rat mammary carcinoma) cells is investigated in a 25-μm liquid layer. Antiphase and coupled oscillation of the tandem microbubble leads to the formation of alternating, directional microjets (with max microstreaming velocity of 10  m/s) and vortices (max vorticity of 350 000  s{-1}) in opposite directions. Localized and directional membrane poration (200 nm to 2  μm in pore size) can be produced by the tandem microbubble in an orientation and proximity-dependent manner, which is absent from a single oscillating microbubble of comparable size and at the same stand-off distance.

Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype

Bacterioplankton of the SAR11 clade are the most abundant microorganisms in marine systems, usually representing 25% or more of the total bacterial cells in seawater worldwide. SAR11 is divided into subclades with distinct spatiotemporal distributions (ecotypes), some of which appear to be specific to deep water. Here we examine the genomic basis for deep ocean distribution of one SAR11 bathytype (depth-specific ecotype), subclade Ic. Four single-cell Ic genomes, with estimated completeness of 55%-86%, were isolated from 770 m at station ALOHA and compared with eight SAR11 surface genomes and metagenomic datasets. Subclade Ic genomes dominated metagenomic fragment recruitment below the euphotic zone. They had similar COG distributions, high local synteny and shared a large number (69%) of orthologous clusters with SAR11 surface genomes, yet were distinct at the 16S rRNA gene and amino-acid level, and formed a separate, monophyletic group in phylogenetic trees. Subclade Ic genomes were enriched in genes associated with membrane/cell wall/envelope biosynthesis and showed evidence of unique phage defenses. The majority of subclade Ic-specfic genes were hypothetical, and some were highly abundant in deep ocean metagenomic data, potentially masking mechanisms for niche differentiation. However, the evidence suggests these organisms have a similar metabolism to their surface counterparts, and that subclade Ic adaptations to the deep ocean do not involve large variations in gene content, but rather more subtle differences previously observed deep ocean genomic data, like preferential amino-acid substitutions, larger coding regions among SAR11 clade orthologs, larger intergenic regions and larger estimated average genome size.

Retinal Pigment Epithelial Cell DNA is Damaged by Exposure to Benzo[a]pyrene, a Constituent of Cigarette Smoke.

This study examined the effect of exogenous benzo[ a ]pyrene (BaP), an important constituent of cigarette smoke, on cultured bovine retinal pigment epithelial (RPE) cells. Evidence is presented for its metabolic conversion into benzo[ a ]pyrene diol epoxide (BPDE) and the consequent formation of potentially cytotoxic nucleobase adducts in DNA. Cultured RPE cells were treated with BaP at concentrations in the range of 0–100 µm. The presence of BaP was found to cause inhibition of cell growth and replication. BaP induced the expression of a phase I drug metabolizing enzyme which was identified as cytochrome P450 1A1 (CYP 1A1) by RT–PCR and by Western blotting. Coincident with the increased expression of CYP 1A1, covalent adducts between the mutagenic metabolite BPDE and DNA could be detected within RPE cells by immunocytochemical staining. Additional support for their formation was afforded by nuclease P1 enhanced 32P-postlabelling assays on cellular DNA. Single-cell gel electrophoresis (comet) assays showed that exposure of RPE cells to BaP rendered them markedly more susceptible to DNA damage induced by broad band UVB or blue light laser irradiation. In the case of UVB, this is consistent with the photosensitization of DNA cleavage by nucleobase adducts of BPDE. Collectively, these findings imply that BaP has a significant impact on RPE cell pathophysiology and suggest mechanisms whereby exposure to cigarette smoke might cause RPE dysfunction and cell death, thus possibly contributing to degenerative disorders of the retina.