Morphological segmentation analysis and texture-based support vector machines classification on mice liver fibrosis microscopic images

Background To reduce the intensity of the work of doctors, pre-classification work needs to be issued. In this paper, a novel and related liver microscopic image classification analysis method is proposed. Objective For quantitative analysis, segmentation is carried out to extract the quantitative information of special organisms in the image for further diagnosis, lesion localization, learning and treating anatomical abnormalities and computer-guided surgery. Methods in the current work, entropy based features of microscopic fibrosis mice’ liver images were analyzed using fuzzy c-cluster, k-means and watershed algorithms based on distance transformations and gradient. A morphological segmentation based on a local threshold was deployed to determine the fibrosis areas of images. Results the segmented target region using the proposed method achieved high effective microscopy fibrosis images segmenting of mice liver in terms of the running time, dice ratio and precision. The image classification experiments were conducted using Gray Level Co-occurrence Matrix (GLCM). The best classification model derived from the established characteristics was GLCM which performed the highest accuracy of classification using a developed Support Vector Machine (SVM). The training model using 11 features was found to be as accurate when only trained by 8 GLCMs. Conclusion The research illustrated the proposed method is a new feasible research approach for microscopy mice liver image segmentation and classification using intelligent image analysis techniques. It is also reported that the average computational time of the proposed approach was only 2.335 seconds, which outperformed other segmentation algorithms with 0.8125 dice ratio and 0.5253 precision

Assembly and Structure of Bacteriophage Capsids and Tails

Phages HK97 and λ are model systems that have aided in the understanding of capsid and tail assembly. HK97 capsid assembly involves the formation of a T=7 icosahedral lattice from 415 monomers of the major capsid protein (mcp), 12 monomers of portal protein, and ~120 copies of the protease. Salt bridges have been found to play a critical role in the assembly pathway, but it is not clear how capsid size is determined. Phage phi1026b is larger than HK97 but the mcp shares 50% sequence similarity with HK97. We hypothesize that comparing the atomic models of phi1026b and HK97 will help illuminate conserved and unique interactions that may play a role in assembly and potentially size determination. By utilizing the FEI Krios microscope, Falcon II camera, and EPU data acquisition software, high-resolution structures were generated which allowed for the generation of atomic models of the phi1026b Prohead I and Head capsids. Differences were observed in the organization of the delta domain, capsomers, and intra-capsomer interactions. The E153-R210 salt bridge that is maintained throughout HK97 capsid assembly is not present between two monomers in the phi1206b Prohead I structure. Phage λ tail assembly proceeds in a stepwise manner in which tail proteins form an initiator complex onto which the major tail tube protein, gpV, polymerizes and is terminated by the tail termination protein, gpU. The tube domain of gpV has been studied in its monomeric form using NMR but the in vivo form has not been visualized. By generating a subnanometer structure for the λ tail tube, we are able to show how gpV undergoes structural changes to facilitate assembly. Also, a putative initiator complex comprised of the tail proteins gpG, gpT, gpH, gpM, gpL, gpI, and gpJ was isolated and characterized using microscopy. Finally, a subnanometer structure for the λ tail tip complex (TTC) is presented providing some initial insight into the organization of the λ TTC. Overall, this research intends to optimize structure determination with the cryo-EM, thereby facilitating studies on the structure and assembly of large viruses

A novel image analysis approach to characterise the effects of dietary components on intestinal morphology and immune system in Atlantic salmon

The intestinal tract of salmonids provides a dynamic interface that not only mediates nutrient uptake but also functions as the first line of defence against ingested pathogens. Exposure of the immune system to beneficial microorganisms and different dietary immunostimulants via the intestine has been shown to prime the immune system and help in the development of immune competence. Furthermore, the morphology and function of teleostean intestines are known to respond to feed components and to ingested and resident bacterial communities. Histological appraisal is still generally considered to be the gold standard for sensitive assessment of the effects of such dietary modulation. The aim of the present study was to improve understanding of salmonid intestinal function, structure and dynamics and to use the knowledge gained to develop a model for analysis, which would allow intestinal health to be assessed with respect to different intestinal communities and feed components. Virtual histology, the process of assessing digital images of histological slides, is gaining momentum as an approach to supplement traditional histological evaluation methodologies and at the same time, image analysis of digitised histological sections provides a practical means for quantifiable assessment of structural and functional changes in tissues, being both objective and reproducible. This project focused on the development of a rapid, practical analytical methodology based on advanced image analysis, that was able to measure and characterise a range of features of the intestinal histology of Atlantic salmon in a quantitative manner. In the first research chapter, the development of a novel histological assessment system based upon advanced image analysis was described, this being developed with the help of a soybean feed model known to induce enteropathy in Atlantic salmon. This tool targeted the evaluation of the extent of morphological changes occurring in the distal intestine of Atlantic salmon following dietary modulation. The final analytical methodology arrived at, could be conducted with minimal user-interaction, allowing rapid and objective assessment of 12 continuous variables per histological frame analysed. The processing time required for each histological frame was roughly 20-25 min, which greatly improved the efficiency of conducting such a quantitative assessment with respect to the time taken for a subjective semi-quantitative alternative approach. Significant agreement between the fully automated and the manual morphometric image segmentation was achieved, however, the strength of this quantitative approach was enhanced by the employment of interactive procedures, which enabled the operator / observer to rectify preceding automated segmentation steps, and account for the specimen’s variations. Results indicated that image analysis provided a viable alternative to a pathologist’s manual scoring, being more practical and time-efficient. In the second research chapter, feeding Atlantic salmon a high inclusion level of unrefined SBM (25 %) produced an inflammatory response in the distal intestine as previously described by other authors. The model feed trial successfully generated differentiable states, although these were not, for the most part, systemically differentiable through the majority of standard immunological procedures used, being only detectable morphologically. Quantitation of morphometric parameters associated with histological sections using the newly developed image analysis tool successfully allowed identification of major morphological changes. Image analysis was thus shown to provide a powerful tool for describing the histomorphological structure of Atlantic salmon distal intestine. In turn, the semi-automated image analysis methods were able to distinguish normal intestinal mucosa from those affected by enteritis. While individual parameters were less discriminatory, use of multivariate techniques allowed better discrimination of states and is likely to prove the most productive approach in further studies. Work described in the third research chapter sought to validate the semi-automated image analysis system to establish that it was measuring the parameters it was purported to be measuring, and to provide reassurance that it could reliably measure pre-determined features. This study, using the same sections for semi-quantitative and quantitative analyses, demonstrated that the quantitative indices performed well when compared to analogous semi-quantitative descriptive parameters of assessment for enteritis prognosis. The excellent reproducibility and accuracy performance levels indicated that the image analysis system was a useful and reliable morphometric method for the quantification of SB-induced enteritis in salmon. Other characteristics such as rapidity, simplicity and adaptability favour this method for image analysis, and are particularly useful where less experienced interpreters are performing the analysis. The work described in the fourth research chapter characterised changes in the morphology of the intestinal epithelial cells occurring as a result of dietary modulation and aspects of inflammatory infiltration, using a selected panel of enzyme and IHC markers. To accomplish this, image analysis techniques were used to evaluate and systematically optimise a quantitative immunolabelling assessment protocol. Digital computer-assisted quantification of labelling for cell proliferation and regeneration; programmed cell death or apoptosis; EGCs and t-cell like infiltrates; mobilisation of stress-related protein regenerative processes and facilitation of nutrient uptake and ion transport provided encouraging results. Through the description of the intestinal cellular responses at a molecular level, such IHC expression profiling further characterised the inflammatory reaction generated by the enteropathic diet. In addition, a number of potential diagnostic parameters were described for fish intestinal health e.g. the relative levels of antigenicity and the spatial distribution of antigens in tissues. Work described in the final research chapter focused on detailed characterisation of intestinal MCs / EGCs in order to try to elucidate their functional role in the intestinal immune responses. Through an understanding of their distribution, composition and ultrastructure, the intention was to better characterise these cells and their functional properties. The general morphology, histochemical characteristics and tissue distribution of these cells were explored in detail using histochemical, IHC and immunogold staining / labelling, visualised using light, confocal and TEM microscopy. Despite these extensive investigations, their physiological function and the content of their granules still remain somewhat obscure, although a role as immunodulatory cells reacting to various exogeneous signals through a finely regulated process and comparable to that causing the degranulation of mammalian MCs is suggested. The histochemical staining properties demonstrated for salmonid MCs / EGCs seem to resemble those of mammalian mucosal mast cells, with both acidophilic and basophilic components in their granules, and a granule content containing neuromodulator / neurotransmitter-peptides such as serotonin, met-enkephalin and substance-p. Consequently, distinguishable bio-chromogenic markers have been identified that are of utility in generating a discriminatory profile for image analysis of such cells

Genomic comparison of novel Staphylococcus aureus bacteriophage and their anti-biofilm properties against MRSA sequence type 22 and 36

Staphylococcus aureus (including methicillin-resistant S. aureus - MRSA) remains a leading cause of both nosocomial and community acquired infections globally and despite constant improvement efforts to patient safety within healthcare, it still remains associated with considerable rates of morbidity and mortality. S. aureus is a common cause of biofilm-associated infections observed in chronic wounds, exhibiting a reduced susceptibility to the action of conventional antimicrobial agents and are often difficult to eradicate. The acquisition of resistance to almost any antibiotic with reference to MRSA has greatly reduced the number of alternative antimicrobial agents effective in the treatment of infections. Current development pipeline for new classes of antibiotics are greatly limited, requiring new, alternative approaches for therapeutic and prophylactic intervention in attempt to effectively control and overcome this current global health threat. Bacteriophage therapy exploits the natural killing ability of lytic bacteriophage (phage) as a means of controlling multidrug-resistant pathogenic bacteria. The utility of phage and their derivatives has been shown to effectively reduced the biofilms of major MRSA clones in vitro and in vivo. Global MRSA infections are caused by highly-successful isolates from a small number of epidemic lineages (clones). ST22 and ST36 are two of the most prevalent clones with global impact and largely responsible for the national epidemic of MRSA infections within UK healthcare system throughout the mid-1990s up until the mid-2000s. Understanding the phenotypic and genotypic characteristics of these clones in relation to the ability of bacteriophage to infect and disrupt established biofilms has yet to be explored. In this study, a total of 46 novel obligately lytic phage were isolated from wastewater samples by utilising a modified Staphylococcus carnosus TM300 isolate with expressed S. aureus wall teichoic acids to aid in phage adsorption. The addition of 32 more phage from our current laboratory stocks helped to establish a collection of 78 phage that were screened against a panel of 185 genetically diverse S. aureus, consisting of major clonal groups with high prevalence within the UK and United States, including 43 ST22 and 24 ST36 strains. The majority of the members displayed a wide host range against our panel. Based on this, the four most effective (wide host-range) phage were assessed for their anti-biofilm properties in polystyrene plates biofilm assays produced using four ST22 and four ST36 isolates. Treatment of mature biofilms was shown to significantly reduce biofilm biomass and viable cell counts. However these assays selected for the emergence of phage resistant mutants. Whole genome sequencing was performed on 22 phage isolates and these were found to share a high degree of similarity to genomes of 38 previously classified Twortvirinae phages represented in GenBank. Comparisons of these 60 phage genomes found a surprisingly high level of genetic diversity. Pairwise distances resolved groups of phage in distinct clusters representing individual genera within the Twortvirinae subfamily. Pan-genome analysis identified no single gene present amongst all phage genomes, however phage displayed a core genome amongst other members of the cluster. The structural homology tool HHpred was used to predict the protein structure of genes encoding for lytic enzymes among our phage genomes. We found that all phage encode a protein that shares high structural similarity to the same CHAP domain protein, a catalytic domain of endolysins employed by phage to degrade the bacterial host cell wall in order thus, mediate cell lysis. Suggesting that the all phage most likely share the same catalytic N-terminal endopeptidase domain of endolysins which have a modular domain structure. Interestingly, endolysins have been proposed as possible candidates for the control of antibiotic resistant S. aureus infections

Diversity and Activity of Roseobacters and Roseophage

Bacteria of the Roseobacter lineage are dominant bacterioplankton in coastal systems and contribute significantly to secondary production in oceanic environments. Generalities of Roseobacter ecology, diversity, and distributions are known, but the intraspecific differences between species and their dynamics over short temporal periods is not well understood. Bacteriophage that infect Roseobacters (‘roseophage’) have the potential to shunt secondary production into the dissolved carbon pool and through the process of infection alter Roseobacter physiology. Despite their significance, little effort was made prior to the onset of this study to characterize roseophage. Using culture dependent and independent approaches, I describe the diversity and activity of Roseobacters and roseophage from two distinct coastal environments. Chapter 2 describes the development of an alternative method to enumerate viruses using epifluorescence microscopy that not only reduces sample processing costs, but also the total volume of sample required. A novel species of the Roseobacter lineage (Marivita roseacus) is proposed in Chapter 3. M. roseacus is unique in its needle-like morphology, forming long, relatively inflexible chains of cells. The Marivita genus is characterized by a distinct ecology, being closely associated with algae, resistant to grazing, and present in numerous marine and saline environments. Chapter 4 details the use of deep-amplicon sequencing (16S rDNA) to describe bacterial succession patterns during a mesocosm algal bloom, revealing the temporal dynamics of ~100 distinct phylotypes. A multivariate analysis showed that temporal portioning amongst the bacterial community was occurring at both high and low taxonomic levels. Chapter 5 details the isolation and genomic characterization of roseophage and describes their ecology using publically available metagenomic databases collected from throughout the world. Four distinct phage were isolated and sequenced including an N4-like strain, a novel Siphoviridae, and two temperate Podoviridae. The two temperate phage were practically identical at the nucleotide level, except for a 3000 bp putative replication module, which showed no homology between the two. Overall, this dissertation suggests that ecological partitioning within the Roseobacter lineage is occurring at and arguably below traditional species level taxonomic classifications and microdiversity amongst closely related marine bacteria is likely the norm rather than the exception

Advanced Instrumentation and Methodology Related to Cryoultramicrotomy: A Review

This review is concerned with the considerable progress in the field of cryo-ultramicrotomy (cryofixation, cryosectioning, investigation and analysis of cryosections) during recent years. This progress includes both more efficient instrumentation and methodology. The article is mainly directed to the investigation and analysis of frozen-hydrated sections in the low dose cryo-transmission electron microscopy (TEM) and cryo-energy filtered TEM (EFTEM). A general survey is followed by an evaluation of the different relevant procedures. Both cryo-ultramicrotomy for macromolecular cytochemistry (Tokuyasu technique) and cryo-ultramicrotomy for element analysis are only shortly mentioned without discussion of the chemical and analytical approach. Because of lack of first hand experience, cryo-sectioning for X-ray microanalysis in the frozen-hydrated state according to Hall and Gupta is not included into this review. The methods and instruments required for ultrathin sectioning at low temperatures are described and discussed in detail. This concerns the preceding cryofixation, the cryosectioning itself with special emphasis to the required stability and precision of the cryo-ultramicrotome, the characteristics of the knives, the charging phenomena due to sectioning and the subsequent TEM investigation including EFTEM with electron spectroscopic imaging (ESI) and the available accessories for digital low dose registration of signals

Defining the mechanism of Yersinia entomophaga MH96 exoprotein release : a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy

There is an increasing need in New Zealand for the development of new solutions and improvement of currently available biological control agents of pasture pests. In the last two decades, the entomopathogen Yersinia entomophaga was isolated, characterised, and defined as an economically viable agent for use in biopesticides against pasture pests, such as the New Zealand grass grub Costelytra giveni, the black beetle Hetreronychus arator, and other insects from the orders Coleoptera and Lepidoptera. Y. entomophaga produces an ABC toxin complex, called the Yen-TC, which is its main orally active virulence factor. Although previous studies have revealed how the toxin is affecting the host, the mechanism for Yen-TC production and cellular release have yet to be fully elucidated. Furthermore, the roles of other virulence factors of Y. entomophaga and their effects and mechanisms of cellular release are yet to be determined. In silico analysis of the draft genome sequence revealed several gene clusters encoding for virulence factors such as Rhs with predicted function in the type 6 secretion system, YenT as a possible heat-stable enterotoxin, and PirAB, a haemocoelic-active toxin. This study aimed to identify regulators of exoproteome release in Y. entomophaga MH96. The Yersinia entomophaga region of exoproteome release (YeRER) was identified by an exoproteome screening assay developed in this study. The YeRER is comprised of the transcriptional regulator RoeA, a regulatory ncRNA, and the Yersinia lysis cassette (YLC). Transcriptomics, mutagenesis, and trans complementation experiments were used to elucidates the importance of YeRER in Y. entomophaga protein release and changes in cell morphology. The global regulator, RoeA, strictly down-regulates the YLC expression which is involved in protein release and vesicle formation. Expression levels of the encoded secretion systems in MH96, T1SS, T3SS, T3SS2, and T6SS are not under the control of YeRER or quorum sensing (QS), which are involved in protein secretion. The T2SS expression is increased in roeA and QS mutants, which in turn showed reduced global exoproteome concentration. It is unlikely that the MH96 secretion systems are involved in MH96 exoproteome production in vitro. While underlying mechanisms have yet to be investigated, this study strongly suggests that exoproteins such as the Yen-TC are secreted by membrane vesicles which are induced by activation of a holin-endolysin complex

Optimization and Application of Chromosome In Situ Suppression Hybridization

The overall aim of this project was to develop the technique of chromosomal in situ suppression (CISS) hybridization using whole chromosome specific libraries (chromosome painting) and to apply it to the investigation of diagnostic problems in clinical cytogenetics. Initially to gain experience with non-isotopic in situ hybridization, repetitive target probes DYS59 (GMY10) and DYS58 (GMGY7) were used. This provided experience in labelling of probe with biotin, hybridization and detection conditions (alkaline phosphatase detection) and analysis of results. The technique was reliable and sensitive and was applied to map the gene for angiotensinogen to 1q42. In the later part of this initial work, a fluorescence detection technique using fluoresceinated avidin and goat biotinylated anti-avidin was applied to confirm an isochromosomes Yp and Yq using DYS59 (GMGY10) and DYS58 (GMGY7) probes. The study then progressed into the development of the chromosome painting technique. Difficulties were encountered in preparing the working library probe from the chromosome 21 specific library and a major part of the work involved solving these problems. The libraries were found to be less concentrated than indicated by the supplier. Consequently, the amplification and purification following established protocols failed to produce a concentrated DNA library in the phage. However, a good yield of the DNA library was achieved by using trypticase in the culture media and high purity agarose as the top agar during the amplification. Labelling of the library by nick-translation and random priming did not achieve decoration of the whole chromosome 21 but direct labelling of Biotin-ll-dUTP by polymerase chain reaction (PCR) amplification was found to be efficient and overcame the problem of non-homogenous painting of the target chromosome. This direct labelling approach had difficulties in the cleaning and concentration of the PCR product. These were overcome by cleaning with Sephadex G-50 column chromatography and freeze drying of eluate. Once homogeneous painting had been achieved the probe was applied for chromosome painting. Many problems and parameters for the optimum working conditions were identified in this part of study. These are either independent or/and related to various conditions involved during all stages of the technique. The maximum final concentration of the DNA mixture per slide was 10ug/10ul and increasing the ratio of the probe and/or the unlabelled DNA did not improve either the quality of suppression or the hybridization signal. Addition of human cot 1-DNA in 1 to 4 ratio with total human DNA gave better suppression. Denaturation of labelled probe and competitor DNA mixture was optimum at 75C for 8 minutes and for optimum preannealling, the mixture was prehybridized for a minimum of 60 minutes at 37C. Slides were only treated with RNase when necessary and not with Proteinase K as the latter tended to wash the cells off the slide. Denaturation of the slides was carried out at 70-75C in 70% formamide/2XSSC for a maximum of 8 minutes. At temperature of 80C the chromosome morphology was found to be distorted. Hybridization when carried out at 37C for 15 to 20 hours showed good hybridization with chromosome morphology undisturbed. Hybridization at 42-45C showed crystallization and heavy background deposits. Posthybridization washing in three changes of 50% formamide/2XSSC at 45C was found to be optimal in producing a clean background. In between detection washing using 0.1M sodium phosphate buffer with 0.1% Nonidet P-40 carried out at room temperature is sufficient to remove excess stain as compared to other washing buffers such as 2XSSC or 4XSSC containing Triton-X or Tween 20. Detection was carried out at room temperature for 15-20 minutes and any slide dried during this stage produced high autofluorescence of fluoresceinated avidin which was difficult to remove by washing. A single amplification cycle was sufficient to enhance the decoration of chromosome 21. Prebanding of slides prior to hybridization did not affect the target chromosomes, however, incomplete destaining did hinder probe penetration and interfere with counterstaining. It was found that refixing of slides (either new or old slides) in methanol:acetic acid (3:1) before denaturation tended to improve the hybridization result as well as reducing background signal. In general, the technical difficulties were related to either probe preparation, poor hybridization, non-homogeneous painting or high background but with modifications of the parameters as detailed above the method was shown to be reliable and reproducible. Chromosomes obtained from phytohaemagglutinin (PHA) stimulated blood cultures were used during the initial phase. Subsequently, painting was successfully performed on cytogenetically normal metaphase and prometaphase samples of cultured amniocytes, lymphoblastoid cell lines, chorionic villus samples (CVS) and bone marrow preparations. The results showed that all normal chromosome 21s in all types of preparation except direct chorionic villus sample (CVS) were intensely painted and distinctly recognisable. However, results with interphase nuclei were not encouraging. The signals produced were not consistent enough to produce as reliable results. Twelve cases with cytogenetic abnormalities involving the chromosome 21 were investigated using chromosome painting. These results proved that chromosome painting can be used for rapid identification of individual chromosomes and is complementary and confirmatory to conventional karyotyping and as such is predicted to have a future routine diagnostic role in clinical cytogenetics in additions to its research applications

Structural investigation of two viral proteins involved in DNA-packaging

Viral DNA-packaging motors are molecular machines that enable viruses to replicate by providing the means of storing the viral genome into empty procapsids. In double stranded DNA bacteriophages the molecular motor is comprised by three elements: the portal protein and the small and large terminases. The portal protein nucleates polymerisation of the capsid and scaffold proteins, initiating procapsid assembly, besides allowing passage of DNA. The small terminase recognises the viral genome and presents it to the large terminase which possesses both nuclease activity to cleave concatemeric DNA at the initiation of the packaging, and ATPase activity to drive translocation. Although currently several X-ray structures for the different components of the motor from different bacteriophages are available fundamental questions regarding the DNA recognition mechanism, stoichiometry and orientation of the motor components in vivo and the mechanism of ATP-driven DNA-translocation remain. This project focused on elucidating the X-ray crystal structures of (i) the major capsid protein, from Bacillus subtilis bacteriophage SPP1 and (ii) the small terminase protein from Thermus thermophilus bacteriophage G20C. Several constructs of the SPP1 capsid protein, including truncations at the N- and C-termini, single and double mutants and engineered proteins were generated in order to produce a protein suitable for crystallisation. Mutant uG13P;T104Y;A261W was the only construct that produced native and Se-Met crystals. The X-ray structure of the SPP1 capsid protein was determined at 3.0 Å resolution by single wavelength anomalous diffraction. The structure exhibited the HK97-fold consisting of the axial and peripheral domains and the extended E-loop. The X-ray structure of the small terminase from phage G20C was solved at 2.5 Å resolution by single wavelength anomalous diffraction. The structure consisted of circular nine-mers where the N- terminal domains of each subunit reside at the periphery of the assembly and the C-terminal oligomerisation domains form a central channel. The conserved structural features between small terminases suggest that the DNA-recognition mechanism might be conserved. DNA-binding experiments demonstrated that the G20C small terminase binds to viral and non-viral DNA with both the N- and C- terminal domains playing an important role. The structural information generated from these two elements of SPP1 and G20C bacteriophages provides insight into two different aspects of the assembly process: (i) how the capsid protein’s conformational plasticity may assist the assembly and (ii) DNA-recognition during virus particle construction. This information is critical for understanding similar processes in other viruses, in particular, in the evolutionarily related herpes viruses