Desiccation tolerance

Despite the fundamental significance of desiccation in determining the distributions and activities of living organisms, there is virtually no insight as to the state of the cytoplasm of an air-dried, or even a wet, cell. In bacterial cells that have been subjected to air-drying the evaporation of free cytoplasmic water (Vf) can be instantaneous, and an equilibrium between cell-bound water (VQ and the environmental water (vapor) potential (Ψwv) may be achieved very rapidly. In the air-dried state some bacteria survive only for seconds, others can tolerate desiccation for thousands, perhaps for millions, of years. The means by which certain cells, the anhydrobiotes, overcome and then tolerate acute water deficit remains one of the most intractable problems in cell biology. One such anhydrobiote, the cyanobacterium Nostoc commune, is cosmopolitan, its colonies form visually-conspicuous and abundant growths in situ, and it constitutes an ecologically-significant component of terrestial nitrogen-fixing communities. The cyanobacteria are phylogenetically-significant organisms that provide model systems for the study of a broad range of problems in cell biology. The studies described in this thesis established the molecular ecology and cell biology of Nostoc commune, and they provide a chronicle of the development of this microorganism as the prokaryotic model for the anhydrobiotic cell. In the design of experiments to investigate this problem the bias was, and remains, this: to understand desiccation tolerance, understand an organism that tolerates desiccation. The thesis documents an investigation into the consequences of acute cell-water deficit and the cellular basis for desiccation tolerance. An eclectic approach has been adopted to study desiccation tolerance and it includes the application of techniques of cell biology, biochemistry, microbiology, molecular biology, structural biology and biophysics

Review on Nematode Molecular Diagnostics: From Bands to Barcodes

Molecular methods of identification provide accurate, reliable diagnostic approaches for the identification of plant-parasitic nematodes. The promising and attractive results have generated increasing demands for applications in new fields and for better performing techniques. Initially, the techniques were used solely for taxonomic purposes, but increasingly became popular as a component of diagnostic information. Diagnostic procedures are now available to differentiate the plant-pathogenic species from related but non-pathogenic species. The microscopic size of plant parasitic nematodes poses problems and techniques have been developed to enrich samples to obtain qualitative and quantitative information on individual species. In addition, techniques are available to evaluate single nematodes, cysts or eggs of individual species in extracts from soil and plant tissue. DNA or RNA-based techniques are the most widely used approaches for identification, taxonomy and phylogenetic studies, although the development and use of other methods has been, and in some cases still is, important. DNA barcoding and the extraction of DNA from preserved specimens will aid considerably in diagnostic information. . In addition, further review is needed to identify all recovered nematode and evaluation of promising treatments for use in integrated disease management strategy to manage not only regulated species such as the potato cyst nematodes Globodera rostochiensis and Globodera pallida, and root-knot nematode Meloidogyne. chitwoodi, but also other related nematode diseases of plant. Key Words: Barcoding, Diagnosis, DNA, Molecular, Nematode, RNA

Molecular detection and identification of parasites involved in human disease

The detection and identification of disease-causing parasites via DNA and the polymerase chain reaction (PCR) offers a new approach for quick and efficient disease diagnosis. Three nucleic acid based technologies have been developed and optimized for the identification of six species of parasites that cause disease, and designed to amplify small fragments of DNA likely to remain in archaeological material. Two methods involve a species-specific identification by examining four gene fragments, one of which has been designed as a multiplex PCR to minimize the time required for diagnosis. The third method involves the use of one species-specific gene target per parasite in a multiplex PCR for the detection of multiple parasites in one reaction to expedite the testing process and confirmation of the disease in question in a clinical setting. Sensitivity and specificity have been proven through the use of a 1/1000 diluted DNA template and sequencing of products confirmed the presence o f each species analyzed. With increased sensitivity, a less invasive sampling procedure would be required from the patient. Also, the earlier the presence of parasites in the tissues is detected, the earlier the diagnosis would occur. Tissues that contain very few parasites and had been classified as previously unusable for diagnosis or tissues from older medical cases can be analyzed for the presence of disease and the identification of causative species. The successful application of the singleplex PCR technique to Plasmodium falciparum and Leishmania degraded DNA samples leads to the feasibility of success for the multiplex PCR techniques involving degraded DNA analyses. Therefore, this methodology may be applied to samples of unknown disease content to either include or exclude the agents of disease. Future directions may extend these analyses to include a range of other agents of disease, thus allowing for the determination of disease antiquity through degraded DNA analyses

Ancient DNA in Archaeologically Charred Zea Mays L: Prospects and Limitations

Plant remains are an integral part of any archaeological investigation given the large role they play in ancient subsistence economies, medicinal practices, technologies and folklore. However, despite new developments in ancient genetics, research in plant ancient DNA (aDNA) is a relatively young and untouched discipline accounting for less than 7% of all aDNA analyses published in academic literature. As a result, paleoethnobotanists, archaeologists and geneticists have not understood the feasibility and limitations of each other’s field. Few are aware that DNA extraction from charred plant remains is rare and without any kind of standard or working protocol. The possibilities of retrieving aDNA from charred Zea mays L. is considered in this study using modern maize for polymerase chain reaction (PCR) optimization and combining purification methods on ancient samples (1150-1250 AD), resolving the question of whether or not archaeologically charred plants are a viable source for genetic material. The confirmed positive results generate questions about the added-value of maize and how knowledge of genetic attributes can contribute to the growing field of archaeology and ethnobiology while demonstrating the value of these findings as they pertain to the treatment of charred floral remains by archaeologists and First Nation communities

A serological study of some cauliflower mosaic virus isolates

Bibliography: pages 134-149.Enzyme-linked immunosorbent assay (ELISA) was used successfully to detect cauliflower mosaic virus (CaMV) in crude leaf extracts. Small serological differences between CaMV isolates could be shown by ELISA and serum cross-absorption. Serological reactivity of CaMV was found to depend on the proteolytic degradation state of the virus coat protein so making it impossible to establish definite serological relationships among the virus isolates tested. Proteolysis during purification of CaMV could not be entirely eliminated. The coat protein of CaMV was shown to be glycosylated by the specific binding of labelled Concanavalin A. The role of carbohydrate residues in CaMV serological reactivity was evaluated

Development of sterilisation strategies for decellularised peripheral nerve grafts

Peripheral nerve injuries represent one of the leading causes of disability globally; due to limitations of current therapies, there is a clear clinical need for a novel peripheral nerve graft . Decellularised porcine peripheral nerves may represent a suitable material; however, they would require terminal sterilisation prior to use. The main aim of this study was to identify a sterilisation method which minimally impacted upon decellularised nerve extracellular matrix (ECM) structure, biochemical composition, biomechanical properties and biocompatibility. Peracetic acid (PAA) solution 0.1 % (v/v) caused disruption to the structure of the endoneurium and reductions in the intensity of antibody labelling for basement membrane components, particularly collagen IV (with increased severity after a 12 month storage period). Supercritical carbon dioxide (SCCO2) treatment (with an additive solution containing 13.5 - 18.5 % [v/v] PAA and 4.5 - 6 % [v/v] H2O2) under standard conditions induced similar effects to the histioarchitecture and significant alterations to tensile mechanical properties; processing of the tissue whilst submerged in phosphate-buffered saline appeared to protect against such effects. Treatment with gamma radiation (25 – 28 kGy) and E Beam (33 – 37 kGy) mediated retention of the decellularised nerve ECM structure, and changes to the localisation and intensity of labelling for basement membrane components were minimal compared with those observed following treatment with PAA solution or SCCO2 under standard conditions. However, gamma radiation caused the stiffness of the nerves to increase. Contact culture experiments did not yield evidence of cytotoxicity following any of the sterilisation methods; furthermore, the sterilised nerves did not induce any significant changes in the secretion of key cytokines by a murine macrophage cell line. These data indicate that E Beam could be an optimal sterilisation method for use with the decellularised nerves, notwithstanding preliminary results indicating that SCCO2 processing under submerged conditions may enable superior basement membrane preservation

Biomarkers Discovery: The Benefit of the Study Exosomes Originated from Merkel Cell Carcinoma Cell Lines

Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine form of skin cancer with a rising incidence and a high mortality rate. The risk of developing Merkel cell carcinoma increased among a large number of immunosuppressed patients. The origin of cancer not known and the pathogenesis of MCC not fully understood. Two causes can initiate MCC tumorigenesis, such as accumulation of UV-induced mutations in the MCPyV-negative MCCs and Merkel cell polyomavirus (MCPyV)-encoded genes in MCPyV-positive tumor. Approximately 80% of all MCCs are positive for viral DNA of MCPyV. Immune therapy is a promising treatment for MCC patients, but it has failed to arrest the cancer progression. Precision medicine is needed. Precision medicine is a core of highly specific biomarkers disclose information for diagnosis, prognosis, and therapy. The usefulness of a biomarker lies in its ability to provide an early indication of a disease and its progression, and it should be easy to detect and measure. Our research aim was to investigate by high-throughput approaches for screening and analysis of two different types of MCC cell lines and their exosomes. Our proteomic investigation result showed that MCPyV-negative and –positive Merkel cell lines’ exosomes contain several proteins associated with tumor cell motility and metastasis. We identified a list of vesicular proteins derived from the extracellular region, which upregulated in exosome from MCPyV-negative MCC cell lines compare to MCPyV-positive MCC cell lines. We did the next-generation sequencing screening of exosomal small RNA from two groups of MCC cell lines. Main findings investigated in samples from healthy donors and MCC patients. The result showed the exosomal miR-222-3p presence in all type of samples derived from MCC cell lines, healthy donors and patients. The miR-222-3p selectively sorted in exosomes. The target genes’ screening indicates that the exosomal miR-222-3p play pleiotropic role dependent on recipient cells in health and disease. Then, the proteomic investigation and integrated analyses revealed the MCPyV-negative MCC cell lines loss the DNA, RNA and protein synthesis and their regulation system activity, and have an unusual event of protein expression at cell proliferation and post-translational modification sites. These may lead to transcription-associated mutation (TAM) and transcription-associated recombination (TAR), which gave a rise a high mutational burden. The MCPyV-positive MCC cell lines showed upregulated expression of proteins involved in DNA transcription initiation, termination, modification, and repair, harnesses of polyomaviruses for DNA integration. Following upregulated proteins of RNA, protein synthesis and post-translational modification machinery such as the protein acylation culminates in the viral proteins and genome synthesis. However, a fixed exosome-ER accession ability and a low activity on endocytosis and exocytosis sites indicate to reduce the chance of MCPyV spreading. Finally, transcriptomic and proteomic approaches are powerful tools for cell phenotyping and biomarkers discovery

African Animal Trypanosomiasis: Immunochemical Studies of the Trypanocide, Isometamidium Chloride

African animal trypanosomiasis affects about half of the habitable land in Africa hampering improvements in livestock production and mixed agriculture. Though the annual economic losses have been difficult to quantify, the disease is a major economic handicap to the continent and denies its ever-growing population much needed food and livestock products. In this study, chapter one provides a review of African animal trypanosomiasis with regard to its aetiology, hosts, geographical location and economic importance, life cycle and transmission, pathogenesis, clinical signs, diagnosis and control. Of the various methods which are employed in controlling the disease, chemotherapy and chemoprophylaxis remain the most widespread. However, the trypanocidal drugs in use today have been used for over 30 years and problems such as drug resistance appear to be increasingly reported. The remaining chapters deal with various immunochemical studies of isometamidium chloride, the main trypanocidal drug used in the treatment and control of the disease. In chapter two, details of purifying an anti-isometamidium antibody produced in sheep and its use in the quantification of isometaraidium chloride in sera of treated cattle by enzyme linked immunosorbent assay (ELISA) are given. The levels of free and bound isometamidium in sera of treated cattle were assessed by employing trichloroacetic acid precipitation and ultrafiltration methods. It was found that, in cattle treated by an intramuscular injection of isometamidium chloride, about 95-99% of the circulating drug is bound to serum proteins and very little if any of it is free and unbound. In chapter three, details of a "Chemiluminescent Western-blot technique" are given. This technique was used in the characterisation of the serum proteins that bind isometamidium. These proteins were shown to be about 122, 140 and 155 kD in cattle and about 300 kD in mice and goat. The major isometamidium binding proteins in cattle appear to be serum albumin. The significance of such bindings in treated animals are discussed. The possibility of developing and using a new technique, "Chemiluminescent dot-blot" for the measurement of isometamidium in sera of treated animals is examined in chapter four. With improvements this new technique may offer a simple, quick, less expensive and sensitive method for the quantification of isometamidium chloride in body fluids under field conditions. Chapter five gives the general conclusions drawn from this study and future areas of immunochemical research of isometamidium chloride are suggested