Screening, optimization and extraction of polyhydroxyalkanoates and peptidoglycan from Bacillus megaterium

Bioplastics are polymers (such as polyesters) produced from bacterial fermentations that are biodegradable and nonhazardous. They are produced by a wide variety of bacteria and are made only when stress conditions allow, such as when nutrient levels are low, more specifically levels of nitrogen and oxygen. These stress conditions cause certain bacteria to build up excess carbon deposits as energy reserves in the form of polyhydroxyalkanoates (PHAs). PHAs can be extracted and formed into actual plastic with the same strength of conventional, synthetic-based plastics without the need to rely on foreign petroleum. The overall goal of this project was to select for a bacteria that could grow on sugars found in the lignocellulosic biomass, and get the bacteria to produce PHAs and peptidoglycan. Once this was accomplished the goal was to extract PHAs and peptidoglycan in order to make a stronger more rigid plastic, by combing them into a co-polymer. The individual goals of this project were to: (1) Select and screen bacteria that are capable of producing PHAs by utilizing the carbon/energy sources found in lignocellulosic biomass; (2) Maximize the utilization of those sugars present in woody biomass in order to produce optimal levels of PHAs. (3) Use room temperature ionic liquids (RTILs) in order to separate the cell membrane and peptidoglycan, allowing for better extraction of PHAs and more intact peptidoglycan. B. megaterium a Gram-positive PHA-producing bacterium was selected for study in this project. It was grown on a variety of different substrates in order to maximize both its growth and production of PHAs. The optimal conditions were found to be 30°C, pH 6.0 and sugar concentration of either 30g/L glucose or xylose. After optimal growth was obtained, both RTILs and enzymatic treatments were used to break the cell wall, in order to extract the PHAs, and peptidoglycan. PHAs and peptidoglycan were successfully extracted from the cell, and will be used in the future to create a new stronger co-polymer. Peptidoglycan recovery yield was 16% of the cells’ dry weight.

A randomised controlled trial of spinal manipulative therapy in acute low back pain

OBJECTIVE: To determine whether treatment with spinal manipulative therapy (SMT) administered in addition to standard care is associated with clinically relevant early reductions in pain and analgesic consumption. METHODS: 104 patients with acute low back pain were randomly assigned to SMT in addition to standard care (n = 52) or standard care alone (n = 52). Standard care consisted of general advice and paracetamol, diclofenac or dihydrocodeine as required. Other analgesic drugs or non-pharmacological treatments were not allowed. Primary outcomes were pain intensity assessed on the 11-point box scale (BS-11) and analgesic use based on diclofenac equivalence doses during days 1-14. An extended follow-up was performed at 6 months. RESULTS: Pain reductions were similar in experimental and control groups, with the lower limit of the 95% CI excluding a relevant benefit of SMT (difference 0.5 on the BS-11, 95% CI -0.2 to 1.2, p = 0.13). Analgesic consumptions were also similar (difference -18 mg diclofenac equivalents, 95% CI -43 mg to 7 mg, p = 0.17), with small initial differences diminishing over time. There were no differences between groups in any of the secondary outcomes and stratified analyses provided no evidence for potential benefits of SMT in specific patient groups. The extended follow-up showed similar patterns. CONCLUSIONS: SMT is unlikely to result in relevant early pain reduction in patients with acute low back pain.

Ultrastructural changes in otoconia of osteoporotic rats

The etiology of benign paroxysmal positional vertigo (BPPV) remains obscure in many cases and women are affected more often than men. A recent prospective study, performed in women >50 years of age suffering from recurrent BPPV, showed associated osteopenia or osteoporosis in a large percentage of these patients. These results suggested the possible relationship between recurrent BPPV and a decreased fixation of calcium in bone in women >50 years. To test this hypothesis, an experimental study was performed in adult female rats. Utricular otoconia of female rats in which osteopenia/osteoporosis was induced by bilateral ovariectomy (OVX) were compared to those of sham-operated adult females rats (SHAM), as control group. FIRST STUDY: The morphology of theutricles of OVX and SHAM rats was analyzed with scanning electron microscopy. In osteopenic/osteoporotic rats, the density of otoconia (i.e. the number of otoconia per unit area) was decreased (p = 0.036)and their size was increased (p = 0.036) compared to the control group. SECOND STUDY: To test the role of calcium turnover in such morphological changes, utricular otoconia of 2 other groups of OVX and SHAM rats, previously injected with calcein subcutaneously, were examined by conventional and epifluorescence microscopy. In epifluorescence microscopy, labeling with calcein showed no significant fluorescence in either group. This finding was interpreted as a lack of external calcium turnover into otoconia of adult female rats. The ultrastructural modifications of otoconia in osteopenic/osteoporotic female adult rats as well as the role of estrogenic receptors in the inner ear are discussed. The possible pathophysiological mechanisms which support the relationship between recurrent BPPV in women and the disturbance of the calcium metabolism of osteopenia/osteoporosis are debated.

Immobilizing bacteriorhodopsin on a single electron transistor

As awareness of potential human and environmental impacts from toxins has increased, so has the development of innovative sensors. Bacteriorhodopsin (bR) is a light activated proton pump contained in the purple membrane (PM) of the bacteria Halobacterium salinarum. Bacteriorhodopsin is a robust protein which can function in both wet and dry states and can withstand extreme environmental conditions. A single electron transistor(SET) is a nano-scale device that exploits the quantum mechanical properties of electrons to switch on and off. SETs have tremendous potential in practical applications due to their size, ultra low power requirements, and electrometer-like sensitivity. The main goal of this research was to create a bionanohybrid device by integrating bR with a SET device. This was achieved by a multidisciplinary approach. The SET devices were created by a combination of sputtering, photolithography, and focused ion beam machining. The bionanomaterial bacteriorhodopsin was created through oxidative fermentation and a series of transmembrane purification processes. The bR was then integrated with the SET by electrophoretic deposition, creating a bionanohybrid device. The bionanohybrid device was then characterized using a semiconductor parametric analyzer. Characterization demonstrated that the bR modulated the operational characteristics of the SET when bR was activated with light within its absorbance spectrum. To effectively integrate bacteriorhodopsin with microelectromechanical systems (MEMS) and nanoelectromechanical systems (NEMS), it is critical to know the electrical properties of the material and to understand how it will affect the functionality of the device. Tests were performed on dried films of bR to determine if there is a relationship between inductance, capacitance, and resistance (LCR) measurements and orientation, light-on/off, frequency, and time. The results indicated that the LCR measurements of the bR depended on the thickness and area of the film, but not on the orientation, as with other biological materials such as muscle. However, there was a transient LCR response for both oriented and unoriented bR which depended on light intensity. From the impedance measurements an empirical model was suggested for the bionanohybrid device. The empirical model is based on the dominant electrical characteristics of the bR which were the parallel capacitance and resistance. The empirical model suggests that it is possible to integrate bR with a SET without influencing its functional characteristics.

Divergence along a steep ecological gradient in lake whitefish (Coregonus sp.)

To understand mechanisms structuring diversity in young adaptive radiations, quantitative and unbiased information about genetic and phenotypic diversity is much needed. Here, we present the first in-depth investigation of whitefish diversity in a Swiss lake, with continuous spawning habitat sampling in both time and space. Our results show a clear cline like pattern in genetics and morphology of populations sampled along an ecological depth gradient in Lake Neuchâtel. Divergent natural selection appears to be involved in shaping this cline given that trait specific P(ST)-values are significantly higher than F(ST)-values when comparing populations caught at different depths. These differences also tend to increase with increasing differences in depth, indicating adaptive divergence along a depth gradient, which persists despite considerable gene flow between adjacent demes. It however remains unclear, whether the observed pattern is a result of currently stable selection-gene flow balance, incipient speciation, or reverse speciation due to anthropogenic habitat alteration causing two formerly divergent species to collapse into a single gene pool.