Interactions Between Light and Production of Microcystins in the Toxic Cyanobacterium Microcystis

Cyanobacterial harmful algal blooms (cHABs) are characterized by the formation of toxins that can impact animal health, cause water quality issues, and recreational hazards. Microcystis, a common genus of cyanobacteria, produces the potent protein phosphatase inhibitor microcystins. Microcystins are nitrogen-rich and have an associated metabolic cost for production. Some outstanding questions in the study of cHABs is why are microcystins produced, what are the benefits of toxin formation, and why only some Microcystis strains produce microcystins? We examined a potential biochemical role for microcystins in the cyanobacterial photosystem regulation in response to various light conditions. Single-celled culture strains of toxic and non- toxic Microcystis aeruginosa were grown under different light irradiances. High-light conditions caused light stress based on decreased photosynthetic efficiency. Cells responded over 2-3 days by decreasing their chlorophyll and phycobilisome content per cell in unison. Looking at a natural system over a diurnal cycle of changing light intensities, Microcystis responded to high-light environments via vertical migration deeper in the water column to avoid light stress. In one culture and in situ, there were no changes in microcystins concentration per cell, but one strain showed decreased microcystins under low-light. Therefore, microcystins protein phosphatase activity may not be involved with phosphorylation/dephosphorylation under high-light. High-light environments are also associated reactive oxygen species (ROS) production. We investigated if microcystins were linked to ROS sensitivity by examining how three chlorophytes and seven cyanobacteria responded to the ROS compound hydrogen peroxide (H2O2). There was no evidence that toxic cyanobacteria were more sensitive to ROS than non-toxic cyanobacteria or chlorophytes. Addition of H2O2 did not change the microcystins concentration per cell. While these experiments did not elucidate the biochemical function of microcystins in Microcystis, they provided valuable information for water quality managers. cHAB monitoring programs must carefully consider vertical migration away from the surface during high-light conditions. The use of H2O2 as a control mechanism may not selectively remove toxic cyanobacteria. Despite visual similarities, Microcystis is composed of diverse species with a wide-range of responses to light and ROS. Care must be taken when applying conclusions using a limited number of strains to broader populations

Cortical N-methyl-d-aspartate receptor in schizophrenia: Characterising molecular changes at the cellular and sub-cellular level

Schizophrenia is a mental disorder that affects approximately 1% of the world’s population. The N-methyl-d-aspartate receptor (NMDAR) seems likely to play a prominent role in schizophrenia; as when NMDAR antagonists are introduced, psychosis occurs in individuals with no mental disorders. This thesis explores how NMDAR may be endogenously changed in the brains of people with schizophrenia. By using tissue fractionation, discussed in chapter 3, to examine the post-synaptic density (PSD) specifically one can specifically examine the NMDAR protein at the synapse of the neuron. Also, one can examine the NMDAR mRNA level in the brains of people with schizophrenia compared to controls. My Masters’ thesis research covers four broad areas, namely subcellular tissue fractionation of human cerebral cortex, the determination of the NMDAR subunit NR1 protein levels specifically in the PSD, analysis of NR1 mRNA transcript across cortical lamina, and the quantification of NMDAR NR1 mRNA at the cellular level. It is hypothesized that the NMDAR NR1 protein will be decreased in the PSD and mRNA will be decreased in neurons in schizophrenia patients. There are four main aims of this study. The first aim is to extract the postsynaptic density fraction from human brains in the BA10 region. Using the New South Wales Tissue Resource Centre Cohort (37 schizophrenia patients and 37 controls). Tissue fractionation, discussed in chapter two, was completed in order to obtain total homogenate and PSD-enriched fractions from the PFC [Brodmann’s Area (BA) 10]) from individuals with schizophrenia and controls. The second aim was to measure NMDAR NR1 protein levels in schizophrenia compared to controls in the total homogenate and PSD associated homogenate from the BA10 region. The amounts of NMDAR NR1 protein were determined using Western blotting techniques, described in chapter four. The third aim was to examine examining the laminar expression of NMDAR NR1 mRNA in BA46 in schizophrenia compared to controls using a riboprobe GRIN1 (human genome designation for the NMDAR NR1 subunit). Film from in situ hybridization was used to determine the amount of mRNA from NMDAR NR1 in each cortical layer in the BA46 in patients with schizophrenia and controls, discussed in chapter five. The fourth aim was to measure the cellular expression of NMDAR NR1 mRNA in BA46 in schizophrenia compared to controls in three laminar layers. In addition, silver grain analysis (corresponding to the amount of NMDAR NR1 mRNA/cells) of individual large and small neurons was analysed to determine if changes in mRNA could be localised to specific cell types within cortical layer V. mRNA for the NR1 subunit was studied in the PFC using in situ hybridization, discussed in chapter six.Through the process of Western blotting, it was concluded that our fractionation samples containing the synapse were indeed enriched for proteins (including NR1) known to be enriched in the PSD. From there, analysis of the PSD fractions revealed a statistically significant decreased NR1 protein (39%) in the PSD in people with schizophrenia compared to controls. There were no significant diagnostic differences in NR1 mRNA expression when analysing the individual layers of the cortex from the autoradiographic films. However, when delving deeper and looking at individual neurons, I found that pyramidal neurons were significantly lower in NR1 mRNA expression in people with schizophrenia, and thus may play a role in the hypoglutamatergia in schizophrenia. Future work required would be to determine which neurons are responsible for the NR1 protein changes at the PSD. A comprehensive examination of NR1 mRNA expression across more cortical layers at a microscopic level in BA46 is warranted

Increased autoantibodies against brain tissue in schizophrenia

The present study investigates the short- and long-term outcomes of a computer-assisted cognitive remediation (CACR) program in adolescents with psychosis or at high risk. 32 adolescents participated in a blinded 8-week randomized controlled trial of CACR treatment compared to computer games (CG). Clinical and neuropsychological evaluations were undertaken at baseline, at the end of the program and at 6-month. At the end of the program (n = 28), results indicated that visuospatial abilities (Repeatable Battery for the Assessment of Neuropsychological Status, RBANS; P = .005) improved signifi cantly more in the CACR group compared to the CG group. Furthermore, other cognitive functions (RBANS), psychotic symptoms (Positive and Negative Symptom Scale) and psychosocial functioning (Social and Occupational Functioning Assessment Scale) improved signifi cantly, but at similar rates, in the two groups. At long term (n = 22), cognitive abilities did not demonstrated any amelioration in the control group while, in the CACR group, signifi cant long-term improvements in inhibition (Stroop; P = .040) and reasoning (Block Design Test; P = .005) were observed. In addition, symptom severity (Clinical Global Improvement) decreased signifi cantly in the control group (P = .046) and marginally in the CACR group (P = .088). To sum up, CACR can be successfully administered in this population. CACR proved to be effective over and above CG for the most intensively trained cognitive ability. Finally, on the long-term, enhanced reasoning and inhibition abilities, which are necessary to execute higher-order goals or to adapt behavior to the ever-changing environment, were observed in adolescents benefi ting from a CACR

Episodic Decrease in Temperature Increases mcy Gene Transcription and Cellular Microcystin in Continuous Cultures of Microcystis aeruginosa PCC 7806

Microcystins produced during harmful cyanobacterial blooms are a public health concern. Although patterns are emerging, the environmental cues that stimulate production of microcystin remain confusing, hindering our ability to predict fluctuations in bloom toxicity. In earlier work, growth at cool temperatures relative to optimum (18°C vs. 26°C) was confirmed to increase microcystin quota in batch cultures of Microcystis aeruginosa NIES-843. Here, we tested this response in M. aeruginosa PCC 7806 using continuous cultures to examine temporal dynamics and using RNA-sequencing to investigate the physiological nature of the response. A temperature reduction from 26 to 19°C increased microcystin quota ∼2-fold, from an average of ∼464 ag μm–3 cell volume to ∼891 ag μm–3 over a 7–9 d period. Reverting the temperature to 26°C returned the cellular microcystin quota to ∼489 ag μm–3. Long periods (31–42 d) at 19°C did not increase or decrease microcystin quota beyond that observed at 7–9 d. Nitrogen concentration had little effect on the overall response. RNA sequencing indicated that the decrease in temperature to 19°C induced a classic cold-stress response in M. aeruginosa PCC 7806, but this operated on a different timescale than the increased microcystin production. Microcystin quota showed a strong 48- to 72-h time-lag correlation to mcy gene expression, but no correlation to concurrent mcy expression. This work confirms an effect of temperature on microcystin quota and extends our understanding of the physiological nature of the response