Development of a small shuttle plasmid for use in oral Veillonella and initial appraisal of potential for fluorescence-based applications.

Oral Veillonella species are among the early colonizers of the human oral cavity. We constructed a small, single-selectable-marker shuttle plasmid, examined its ability to be transformed into diverse oral Veillonella strains, and assessed its potential use for expressing a gene encoding an oxygen-independent fluorescent protein, thus generating a fluorescent Veillonella parvula strain. Because tetracycline resistance is common in Veillonella, we replaced genes encoding ampicillin- and tetracycline-resistance in a previously described shuttle plasmid (pBSJL2) with a chloramphenicol acetyltransferase gene. The resulting plasmid pCF1135 was successfully introduced into four strains representing V. parvula and V. atypica by either natural transformation or electroporation. We then modified this plasmid to express a gene encoding an oxygen-independent fluorescent protein in V. parvula SKV38. The resulting strain yielded a fluorescence signal intensity approximately 16 times higher than the wild-type in microplate-based fluorimetry experiments. While fluorescence microscopy demonstrated that planktonic cells, colonies, and biofilms of fluorescent V. parvula could also be imaged, photobleaching was a significant issue. In conclusion, we anticipate this genetic system and information provided here will facilitate expanded studies of oral Veillonella species' properties and behavior.

Five state factors control progressive stages of freshwater salinization syndrome.

Factors driving freshwater salinization syndrome (FSS) influence the severity of impacts and chances for recovery. We hypothesize that spread of FSS across ecosystems is a function of interactions among five state factors: human activities, geology, flowpaths, climate, and time. (1) Human activities drive pulsed or chronic inputs of salt ions and mobilization of chemical contaminants. (2) Geology drives rates of erosion, weathering, ion exchange, and acidification-alkalinization. (3) Flowpaths drive salinization and contaminant mobilization along hydrologic cycles. (4) Climate drives rising water temperatures, salt stress, and evaporative concentration of ions and saltwater intrusion. (5) Time influences consequences, thresholds, and potentials for ecosystem recovery. We hypothesize that state factors advance FSS in distinct stages, which eventually contribute to failures in systems-level functions (supporting drinking water, crops, biodiversity, infrastructure, etc.). We present future research directions for protecting freshwaters at risk based on five state factors and stages from diagnosis to prognosis to cure.

Distribution Patterns of Astrocyte Populations in the Human Cortex.

Astrocytes are a major class of glial cell in the central nervous system that have a diverse range of types and functions thought to be based on their anatomical location, morphology and cellular properties. Recent studies highlight that astrocyte dysfunction contributes to the pathogenesis of neurological conditions. However, few studies have described the pattern, distribution and density of astrocytes in the adult human cortex. This study mapped the distribution and density of astrocytes immunolabelled with a range of cytoskeletal and membrane markers in the human frontal cortex. Distinct and overlapping astrocyte populations were determined. The frontal cortex from ten normal control cases (75 ± 9 years) was immunostained with glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase-1 L1 (ALDH1L1), connexin-43 (Cx43), aquaporin-4 (AQP4), and glutamate transporter 1 (GLT-1). All markers labelled populations of astrocytes in the grey and white matter, separate cortical layers, subpial and perivascular regions. All markers were informative for labelling different cellular properties and cellular compartments of astrocytes. ALDH1L1 labelled the largest population of astrocytes, and Cx43-immunopositive astrocytes were found in all cortical layers. AQP4 and GLT-1 labelled distal astrocytic process and end-feet in the same population of astrocytes (98% of GLT-1-immunopositive astrocytes contained AQP4). In contrast, GFAP, the most widely used marker, predominantly labelled astrocytes in superficial cortical layers. This study highlights the diversity of astrocytes in the human cortex, providing a reference map of the distribution of distinct and overlapping astrocyte populations which can be used for comparative purposes in various disease, inflammatory and injury states involving astrocytes.

Coexisting Lewy body disease and clinical parkinsonism in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is associated with a range of clinical phenotypes and shows progressive degeneration of upper and/or lower motor neurons, and phosphorylated 43 kDa TAR DNA-binding protein (pTDP-43) inclusions in motor and non-motor pathways. Parkinsonian features have been reported in up to 30% of ALS patients, and Lewy bodies, normally associated with Lewy body disease (LBD), have been reported in a small number of ALS cases, with unknown clinical relevance. This study investigates the prevalence of clinically relevant LBD in a prospectively studied ALS cohort to determine whether concomitant pathology contributes to the clinical heterogeneity. METHODS: All ALS cases held by the New South Wales Brain Bank (n = 97) were screened for coexisting LBD consistent with clinical disease (Braak ≥ stage IV). Relevant clinical and genetic associations were determined. RESULTS: Six cases had coexisting LBD Braak ≥ stage IV pathology. The age at symptom onset (69 ± 7 years) and disease duration (4 ± 3 years) in ALS cases with coexisting LBD did not differ from ALS cases. Three patients had lower limb onset and two patients had bulbar onset. Two patients developed the clinical features of Parkinson's disease, with one receiving a dual diagnosis. All cases had no known relevant family history or genetic abnormalities. CONCLUSION: The prevalence of clinically relevant LBD pathology in ALS is higher than in the general population, and has implications for clinical and neuropathological diagnoses and the identification of biomarkers.