Mechanistic impacts of bacterial diet on dopaminergic neurodegeneration in a Caenorhabditis elegans α-synuclein model of Parkinson's disease.

Failure of inherently protective cellular processes and misfolded protein-associated stress contribute to the progressive loss of dopamine (DA) neurons characteristic of Parkinson's disease (PD). A disease-modifying role for the microbiome has recently emerged in PD, representing an impetus to employ the soil-dwelling nematode, Caenorhabditis elegans, as a preclinical model to correlate changes in gene expression with neurodegeneration in transgenic animals grown on distinct bacterial food sources. Even under tightly controlled conditions, hundreds of differentially expressed genes and a robust neuroprotective response were discerned between clonal C. elegans strains overexpressing human alpha-synuclein in the DA neurons fed either one of only two subspecies of Escherichia coli. Moreover, this neuroprotection persisted in a transgenerational manner. Genetic analysis revealed a requirement for the double-stranded RNA (dsRNA)-mediated gene silencing machinery in conferring neuroprotection. In delineating the contribution of individual genes, evidence emerged for endopeptidase activity and heme-associated pathway(s) as mechanistic components for modulating dopaminergic neuroprotection.

Therapeutic genetic variation revealed in diverse Hsp104 homologs.

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in C. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.

Modeling neurodegeneration in Caenorhabditiselegans.

The global burden of neurodegenerative diseases underscores the urgent need for innovative strategies to define new drug targets and disease-modifying factors. The nematode Caenorhabditis elegans has served as the experimental subject for multiple transformative discoveries that have redefined our understanding of biology for ∼60 years. More recently, the considerable attributes of C. elegans have been applied to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease and Huntington's disease. Transgenic nematodes with genes encoding normal and disease variants of proteins at the single- or multi-copy level under neuronal-specific promoters limits expression to select neuronal subtypes. The anatomical transparency of C. elegans affords the use of co-expressed fluorescent proteins to follow the progression of neurodegeneration as the animals age. Significantly, a completely defined connectome facilitates detailed understanding of the impact of neurodegeneration on organismal health and offers a unique capacity to accurately link cell death with behavioral dysfunction or phenotypic variation in vivo Moreover, chemical treatments, as well as forward and reverse genetic screening, hasten the identification of modifiers that alter neurodegeneration. When combined, these chemical-genetic analyses establish critical threshold states to enhance or reduce cellular stress for dissecting associated pathways. Furthermore, C. elegans can rapidly reveal whether lifespan or healthspan factor into neurodegenerative processes. Here, we outline the methodologies employed to investigate neurodegeneration in C. elegans and highlight numerous studies that exemplify its utility as a pre-clinical intermediary to expedite and inform mammalian translational research.

Molecular Detection of Spotted Fever Group Rickettsiae (Rickettsiales: Rickettsiaceae) in Dermacentor variabilis (Acari: Ixodidae) Collected Along the Platte River in South Central Nebraska.

Dermacentor variabilis is the predominant tick species in Nebraska and is presumed to be the primary vector of Rickettsia rickettsii associated with cases of Rocky Mountain spotted fever (RMSF). Interestingly, RMSF cases in Nebraska have increased on a year-to-year basis, yet the prevalence of R. rickettsii in D. variabilis ticks has not been established for Nebraska. Here we sought to set a baseline for the prevalence of R. rickettsii and other spotted fever group (SFG) rickettsiae harbored by D. variabilis ticks. Over a 3-yr period, D. variabilis were collected along the Platte River in south central Nebraska. Individual tick DNA was analyzed using endpoint PCR to identify ticks carrying SFG rickettsiae. In total, 927 D. variabilis were analyzed by PCR and 38 (4.1%) ticks tested positive for SFG rickettsiae. Presumptive positives were sequenced to identify the Rickettsia species, of which 29 (76%) were R. montanensis, 5 (13%) were R. amblyommatis, 4 (11%) were R. bellii, and R. rickettsii was not detected. These data indicate that R. rickettsii is likely at a low prevalence in south central Nebraska and spillover of R. amblyommatis into D. variabilis is likely occurring due to the invasive lone star tick (Amblyomma americanum). In addition, our data suggest that R. montanensis and R. amblyommatis could be associated with the increase in SFG rickettsiae infections in Nebraska. This information will be of value to clinicians and the general public for evaluating diagnosis of disease- and risk-associated environmental exposure, respectively.

Clinical applications of capillary electrophoresis based immunoassays.

Immunoassays have long been an important set of tools in clinical laboratories for the detection, diagnosis, and treatment of disease. Over the last two decades, there has been growing interest in utilizing CE as a means for conducting immunoassays with clinical samples. The resulting method is known as a CE immunoassay. This approach makes use of the selective and strong binding of antibodies for their targets, as is employed in a traditional immunoassay, and combines this with the speed, efficiency, and small sample requirements of CE. This review discusses the variety of ways in which CE immunoassays have been employed with clinical samples. An overview of the formats and detection modes that have been employed in these applications is first presented. A more detailed discussion is then given on the type of clinical targets and samples that have been measured or studied by using CE immunoassays. Particular attention is given to the use of this method in the fields of endocrinology, pharmaceutical measurements, protein and peptide analysis, immunology, infectious disease detection, and oncology. Representative applications in each of these areas are described, with these examples involving work with both traditional and microanalytical CE systems.