Benjamin T. Aldrich

CHANGES IN EXPRESSION OF SENSORY ORGAN-SPECIFIC microRNAs IN RAT DORSAL ROOT GANGLIA IN ASSOCIATION WITH MECHANICAL HYPERSENSITIVITY INDUCED BY SPINAL NERVE LIGATION

Chronic neuropathic pain caused by peripheral nerve injury is associated with global changes in gene expression in damaged neurons. To understand the molecular mechanisms underlying neuropathic pain, it is essential to elucidate how nerve injury alters gene expression and how the change contributes to the development and maintenance of chronic pain. MicroRNAs are non-protein-coding RNA molecules that regulate gene expression in a wide variety of biological processes mainly at the level of translation. This study investigated the possible involvement of microRNAs in gene regulation relevant to neuropathic pain. The analyses focused on a sensory organ-specific cluster of microRNAs that includes miR-96, -182, and -183. Quantitative real-time polymerase chain reaction (qPCR) analyses confirmed that these microRNAs were highly enriched in the dorsal root ganglion (DRG) of adult rats. Using the L5 spinal nerve ligation (SNL) model of chronic neuropathic pain, we observed a significant reduction in expression of these microRNAs in injured DRG neurons compared to controls. In situ hybridization and immunohistochemical analyses revealed that these microRNAs are expressed in both myelinated (N52 positive) and unmyelinated (IB4 positive) primary afferent neurons. They also revealed that the intracellular distributions of the microRNAs in DRG neurons were dramatically altered in animals with mechanical hypersensitivity. Whereas microRNAs were uniformly distributed within the DRG soma of non-allodynic animals, they were preferentially localized to the periphery of neurons in allodynic animals. The redistribution of microRNAs was associated with changes in the distribution of the stress granule (SG) protein, T-cell intracellular antigen 1 (TIA-1). These data demonstrate that SNL induces changes in expression levels and patterns of miR-96, -182, and -183, implying their possible contribution to chronic neuropathic pain through translational regulation of pain-relevant genes. Moreover, SGs were suggested to be assembled and associated with microRNAs after SNL, which may play a role in modification of microRNA-mediated gene regulation in DRG neurons.

Identification of Termite Species and Subspecies of the Genus Zootermopsis Using Near-Infrared Reflectance Spectroscopy

Abstract Dampwood termites of the genus Zootermopsis (Isoptera: Termopsidae) are an abundant group of basal termites found in temperate forests of western North America. Three species are currently recognized in the genus and one of these species is subdivided into two subspecies. Although morphological and genetic characters are useful in differentiating among the three species and the two subspecies, respectively, only hydrocarbon analysis can enable differentiation both among the three species and the two subspecies. Due to the limitations of hydrocarbon analysis, such as the need for fresh specimens, alternative methods that could rapidly and accurately identify Zootermopsis would be useful. Using a partial least squares analysis of near-infrared spectra, each of the Zootermopsis species and subspecies were identified with greater than 95% and 80% accuracy, respectively. Neural network analysis of the near-infrared spectra successfully enabled the identification of the species and subspecies with greater than 99% accuracy. The inexpensive, reproducible, and rapid nature of near-infrared spectroscopy makes it a viable alternative to morphological, hydrocarbon, or genetic analysis for identifying Zootermopsis.

UNC79 and UNC80, putative auxiliary subunits of the NARROW ABDOMEN ion channel, are indispensable for robust circadian locomotor rhythms in Drosophila.

In the fruit fly Drosophila melanogaster, a network of circadian pacemaker neurons drives daily rhythms in rest and activity. The ion channel NARROW ABDOMEN (NA), orthologous to the mammalian sodium leak channel NALCN, functions downstream of the molecular circadian clock in pacemaker neurons to promote behavioral rhythmicity. To better understand the function and regulation of the NA channel, we have characterized two putative auxiliary channel subunits in Drosophila, unc79 (aka dunc79) and unc80 (aka CG18437). We have generated novel unc79 and unc80 mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from na mutant phenotypes. Tissue-specific RNA interference and rescue analyses indicate that UNC79 and UNC80 likely function within pacemaker neurons, with similar anatomical requirements to NA. We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels. Yet despite this relationship, we find that the requirement for unc79 and unc80 in circadian rhythmicity cannot be bypassed by increasing NA protein expression, nor can these putative auxiliary subunits substitute for each other. These data indicate functional requirements for UNC79 and UNC80 beyond promoting channel subunit expression. Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain. Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

The amnesiac gene is involved in the regulation of thermal nociception in Drosophila melanogaster

Abstract: Nociception is a mechanism fundamental to the ability of animals to avoid noxious stimuli capable of causing serious tissue damage. It has been established that in the fruit fly Drosophila melanogaster, the transient receptor potential (TRP) channel encoded by the painless gene (pain) is required for detecting thermal and mechanical noxious stimuli. Little is known, however, about other genetic components that control nociceptive behaviors in Drosophila. The amnesiac gene (amn), which encodes a putative neuropeptide precursor, is important for stabilizing olfactory memory, and is involved in various aspects of other associative and nonassociative learning. Previous studies have indicated that amn also regulates ethanol sensitivity and sleep. Here the authors show that amn plays an additional critical role in nociception. Their data show that amn mutant larvae and adults are significantly less responsive to noxious heat stimuli (greater than ∼40°C) than their wild-type counterparts. The phenotype of amn mutants in thermal nociception, which closely resembles that of pain mutants, was phenocopied in flies expressing amn RNAi, and this phenotype was rescued by the expression of a wild-type amn transgene. These results provide compelling evidence that amn is a novel genetic component of the mechanism that regulates thermal nociception in Drosophila.

Species-Specific Allozyme Markers for Appalachian Wood-Feeding Cockroaches (Dictyoptera: Cryptocercidae)

Members of the genus Cryptocercus are wood-feeding cockroaches that live in the temperate forests. Nine species are recognized in the genus worldwide: two in eastern Eurasia, two in China, and five in the United States. Within the United States, one species occurs in the Pacific Northwest and four occur in the Appalachian Mountains. Previous studies have revealed the presence of potential zones of overlap in distribution among the Appalachian species, raising the possibility of hybridization among them. Differences in mitochondrial DNA have previously been identified for the Appalachian species. However, to identify hybrid individuals one or more species-specific, codominant nuclear markers are required. Therefore, our objective was to undertake allozyme analysis of enzymatic loci to identify fixed, species-specific alleles for the four Appalachian species. We assayed a mean of 42 individuals each from 16 sites for allozyme variation for the four species. At 6 of the 33 loci examined, fixed alternate alleles were identified; a combination of 2 loci enabled the identification of all four species. To identify hybrids in the field, we examined 42 individuals each from 13 sites in which two or more of the above species occur in close proximity for presence of heterozygous individuals at one or more of the six fixed loci. No heterozygous individuals were found suggesting the lack of hybridization among the Appalachian species.

Mitochondrial and Morphologic Alterations in Native Human Corneal Endothelial Cells Associated With Diabetes Mellitus

Purpose To characterize changes in the energy-producing metabolic activity and morphologic ultrastructure of corneal endothelial cells associated with diabetes mellitus. Methods Transplant suitable corneoscleral tissue was obtained from donors aged 50 to 75 years. We assayed 3-mm punches of endothelium-Descemet membrane for mitochondrial respiration and glycolysis activity using extracellular flux analysis of oxygen and pH, respectively. Transmission electron microscopy was used to assess qualitative and quantitative ultrastructural changes in corneal endothelial cells and associated Descemet membrane. For purposes of analysis, samples were divided into four groups based on a medical history of diabetes regardless of type: (1) nondiabetic, (2) noninsulin-dependent diabetic, (3) insulin-dependent diabetic, and (4) insulin-dependent diabetic with specified complications due to diabetes (advanced diabetic). Results In total, 229 corneas from 159 donors were analyzed. Insulin-dependent diabetic samples with complications due to diabetes displayed the lowest spare respiratory values compared to all other groups (P ≤ 0.002). The remaining mitochondrial respiration and glycolysis metrics did not differ significantly among groups. Compared to nondiabetic controls, the endothelium from advanced diabetic samples had alterations in mitochondrial morphology, pronounced Golgi bodies associated with abundant vesicles, accumulation of lysosomal bodies/autophagosomes, and focal production of abnormal long-spacing collagen. Conclusions Extracellular flux analysis suggests that corneal endothelial cells of donors with advanced diabetes have impaired mitochondrial function. Metabolic findings are supported by observed differences in mitochondrial morphology of advanced diabetic samples but not controls. Additional studies are needed to determine the precise mechanism(s) by which mitochondria become impaired in diabetic corneal endothelial cells.

Assessing the Impact of Diabetes Mellitus on Donor Corneal Endothelial Cell Density

Purpose: To quantify changes in endothelial cell density (ECD) of donor corneal tissue in relation to the presence or absence of a medical history of diabetes mellitus diagnosis, treatment, and complications. Methods: A retrospective review was performed for all corneas collected at Iowa Lions Eye Bank between January 2012 and December 2015. For purposes of analysis, donor corneas were divided into 4 groups: nondiabetic, non–insulin-dependent diabetic, insulin-dependent diabetic without medical complications due to diabetes, and insulin-dependent diabetic with medical complications due to diabetes. ECD values (obtained through specular microscopy) and transplant suitability for endothelial transplantation (determined by the standard protocol of the eye bank) were compared among groups using linear mixed model analysis. Results: In total, 4185 corneas from 2112 donors were included for analysis. Insulin-dependent diabetic samples with medical complications due to diabetes (N = 231 from 119 donors) showed lower ECD values compared with nondiabetic samples (−102 cells/mm2, P = 0.049) and non–insulin-dependent diabetic samples (−117 cells/mm2, P = 0.031). ECD values did not differ significantly among the remaining groups. The likelihood of suitability for endothelial transplantation did not differ among all 4 groups. Conclusions: Corneas from donors with insulin-dependent diabetes mellitus and medical complications resulting from the disease have lower mean ECD values compared with other donors. However, our analysis suggests that these corneas are equally likely to be included in the donor pool for corneal transplantation. Additional studies are needed to determine the mechanism(s) contributing to cell loss in donors with advanced diabetes and to assess associated endothelial cell functional impairment.

Incidence of Cystoid Macular Edema After Descemet Membrane Endothelial Keratoplasty as a Staged and Solitary Procedure.

Purpose: To compare the incidence of visually significant postoperative cystoid macular edema (CME) in pseudophakic eyes after Descemet membrane endothelial keratoplasty (DMEK) performed after recent versus remote cataract surgery. Methods: A retrospective chart review was performed of all consecutive eyes that underwent DMEK without concurrent cataract surgery at the University of Iowa between October 2012 and December 2014. The DMEK procedures were classified as staged if performed between 2 weeks and 6 months after cataract surgery and solitary if performed more than 6 months after cataract surgery. Possible confounders, including a history of diabetes in the recipient, were tracked. Macular optical coherence tomography was performed to detect CME 1 month after DMEK if the best-corrected visual acuity was ⩽20/30 with a clear cornea with no other reason for visual compromise. Results: A total of 173 eyes from 140 patients were included in the statistical analysis. Staged DMEK was performed in 88 eyes (50.8%) and solitary DMEK in 85 eyes (49.2%). The incidence of CME was 8.0% (7 of 88 eyes) in the staged DMEK group and 7.1% (6 of 85 eyes) in the solitary DMEK group (P = 0.823). The incidence of CME did not differ significantly between the staged and solitary DMEK groups regardless of the recipient diabetic status. All cases of CME resolved within 6 months on topical therapy. Conclusions: The incidence of postoperative CME after DMEK is similar in the setting of recent or remote cataract surgery.

Physiological, anatomical, and behavioral changes after acoustic trauma in Drosophila melanogaster

Significance Noise-induced hearing loss (NIHL) is an important health issue, yet its mechanisms and potential treatments remain unclear. We present the first study of NIHL in the fruit fly Drosophila, which has many advantages as an NIHL model. We examined auditory function and structure after exposing flies to acoustic trauma. Trauma impairs auditory system function and changes neural mitochondria size, suggesting metabolic stress. In mutant flies with a reduced ability to cope with such stresses, the responses to trauma were more severe and recovery delayed or impaired. Noise-induced hearing loss (NIHL) is a growing health issue, with costly treatment and lost quality of life. Here we establish Drosophila melanogaster as an inexpensive, flexible, and powerful genetic model system for NIHL. We exposed flies to acoustic trauma and quantified physiological and anatomical effects. Trauma significantly reduced sound-evoked potential (SEP) amplitudes and increased SEP latencies in control genotypes. SEP amplitude but not latency effects recovered after 7 d. Although trauma produced no gross morphological changes in the auditory organ (Johnston’s organ), mitochondrial cross-sectional area was reduced 7 d after exposure. In nervana 3 heterozygous flies, which slightly compromise ion homeostasis, trauma had exaggerated effects on SEP amplitude and mitochondrial morphology, suggesting a key role for ion homeostasis in resistance to acoustic trauma. Thus, Drosophila exhibit acoustic trauma effects resembling those found in vertebrates, including inducing metabolic stress in sensory cells. This report of noise trauma in Drosophila is a foundation for studying molecular and genetic sequelae of NIHL.

Proteomic analysis of corneal endothelial cell-descemet membrane tissues reveals influence of insulin dependence and disease severity in type 2 diabetes mellitus

The objective of this study was to characterize the proteome of the corneal endothelial cell layer and its basement membrane (Descemet membrane) in humans with various severities of type II diabetes mellitus compared to controls, and identify differentially expressed proteins across a range of diabetic disease severities that may influence corneal endothelial cell health. Endothelium-Descemet membrane complex tissues were peeled from transplant suitable donor corneas. Protein fractions were isolated from each sample and subjected to multidimensional liquid chromatography and tandem mass spectrometry. Peptide spectra were matched to the human proteome, assigned gene ontology, and grouped into protein signaling pathways unique to each of the disease states. We identified an average of 12,472 unique proteins in each of the endothelium-Descemet membrane complex tissue samples. There were 2,409 differentially expressed protein isoforms that included previously known risk factors for type II diabetes mellitus related to metabolic processes, oxidative stress, and inflammation. Gene ontology analysis demonstrated that diabetes progression has many protein footprints related to metabolic processes, binding, and catalysis. The most represented pathways involved in diabetes progression included mitochondrial dysfunction, cell-cell junction structure, and protein synthesis regulation. This proteomic dataset identifies novel corneal endothelial cell and Descemet membrane protein expression in various stages of diabetic disease. These findings give insight into the mechanisms involved in diabetes progression relevant to the corneal endothelium and its basement membrane, prioritize new pathways for therapeutic targeting, and provide insight into potential biomarkers for determining the health of this tissue.