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At least 1,549 records · Page 86

Island of misfit tortoises: waif gopher tortoise health assessment following translocation

Translocation, the intentional movement of animals from one location to another, is a common management practice for the gopher tortoise (Gopherus polyphemus). Although the inadvertent spread of pathogens is a concern with any translocation effort, waif tortoises—individuals that have been collected illegally, injured and rehabilitated or have unknown origins—are generally excluded from translocation efforts due to heightened concerns of introducing pathogens and subsequent disease to naïve populations. However, repurposing these long-lived animals for species recovery is desirable when feasible, and introducing waif tortoises may bolster small populations facing extirpation. The objective of this study was to assess the health of waif tortoises experimentally released at an isolated preserve in Aiken County, SC, USA. Our assessments included visual examination, screening for 14 pathogens using conventional or quantitative polymerase chain reaction (qPCR) and haematological evaluation. Of the 143 individuals assessed in 2017 and 2018, most individuals (76%; n = 109 of 143) had no overt clinical evidence of disease and, when observed, clinical findings were mild. In both years, we detected two known tortoise pathogens, Mycoplasma agassizii and Mycoplasma testudineum, at a prevalence of 10.2–13.9% and 0.0–0.8%, respectively. Additionally, we found emydid Mycoplasma, a bacterium commonly found in box turtles (Terrapene spp.), in a single tortoise that showed no clinical evidence of infection. The presence of nasal discharge was an important, but imperfect, predictor of Mycoplasma spp. infection in translocated tortoises. Hemogram data were comparable with wild populations. Our study is the first comprehensive effort to assess pathogen prevalence and hemogram data of waif gopher tortoises following translocation. Although caution is warranted and pathogen screening necessary, waif tortoises may be an important resource for establishing or augmenting isolated populations when potential health risks can be managed.

60 APPLIED LIFE SCIENCES↗

Divergent Evolution of Early Terrestrial Fungi Reveals the Evolution of Mucormycosis Pathogenicity Factors

Abstract Fungi have evolved over millions of years and their species diversity is predicted to be the second largest on the earth. Fungi have cross-kingdom interactions with many organisms that have mutually shaped their evolutionary trajectories. Zygomycete fungi hold a pivotal position in the fungal tree of life and provide important perspectives on the early evolution of fungi from aquatic to terrestrial environments. Phylogenomic analyses have found that zygomycete fungi diversified into two separate clades, the Mucoromycota which are frequently associated with plants and Zoopagomycota that are commonly animal-associated fungi. Genetic elements that contributed to the fitness and divergence of these lineages may have been shaped by the varied interactions these fungi have had with plants, animals, bacteria, and other microbes. To investigate this, we performed comparative genomic analyses of the two clades of zygomycetes in the context of Kingdom Fungi, benefiting from our generation of a new collection of zygomycete genomes, including nine produced for this study. We identified lineage-specific genomic content that may contribute to the disparate biology observed in these zygomycetes. Our findings include the discovery of undescribed diversity in CotH, a Mucormycosis pathogenicity factor, which was found in a broad set of zygomycetes. Reconciliation analysis identified multiple duplication events and an expansion of CotH copies throughout the Mucoromycotina, Mortierellomycotina, Neocallimastigomycota, and Basidiobolus lineages. A kingdom-level phylogenomic analysis also identified new evolutionary relationships within the subphyla of Mucoromycota and Zoopagomycota, including supporting the sister-clade relationship between Glomeromycotina and Mortierellomycotina and the placement of Basidiobolus as sister to other Zoopagomycota lineages.

59 BASIC BIOLOGICAL SCIENCES↗

Box, stalked, and upside-down? Draft genomes from diverse jellyfish (Cnidaria, Acraspeda) lineages: Alatina alata (Cubozoa), Calvadosia cruxmelitensis (Staurozoa), and Cassiopea xamachana (Scyphozoa)

Abstract Background Anthozoa, Endocnidozoa, and Medusozoa are the 3 major clades of Cnidaria. Medusozoa is further divided into 4 clades, Hydrozoa, Staurozoa, Cubozoa, and Scyphozoa—the latter 3 lineages make up the clade Acraspeda. Acraspeda encompasses extraordinary diversity in terms of life history, numerous nuisance species, taxa with complex eyes rivaling other animals, and some of the most venomous organisms on the planet. Genomes have recently become available within Scyphozoa and Cubozoa, but there are currently no published genomes within Staurozoa and Cubozoa. Findings Here we present 3 new draft genomes of Calvadosia cruxmelitensis (Staurozoa), Alatina alata (Cubozoa), and Cassiopea xamachana (Scyphozoa) for which we provide a preliminary orthology analysis that includes an inventory of their respective venom-related genes. Additionally, we identify synteny between POU and Hox genes that had previously been reported in a hydrozoan, suggesting this linkage is highly conserved, possibly dating back to at least the last common ancestor of Medusozoa, yet likely independent of vertebrate POU-Hox linkages. Conclusions These draft genomes provide a valuable resource for studying the evolutionary history and biology of these extraordinary animals, and for identifying genomic features underlying venom, vision, and life history traits in Acraspeda.

Ohdera, Aki↗

Microbiome Comparison and Pathogen Identification for Three Migrating Passerines Captured During Spring Season in Jordan Using 16S rRNA Sequencing

Jordan is located on an important spot along the Mediterranean and Black Sea Flyway. Hundreds of migratory bird species have been identified stopping over in Jordan during spring and autumn migratory seasons. Compared to mammals and economically important birds, the microbiomes of wild bird species are severely understudied. Gut microbial composition is a valuable source of information that reflects food preferences, foraging behavior, and the risk of pathogen transmission to humans and other animals. In this study, we assessed the microbiome composition of three species of migrating passerines (willow warblers, lesser whitethroats, and common reed warblers) captured during the spring migration stopover in Jordan in 2023. A total of 59 fecal samples were selected evenly from the three species and subjected to 16S sequencing and microbiome analysis. Our objectives were to determine the diversity of bacteria in these three species, assess the amount of intra- and inter-specific variation, and detect pathogenic genera and species that could pose health risks to humans, domestic animals, and wildlife. Bacteria mainly belonged to the phyla Proteobacteria (62%), Actinobacteriota (18%), Firmicutes (13%), Cyanobacteria (5%), and Bacteroidota (1%). The results reveal that lesser whitethroats had the greatest variation in bacterial genus richness, Shannon diversity, and microbial composition compared to willow warblers and common reed warblers. The three bird species harbored several pathogenic genera and species, including Campylobacter, Enterococcus, Escherichia-Shigella, Mycoplasma, Rickettsia, Clostridium perfringens, and Vibrio cholerae. We suggest further investigation to understand the relationship between migratory behavior and their gut microbiome. We advocate for the use of advanced molecular techniques to characterize the pathogens found in migratory birds that might have public and environmental health impacts in addition to economic loss.

59 BASIC BIOLOGICAL SCIENCES↗

Acquisition of Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by Haemaphysalis longicornis (Acari: Ixodidae) through co-feeding with infected Dermacentor variabilis (Acari: Ixodidae) in the laboratory

Abstract Haemaphysalis longicornis (Neumann) is an invasive ixodid tick originating from eastern Asia which recently has become established in the United States. In its native range, this tick can transmit several pathogens to animals and humans, but little is known about its ability to acquire and transmit pathogens native to the United States. Geographic overlap with ticks such as Dermacentor variabilis (Say), a known vector of Rickettsia rickettsii, makes investigation into the interactions between H. longicornis and D. variabilis of interest to the public health community. Previous studies have shown that H. longicornis can serve as a competent vector of R. rickettsii under laboratory settings, but there is little information on its ability to acquire this pathogen via other biologically relevant routes, such as co-feeding. Here, we assess the ability of H. longicornis nymphs to acquire R. rickettsii through co-feeding with infected D. variabilis adults on a vertebrate animal model under laboratory conditions. The median infection prevalence in engorged H. longicornis nymphs across 8 cohorts was 0% with an interquartile range (IQR) of 4.13%. Following transstadial transmission, the median infection prevalence in flat females was 0.7% (IQR = 2.4%). Our results show that co-feeding transmission occurs at low levels in the laboratory between these 2 species. However, based on the relatively low transmission rates, this may not be a likely mechanism of R. rickettsii introduction to H. longicornis.

Entomology↗

Volatiles from the necrophagous fly Cochliomyia macellaria (Diptera: Calliphoridae) as indicators of Salmonella exposure

Blow flies (Diptera: Calliphoridae) are crucial in forensic investigations due to their association with both living and dead humans and other animals. Additionally, their interactions with various resources and potential as vectors of pathogens of humans and other animals, thus, make them potential tools for biosurveillance. This study investigated the potential of monitoring volatile organic compounds (VOCs) emitted by blow flies exposed to Salmonella as a method for pathogen surveillance. Adult blow flies ( Cochliomyia macellaria ) were exposed, or not, to Salmonella enterica . Following exposure, VOCs released by the blow flies were collected and analyzed using gas chromatography-mass spectrometry (GC-MS). Results indicate a treatment by time interaction (P < 0.01). Indicator species analysis identified a single compound significantly associated with S. enterica exposure (P = 0.02), Nonane, 2,2,4,4,6,8,8-heptamethyl, potentially indicating an immune system response. Given a compound indicating exposure was detected, future research should determine if more replicates could detect more differences after Salmonella ingestion. This research highlights the potential of blow flies as biosurveillance tools and the potential value of volatiles for assessing their exposure to pathogens.

59 BASIC BIOLOGICAL SCIENCES↗

Translocations are induced in hematopoietic stem cells after irradiation of fetal mice

Abstract Although mammalian fetuses have been suggested to be sensitive to radiation, an increased frequency of translocations was not observed in blood lymphocytes from atomic bomb (A-bomb) survivors who were exposed to the bomb in utero and examined as adults. Since experiments using hematopoietic cells of mice and rats confirmed this finding, it was hypothesized that either irradiated fetal hematopoietic stem cells (f-HSCs) cannot generate exchange-type chromosomal aberrations or cells bearing induced aberrations are eliminated before the animals reach adulthood. In the present study, pregnant mice (12.5–15.5 days post coitum [dpc]) were irradiated with 2 Gy of X-rays and long-term HSCs (LT-HSCs) were isolated 24 h later. Multicolor fluorescence in situ hybridization (mFISH) analysis of LT-HSC clones proliferated in vitro showed that nine out of 43 (21%) clones from fetuses and 21 out of 41 (51%) clones from mothers bore translocations. These results indicate that cells with translocations can arise in mouse f-HSCs but exist at a lower frequency than in the mothers 24 h after X-ray exposure. Thus, it seems likely that translocation-bearing f-HSCs are generated but subsequently disappear, so that the frequency of lymphocyte translocations may decrease and reach the control level by the time the animals reach adulthood.

60 APPLIED LIFE SCIENCES↗

Optimization of a Lethal, Combat-Relevant Model of Sterile Inflammation in Mice for Drug Candidate Screening

ABSTRACT Introduction Extensive trauma, commonly seen in wounded military Service Members, often leads to a severe sterile inflammation termed systemic inflammatory response syndrome (SIRS), which can progress to multiple organ dysfunction syndrome (MODS) and death. MODS is a serious threat to wounded Service Members, historically causing 10% of all deaths in trauma admissions at a forward deployed combat hospital. The importance of this problem will be exacerbated in large-scale combat operations, in which evacuation will be delayed and care of complex injuries at lower echelons of care may be prolonged. The main goal of this study was to optimize an existing mouse model of lethal SIRS/MODS as a therapeutic screening platform for the evaluation of immunomodulatory drugs. Materials and Methods Male C57BL/6 mice were euthanized, and the bones and muscles were collected and blended into a paste termed tissue–bone matrix (TBX). The TBX at 12.5%–20% relative to body weight of each recipient mouse was implanted into subcutaneous pouches created on the dorsum of anesthetized animals. Mice were observed for clinical scores for up to 48 hours postimplantation and euthanized at the preset point of moribundity. To test effects of anesthetics on TBX-induced mortality, animals received isoflurane or ketamine/xylazine (K/X). In a separate set of studies, mice received TBX followed by intraperitoneal injection with 20 mg/kg or 40 mg/kg Eritoran or a placebo carrier. All Eritoran studies were performed in a blinded fashion. Results We observed that K/X anesthesia significantly increased the lethality of the implanted TBX in comparison to inhaled anesthetics. Although all the mice anesthetized with isoflurane and implanted with 12.5% TBX survived for 24 hours, 60% of mice anesthetized with K/X were moribund by 24 hours postimplantation. To mimic more closely the timing of lethal SIRS/MODS following polytrauma in human patients, we extended observation to 48 hours. We performed TBX dose–response studies and found that as low as 15%, 17.5%, and 20% TBX caused moribundity/mortality in 50%, 80%, and 100% mice, respectively, over a 48-hour time period. With 17.5% TBX, we tested if moribundity/mortality could be rescued by anti-inflammatory drug Eritoran, a toll-like receptor 4 antagonist. Neither 20 mg/kg nor 40 mg/kg doses of Eritoran were found to be effective in this model. Conclusions We optimized a TBX mouse model of SIRS/MODS for the purpose of evaluating novel therapeutic interventions to prevent trauma-related pathophysiologies in wounded Service Members. Negative effects of K/X on lethality of TBX should be further evaluated, particularly in the light of widespread use of ketamine in treatment of pain. By mimicking muscle crush, bone fracture, and necrosis, the TBX model has pleiotropic effects on physiology and immunology that make it uniquely valuable as a screening tool for the evaluation of novel therapeutics against trauma-induced SIRS/MODS.

General & Internal Medicine↗

Multimodal imaging of hemorrhagic transformation biomarkers in an ischemic stroke model.

Abstract Hemorrhagic transformation of ischemic stroke has devastating consequences, with high mortality and poor functional outcomes. Animal models of ischemic stroke also demonstrate the potential for hemorrhagic transformation, which complicates biochemical characterization, treatment studies, and hinders poststroke functional outcomes in affected subjects. The incidence of hemorrhagic transformation of ischemic stroke in animal model research is not commonly reported. The postmortem brain of such cases presents a complex milieu of biomarkers due to the presence of healthy cells, regions of varying degrees of ischemia, dead and dying cells, dysregulated metabolites, and blood components (especially reactive Fe species released from lysed erythrocytes). To improve the characterization of hemorrhage biomarkers on an ischemic stroke background, we have employed a combination of histology, X-ray fluorescence imaging (XFI), and Fourier transform infrared (FTIR) spectroscopic imaging to assess 122 photothrombotic (ischemic) stroke brains. Rapid freezing preserves brain biomarkers in situ and minimizes metabolic artifacts due to postmortem ischemia. Analysis revealed that 25% of the photothrombotic models had clear signs of hemorrhagic transformation. The XFI and FTIR metabolites provided a quantitative method to differentiate key metabolic regions in these models. Across all hemorrhage cases, it was possible to consistently differentiate otherwise healthy tissue from other metabolically distinct regions, including the ischemic infarct, the ischemic penumbra, blood vessels, sites of hemorrhage, and a region surrounding the hemorrhage core that contained elevated lipid oxidation. Chemical speciation of deposited Fe demonstrates the presence of heme-Fe and accumulation of ferritin.

Pushie, M. J. (ORCID:0000000174945427)↗

Genome organization and botanical diversity

Abstract The rich diversity of angiosperms, both the planet's dominant flora and the cornerstone of agriculture, is integrally intertwined with a distinctive evolutionary history. Here, we explore the interplay between angiosperm genome organization and botanical diversity, empowered by genomic approaches ranging from genetic linkage mapping to analysis of gene regulation. Commonality in the genetic hardware of plants has enabled robust comparative genomics that has provided a broad picture of angiosperm evolution and implicated both general processes and specific elements in contributing to botanical diversity. We argue that the hardware of plant genomes—both in content and in dynamics—has been shaped by selection for rather substantial differences in gene regulation between plants and animals such as maize and human, organisms of comparable genome size and gene number. Their distinctive genome content and dynamics may reflect in part the indeterminate development of plants that puts strikingly different demands on gene regulation than in animals. Repeated polyploidization of plant genomes and multiplication of individual genes together with extensive rearrangement and differential retention provide rich raw material for selection of morphological and/or physiological variations conferring fitness in specific niches, whether natural or artificial. These findings exemplify the burgeoning information available to employ in increasing knowledge of plant biology and in modifying selected plants to better meet human needs.

Biochemistry & Molecular Biology↗

SPOTTED-LEAF7 targets the gene encoding β-galactosidase9, which functions in rice growth and stress responses

Abstract β-Galactosidases (Bgals) remove terminal β-D-galactosyl residues from the nonreducing ends of β-D-galactosidases and oligosaccharides. Bgals are present in bacteria, fungi, animals, and plants and have various functions. Despite the many studies on the evolution of BGALs in plants, their functions remain obscure. Here, we identified rice (Oryza sativa) β-galactosidase9 (OsBGAL9) as a direct target of the heat stress-induced transcription factor SPOTTED-LEAF7 (OsSPL7), as demonstrated by protoplast transactivation analysis and yeast 1-hybrid and electrophoretic mobility shift assays. Knockout plants for OsBGAL9 (Osbgal9) showed short stature and growth retardation. Histochemical β-glucuronidase (GUS) analysis of transgenic lines harboring an OsBGAL9pro:GUS reporter construct revealed that OsBGAL9 is mainly expressed in internodes at the mature stage. OsBGAL9 expression was barely detectable in seedlings under normal conditions but increased in response to biotic and abiotic stresses. Ectopic expression of OsBGAL9 enhanced resistance to the rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, as well as tolerance to cold and heat stress, while Osbgal9 mutant plants showed the opposite phenotypes. OsBGAL9 localized to the cell wall, suggesting that OsBGAL9 and its plant putative orthologs likely evolved functions distinct from those of its closely related animal enzymes. Enzyme activity assays and analysis of the cell wall composition of OsBGAL9 overexpression and mutant plants indicated that OsBGAL9 has activity toward galactose residues of arabinogalactan proteins (AGPs). Our study clearly demonstrates a role for a member of the BGAL family in AGP processing during plant development and stress responses.

54 ENVIRONMENTAL SCIENCES↗

New Approach Methodology for Assessing Inhalation Risks of a Contact Respiratory Cytotoxicant: Computational Fluid Dynamics-Based Aerosol Dosimetry Modeling for Cross-Species and In Vitro Comparisons

Regulatory agencies are considering alternative approaches to assessing inhalation toxicity that utilizes in vitro studies with human cells and in silico modeling in lieu of additional animal studies. In support of this goal, computational fluid-particle dynamics models were developed to estimate site-specific deposition of inhaled aerosols containing the fungicide, chlorothalonil, in the rat and human for comparisons to prior rat inhalation studies and new human in vitro studies. Under bioassay conditions, the deposition was predicted to be greatest at the front of the rat nose followed by the anterior transitional epithelium and larynx corresponding to regions most sensitive to local contact irritation and cytotoxicity. For humans, simulations of aerosol deposition covering potential occupational or residential exposures (1–50 µm diameter) were conducted using nasal and oral breathing. Aerosols in the 1–5 µm range readily penetrated the deep region of the human lung following both oral and nasal breathing. Under actual use conditions (aerosol formulations >10 µm), the majority of deposited doses were in the upper conducting airways. Beyond the nose or mouth, the greatest deposition in the pharynx, larynx, trachea, and bronchi was predicted for aerosols in the 10–20 µm size range. Only small amounts of aerosols >20 µm penetrated past the pharyngeal region. Using the ICRP clearance model, local retained tissue dose metrics including maximal concentrations and areas under the curve were calculated for each airway region following repeated occupational exposures. These results are directly comparable with benchmark doses from in vitro toxicity studies in human cells leading to estimated human equivalent concentrations that reduce the reliance on animals for risk assessments.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗

Gene Expression and DNA Methylation Alterations in the Glycine N-Methyltransferase Gene in Diet-Induced Nonalcoholic Fatty Liver Disease-Associated Carcinogenesis

Abstract Nonalcoholic fatty liver disease (NAFLD) is becoming a major etiological risk factor for hepatocellular carcinoma (HCC) in the United States and other Western countries. In this study, we investigated the role of gene-specific promoter cytosine DNA methylation and gene expression alterations in the development of NAFLD-associated HCC in mice using (1) a diet-induced animal model of NAFLD, (2) a Stelic Animal Model of nonalcoholic steatohepatitis-derived HCC, and (3) a choline- and folate-deficient (CFD) diet (CFD model). We found that the development of NAFLD and its progression to HCC was characterized by down-regulation of glycine N-methyltransferase (Gnmt) and this was mediated by progressive Gnmt promoter cytosine DNA hypermethylation. Using a panel of genetically diverse inbred mice, we observed that Gnmt down-regulation was an early event in the pathogenesis of NAFLD and correlated with the extent of the NAFLD-like liver injury. Reduced GNMT expression was also found in human HCC tissue and liver cancer cell lines. In in vitro experiments, we demonstrated that one of the consequences of GNMT inhibition was an increase in genome methylation facilitated by an elevated level of S-adenosyl-L-methionine. Overall, our findings suggest that reduced Gnmt expression caused by promoter hypermethylation is one of the key molecular events in the development of NAFLD-derived HCC and that assessing Gnmt methylation level may be useful for disease stratification.

Borowa-Mazgaj, Barbara↗

Sendai virus intra-host population dynamics and host immunocompetence influence viral virulence during in vivo passage

In vivo serial passage of non-pathogenic viruses has been shown to lead to increased viral virulence, and although the precise mechanism(s) are not clear, it is known that both host and viral factors are associated with increased pathogenicity. Under- or overnutrition leads to a decreased or dysregulated immune response and can increase viral mutant spectrum diversity and virulence. The objective of this study was to identify the role of viral mutant spectra dynamics and host immunocompetence in the development of pathogenicity during in vivo passage. Because the nutritional status of the host has been shown to affect the development of viral virulence, the diet of animal model reflected two extremes of diets which exist in the global population, malnutrition and obesity. Sendai virus was serially passaged in groups of mice with differing nutritional status followed by transmission of the passaged virus to a second host species, guinea pigs. Viral population dynamics were characterized using deep sequence analysis and computational modeling. Histopathology, viral titer and cytokine assays were used to characterize viral virulence. Viral virulence increased with passage and the virulent phenotype persisted upon passage to a second host species. Additionally, nutritional status of mice during passage influenced the phenotype. Sequencing revealed the presence of several non-synonymous changes in the consensus sequence associated with passage, a majority of which occurred in the hemagglutinin-neuraminidase and polymerase genes, as well as the presence of persistent high frequency variants in the viral population. In particular, an N1124D change in the consensus sequences of the polymerase gene was detected by passage 10 in a majority of the animals. In vivo comparison of an 1124D plaque isolate to a clone with 1124N genotype indicated that 1124D was associated with increased virulence.

59 BASIC BIOLOGICAL SCIENCES↗

Irradiation of the kidneys causes pathologic remodeling in the nontargeted heart: A role for the immune system

Cardiac disease is a frequent and significant adverse event associated with radiotherapy for cancer. Identifying the underlying mechanism responsible for radiation injury to the heart will allow interventions to be developed. In the present study, we tested if local kidney irradiation results in remodeling of the shielded, nontargeted heart. One kidney, two kidneys, or the total body of male WAG and Dahl SS rats were irradiated with 10 Gy of X-rays. Local kidney irradiation resulted in systemic hypertension, increased BUN, infiltration of T lymphocytes, natural killer cells, and macrophages into the renal cortex and medulla, and renal fibrosis. Local irradiation of kidneys in WAG rats resulted in remodeling in the nontargeted heart after 120 days, manifested by perivascular fibrosis and increased interventricular septal thickness, but was not seen in Dahl SS rats due to a high baseline level of fibrosis in the sham-irradiated animals. Genetic depletion of T cells mitigated the nephropathy after local kidney irradiation, indicating a role for the immune system in mediating this outcome. Local kidney irradiation resulted in a cascade of pro-inflammatory cytokines and low-molecular weight metabolites into the circulation associated with transmission of signals resulting in pathologic remodeling in the nontargeted heart. A new model is proposed whereby radiation-induced cardiac remodeling in susceptible animals is indirect, with lower hemi body organs such as the kidney exporting factors into the circulation that cause remodeling outside of the irradiated field in the shielded, nontargeted heart. This nontargeted effect appears to be mediated, in part, by the immune system.

60 APPLIED LIFE SCIENCES↗

2022 Prentice Award Lecture: Advancing Retinal Imaging and Visual Function in Patient Management and Disease Mechanisms

Patient-based research plays a key role in probing basic visual mechanisms. Less-well recognized is the role of patient-based retinal imaging and visual function studies in elucidating disease mechanisms, which are accelerated by advances in imaging and function techniques and are most powerful when combined with the results from histology and animal models. A patient's visual complaints can be one key to patient management, but human data are also key to understanding disease mechanisms. Unfortunately, pathological changes can be difficult to detect. Before advanced retinal imaging, the measurement of visual function indicated the presence of pathological changes that were undetectable with existing clinical examination. Over the past few decades, advances in retinal imaging have increasingly revealed the unseen. This has led to great strides in the management of many diseases, particularly diabetic retinopathy and macular edema, and age-related macular degeneration. It is likely widely accepted that patient-based research, as in clinical trials, led to such positive outcomes. Both visual function measures and advanced retinal imaging have clearly demonstrated differences among retinal diseases. Contrary to initial thinking, sight-threatening damage in diabetes occurs to the outer retina and not only to the inner retina. This has been clearly indicated in patient results but has only gradually entered the clinical classifications and understanding of disease etiology. There is strikingly different pathophysiology for age-related macular degeneration compared with photoreceptor and retinal pigment epithelial genetic defects, yet research models and even some treatments confuse these. It is important to recognize the role that patient-based research plays in probing basic visual mechanisms and elucidating disease mechanisms, combining these findings with the concepts from histology and animal models. Thus, this article combines sample instrumentation from my laboratory and progress in the fields of retinal imaging and visual function.

60 APPLIED LIFE SCIENCES↗

Superresolution Diffuse Optical Imaging by Localization of Fluorescence

The multiple scattering of light presents major challenges in realizing useful in vivo imaging at tissue depths of more than about one millimeter, where many answers to health questions lie. Visible through near-infrared photons can be readily and safely detected through centimeters of tissue; however, limited information is available for image formation. One strategy for obtaining images is to model the photon transport and a simple incoherent model is the diffusion equation approximation to the Boltzmann transport equation. Such an approach provides a prediction of the mean intensity of heavily scattered light and hence provides a forward model for optimization-based computational imaging. While diffuse optical imaging methods have received substantial attention, they remain restricted in terms of resolution because of the loss of high-spatial-frequency information that is associated with the multiple scattering of photons. Consequently, only relatively large inhomogeneities, such as tumors or organs in small animals, can be effectively resolved. We introduce a superresolution imaging approach based on point localization in a diffusion framework that enables over two orders of magnitude improvement in the spatial resolution of diffuse optical imaging. The method is demonstrated experimentally by localizing a fluorescent inhomogeneity in a highly scattering slab and characterizing the localization uncertainty. The approach allows imaging through centimeters of tissue with a resolution of tens of microns, thereby enabling cells or cell clusters to be resolved. More generally, this high-resolution imaging approach could be applied with any physical transport or wave model and hence to a broad class of physical problems. Finally, paired with a suitable optical contrast mechanism, as can be realized with targeted fluorescent molecules or genetically modified animals, superresolution diffuse imaging should open alternative dimensions for in vivo applications.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

Seedling Chloroplast Responses Induced by N-Linolenoylethanolamine Require Intact G-Protein Complexes

In animals, several long-chain N-acylethanolamines (NAEs) have been identified as endocannabinoids and are autocrine signals that operate through cell surface G-protein coupled, cannabinoid receptors. Despite the occurrence of N-acylethanolamines in land plants, including non-vascular plants, their precise signaling properties and molecular targets are not well defined. Here we show that the activity of N-linolenoylethanolamine (NAE 18:3) requires an intact G-protein complex. Specifically, genetic ablation of the Gβγ dimer or loss of the full set of atypical Gα subunits strongly attenuates an NAE 18:3-induced de-greening of cotyledons in Arabidopsis seedlings. This effect involves, at least in part, transcriptional regulation of chlorophyll biosynthesis and catabolism genes. In addition, there is feedforward transcriptional control of G-protein signaling components and G-protein interactors. These results are consistent with NAE 18:3 being a lipid signaling molecule in plants with a requirement for G-proteins to mediate signal transduction, a situation similar, but not identical, to the action of NAE endocannabinoids in animal systems.

59 BASIC BIOLOGICAL SCIENCES↗