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Context: In response to the COVID-19 pandemic, Congress passed the American Rescue Plan Act of 2021 (ARPA) that included a historic investment in the public health workforce., Program: Charged with implementing this investment, the U.S. Centers for Disease Control and Prevention (CDC) launched the Public Health Infrastructure Grant (PHIG). PHIG builds on CDC's experience working with state, local, and territorial public health departments and represents a new approach to strengthening the public health workforce., Implementation: Specifically, PHIG incorporates features that allow these public health departments to prioritize and tailor the funding to meet their communities' needs: 1) focus on workforce as core infrastructure, 2) streamlined programmatic and administrative requirements, 3) more equitable funding approach, and 4) enhanced support from national partners and CDC., Discussion: The goal is to optimize the unprecedented opportunity afforded by ARPA and lead to a stronger public health workforce and infrastructure across the United States., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

A long-standing question in vision science is how the three cone photoreceptor types-long (L), medium (M), and short (S) wavelength sensitive-combine to generate our perception of color. Hue perception can be described along two opponent axes: red-green and blue-yellow. Psychophysical measurements of color appearance indicate that the cone inputs to the red-green and blue-yellow opponent axes are M vs. L + S and L vs. M + S, respectively. However, the "cardinal directions of color space" revealed by psychophysical measurements of color detection thresholds following adaptation are L vs. M and S vs. L + M. These cardinal directions match the most common cone-opponent retinal ganglion cells (RGCs) in the primate retina. Accordingly, the cone opponency necessary for color appearance is thought to be established in the cortex. While neurons with the appropriate M vs. L + S and L vs. M + S opponency have been reported in the retina and lateral geniculate nucleus, their existence continues to be debated. Resolving this long-standing debate is necessary because a complete account of the cone opponency in the retinal output is critical for understanding how downstream neural circuits process color. Here, we performed adaptive optics calcium imaging to noninvasively measure foveal RGC light responses in the living Macaca fascicularis eye. We confirm the presence of L vs. M + S and M vs. L + S neurons with noncardinal cone opponency and demonstrate that cone-opponent signals in the retinal output are more diverse than classically thought., (Copyright © 2024 the authors.)

Considerable progress has been made in studying the receptive fields of the most common primate retinal ganglion cell (RGC) types, such as parasol RGCs. Much less is known about the rarer primate RGC types and the circuitry that gives rise to noncanonical receptive field structures. The goal of this study was to analyze synaptic inputs to smooth monostratified RGCs to determine the origins of their complex spatial receptive fields, which contain isolated regions of high sensitivity called "hotspots." Interestingly, smooth monostratified RGCs co-stratify with the well-studied parasol RGCs and are thus constrained to receiving input from bipolar and amacrine cells with processes sharing the same layer, raising the question of how their functional differences originate. Through 3D reconstructions of circuitry and synapses onto ON smooth monostratified and ON parasol RGCs from central macaque retina, we identified four distinct sampling strategies employed by smooth and parasol RGCs to extract diverse response properties from co-stratifying bipolar and amacrine cells. The two RGC types differed in the proportion of amacrine cell input, relative contributions of co-stratifying bipolar cell types, amount of synaptic input per bipolar cell, and spatial distribution of bipolar cell synapses. Our results indicate that the smooth RGC's complex receptive field structure arises through spatial asymmetries in excitatory bipolar cell input which formed several discrete clusters comparable with physiologically measured hotspots. Taken together, our results demonstrate how the striking differences between ON parasol and ON smooth monostratified RGCs arise from distinct strategies for sampling a common set of synaptic inputs., (Copyright © 2024 Patterson et al.)

Zebrafish retinal cone signals shift in spectral shape through larval, juvenile, and adult development as expression patterns of eight cone-opsin genes change. An algorithm extracting signal amplitudes for the component cone spectral types is developed and tested on two thyroxin receptor β2 (trβ2) gain-of-function lines crx:mYFP-2A-trβ2 and gnat2:mYFP-2A-trβ2 , allowing correlation between opsin signaling and opsin immunoreactivity in lines with different developmental timing and cell-type expression of this red-opsin-promoting transgene. Both adult transgenics became complete, or nearly complete, "red-cone dichromats," with disproportionately large long-wavelength-sensitive (LWS)1 opsin amplitudes as compared with controls, where LWS1 and LWS2 amplitudes were about equal, and significant signals from SWS1, SWS2, and Rh2 opsins were detected. But in transgenic larvae and juveniles of both lines it was LWS2 amplitudes that increased, with LWS1 cone signals rarely encountered. In gnat2:mYFP-2A-trβ2 embryos at 5 d postfertilization (dpf), red-opsin immunoreactive cone density doubled, but red-opsin amplitudes (LWS2) increased <10%, and green-opsin, blue-opsin, and UV-opsin signals were unchanged, despite co-expressed red opsins, and the finding that an sws1 UV-opsin reporter gene was shut down by the gnat2:mYFP-2A-trβ2 transgene. By contrast both LWS2 red-cone amplitudes and the density of red-cone immunoreactivity more than doubled in 5-dpf crx:mYFP-2A-trβ2 embryos, while UV-cone amplitudes were reduced 90%. Embryonic cones with trβ2 gain-of-function transgenes were morphologically distinct from control red, blue or UV cones, with wider inner segments and shorter axons than red cones, suggesting cone spectral specification, opsin immunoreactivity and shape are influenced by the abundance and developmental timing of trβ2 expression., Competing Interests: The authors declare no competing financial interests., (Copyright © 2022 Nelson et al.)

Ganglion cells are the projection neurons of the retina. Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and also receive input from rods and cones via bipolar cells and amacrine cells. In primates, multiple types of ipRGCs have been identified. The ipRGCs with somas in the ganglion cell layer have been studied extensively, but less is known about those with somas in the inner nuclear layer, the "displaced" cells. To investigate their synaptic inputs, three sets of horizontal, ultrathin sections through central macaque retina were collected using serial block-face scanning electron microscopy. One displaced ipRGC received nearly all of its excitatory inputs from ON bipolar cells and would therefore be expected to have ON responses to light. In each of the three volumes, there was also at least one cell that had a large soma in the inner nuclear layer, varicose axons and dendrites with a large diameter that formed large, extremely sparse arbor in the outermost stratum of the inner plexiform layer. They were identified as the displaced M1 type of ipRGCs based on this morphology and on the high density of granules in their somas. They received extensive input from amacrine cells, including the dopaminergic type. The vast majority of their excitatory inputs were from OFF bipolar cells, including two subtypes with extensive input from the primary rod pathway. They would be expected to have OFF responses to light stimuli below the threshold for melanopsin or soon after the offset of a light stimulus., (© 2022. The Author(s).)

The detection of motion direction is a fundamental visual function and a classic model for neural computation. In the non-primate retina, direction selectivity arises in starburst amacrine cell (SAC) dendrites, which provide selective inhibition to direction-selective retinal ganglion cells (dsRGCs). Although SACs are present in primates, their connectivity and the existence of dsRGCs remain open questions. Here, we present a connectomic reconstruction of the primate ON SAC circuit from a serial electron microscopy volume of the macaque central retina. We show that the structural basis for the SACs' ability to confer directional selectivity on postsynaptic neurons is conserved. SACs selectively target a candidate homolog to the mammalian ON-sustained dsRGCs that project to the accessory optic system (AOS) and contribute to gaze-stabilizing reflexes. These results indicate that the capacity to compute motion direction is present in the retina, which is earlier in the primate visual system than classically thought., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light by virtue of containing melanopsin which peaks at about 483 nm. However, in primates, ipRGCs also receive color opponent inputs from short-wavelength-sensitive (S) cone circuits that are well-suited to encode circadian changes in the color of the sky that accompany the rising and setting sun. Here, we review the retinal circuits that endow primate ipRGCs with the cone-opponency capable of encoding the color of the sky and contributing to the wide-ranging effects of short-wavelength light on ipRGC-mediated non-image-forming visual function in humans., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In primates, broad thorny retinal ganglion cells are highly sensitive to small, moving stimuli. They have tortuous, fine dendrites with many short, spine-like branches that occupy three contiguous strata in the middle of the inner plexiform layer. The neural circuits that generate their responses to moving stimuli are not well-understood, and that was the goal of this study. A connectome from central macaque retina was generated by serial block-face scanning electron microscopy, a broad thorny cell was reconstructed, and its synaptic inputs were analyzed. It received fewer than 2% of its inputs from both ON and OFF types of bipolar cells; the vast majority of its inputs were from amacrine cells. The presynaptic amacrine cells were reconstructed, and seven types were identified based on their characteristic morphology. Two types of narrow-field cells, knotty bistratified Type 1 and wavy multistratified Type 2, were identified. Two types of medium-field amacrine cells, ON starburst and spiny, were also presynaptic to the broad thorny cell. Three types of wide-field amacrine cells, wiry Type 2, stellate wavy, and semilunar Type 2, also made synapses onto the broad thorny cell. Physiological experiments using a macaque retinal preparation in vitro confirmed that broad thorny cells received robust excitatory input from both the ON and the OFF pathways. Given the paucity of bipolar cell inputs, it is likely that amacrine cells provided much of the excitatory input, in addition to inhibitory input., (© 2021 Wiley Periodicals LLC.)

The United States skin of color population is increasing. Consequently, the importance of skin of color education in dermatology residency programs will continue to grow. Previous data has shown a lack of formal education on skin of color across residency programs. In order to address this identified knowledge gap, we created a curriculum focusing exclusively on skin of color for dermatology residents. The purpose of this pilot study was to examine the effect of a week-long curriculum on the perception of dermatology residents&rsquo; comfort level treating patients of color and to determine if this type of curriculum could be expanded to other dermatology residents. Results demonstrated a significant increase when residents were asked to rate their overall confidence in managing patients with skin of color on a scale from 1 (minimally confident) to 10 (extremely confident) pre- and post-intervention. Overall, 100% of learners felt that their ability to care for patients of color was improved by this curriculum and that a skin of color curriculum should be an annual component of their dermatology academic curriculum. J Drugs Dermatol. 2021;20(7):786-789. doi:10.36849/JDD.6193.

Parasol cells are one of the major types of primate retinal ganglion cells. The goal of this study was to describe the synaptic inputs that shape the light responses of the ON type of parasol cells, which are excited by increments in light intensity. A connectome from central macaque retina was generated by serial blockface scanning electron microscopy. Six neighboring ON parasol cells were reconstructed, and their synaptic inputs were analyzed. On average, they received 21% of their input from bipolar cells, excitatory local circuit neurons receiving input from cones. The majority of their input was from amacrine cells, local circuit neurons of the inner retina that are typically inhibitory. Their contributions to the neural circuit providing input to parasol cells are not well-understood, and the focus of this study was on the presynaptic wide-field amacrine cells, which provided 17% of the input to ON parasol cells. These are GABAergic amacrine cells with long, relatively straight dendrites, and sometimes also axons, that run in a single, narrow stratum of the inner plexiform layer. The presynaptic wide-field amacrine cells were reconstructed, and two types were identified based on their characteristic morphology. One presynaptic amacrine cell was identified as semilunar type 2, a polyaxonal cell that is electrically coupled to ON parasol cells. A second amacrine was identified as wiry type 2, a type known to be sensitive to motion. These inputs likely make ON parasol cells more sensitive to stimuli that are rapidly changing outside their classical receptive fields., (© 2019 Wiley Periodicals, Inc.)

Melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) synchronize our biological clocks with the external light/dark cycle [1]. In addition to photoentrainment, they mediate the effects of light experience as a central modulator of mood, learning, and health [2]. This makes a complete account of the circuity responsible for ipRGCs' light responses essential to understanding their diverse roles in our well-being. Considerable progress has been made in understanding ipRGCs' melanopsin-mediated responses in rodents [3-5]. However, in primates, ipRGCs also have a rare blue-OFF response mediated by an unknown short-wavelength-sensitive (S)-cone circuit [6]. Identifying this S-cone circuit is particularly important because ipRGCs mediate many of the wide-ranging effects of short-wavelength light on human biology. These effects are often attributed to melanopsin, but there is evidence for an S-cone contribution as well [7, 8]. Here, we tested the hypothesis that the S-OFF response is mediated by the S-ON pathway through inhibitory input from an undiscovered S-cone amacrine cell. Using serial electron microscopy in the macaque retina, we reconstructed the neurons and synapses of the S-cone connectome, revealing a novel inhibitory interneuron, an amacrine cell, receiving excitatory glutamatergic input exclusively from S-ON bipolar cells. This S-cone amacrine cell makes highly selective inhibitory synapses onto ipRGCs, resulting in a blue-OFF response. Identification of the S-cone amacrine cell provides the missing component of an evolutionarily ancient circuit using spectral information for non-image forming visual functions., Competing Interests: Declaration of Interests The University of Washington has submitted provisional patent application (628504893) disclosing Systems, Methods, and Devices for Stimulating Circadian Rhythms (Authors: S.S.P., J.A.K., M.N., J.N.)., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The spatial and spectral topography of the cone mosaic set the limits for detection and discrimination of chromatic sinewave gratings. Here, we sought to compare the spatial characteristics of mechanisms mediating hue perception against those mediating chromatic detection in individuals with known spectral topography and with optical aberrations removed with adaptive optics. Chromatic detection sensitivity in general exceeded previous measurements and decreased monotonically for increasingly skewed cone spectral compositions. The spatial grain of hue perception was significantly coarser than chromatic detection, consistent with separate neural mechanisms for color vision operating at different spatial scales.

The human long- and middle-wavelength sensitive cone opsin genes exhibit an extraordinary degree of haplotype diversity that results from recombination mechanisms that have intermixed the genes. As a first step in expression, genes-including the protein coding exons and intervening introns-are transcribed. Next, transcripts are spliced to remove the introns and join the exons to generate a mature message that codes for the protein. Important information necessary for splicing is contained within exons, and is overlaid by the protein code. Intermixing the long- and middle-wavelength sensitive cone opsin genes has disrupted the splicing code, leading to exclusion of some exons from the mature message and is associated with several vision disorders including nearsightedness, cone dystrophy, and color vision deficiencies., Competing Interests: Declaration of Interests: Maureen Neitz and Jay Neitz have significant financial interest in the company Waveshift and its subsidiaries, SightGlass and Visu, and receive royalties from the University of Washington for intellectual property, including patents, licensed to these companies. Sara Patterson: none

Midget retinal ganglion cells (RGCs) make up the majority of foveal RGCs in the primate retina. The receptive fields of midget RGCs exhibit both spectral and spatial opponency and are implicated in both color and achromatic form vision, yet the exact mechanisms linking their responses to visual perception remain unclear. Efforts to develop color vision models that accurately predict all the features of human color and form vision based on midget RGCs provide a case study connecting experimental and theoretical neuroscience, drawing on diverse research areas such as anatomy, physiology, psychophysics, and computer vision. Recent technological advances have allowed researchers to test some predictions of color vision models in new and precise ways, producing results that challenge traditional views. Here, we review the progress in developing models of color-coding receptive fields that are consistent with human psychophysics, the biology of the primate visual system and the response properties of midget RGCs.

Midget retinal ganglion cells (RGCs) are the most common RGC type in the primate retina. Their responses have been proposed to mediate both color and spatial vision, yet the specific links between midget RGC responses and visual perception are unclear. Previous research on the dual roles of midget RGCs has focused on those comparing long (L) vs. middle (M) wavelength sensitive cones. However, there is evidence for several other rare midget RGC subtypes receiving S-cone input, but their role in color and spatial vision is uncertain. Here, we confirm the existence of the single S-cone center OFF midget RGC circuit in the central retina of macaque monkey both structurally and functionally. We investigated the receptive field properties of the S-OFF midget circuit with single cell electrophysiology and 3D electron microscopy reconstructions of the upstream circuitry. Like the well-studied L vs. M midget RGCs, the S-OFF midget RGCs have a center-surround receptive field consistent with a role in spatial vision. While spectral opponency in a primate RGC is classically assumed to contribute to hue perception, a role supporting edge detection is more consistent with the S-OFF midget RGC receptive field structure and studies of hue perception.

There are four cone morphologies in zebrafish, corresponding to UV (U), blue (B), green (G), and red (R)-sensing types; yet genetically, eight cone opsins are expressed. How eight opsins are physiologically siloed in four cone types is not well understood, and in larvae, cone physiological spectral peaks are unstudied. We use a spectral model to infer cone wavelength peaks, semisaturation irradiances, and saturation amplitudes from electroretinogram (ERG) datasets composed of multi-wavelength, multi-irradiance, aspartate-isolated, cone-PIII signals, as compiled from many 5- to 12-day larvae and 8- to 18-month-old adult eyes isolated from wild-type (WT) or roy orbison (roy) strains. Analysis suggests (in nm) a seven-cone, U-360/B1-427/B2-440/G1-460/G3-476/R1-575/R2-556, spectral physiology in WT larvae but a six-cone, U-349/B1-414/G3-483/G4-495/R1-572/R2-556, structure in WT adults. In roy larvae, there is a five-cone structure: U-373/B2-440/G1-460/R1-575/R2-556; in roy adults, there is a four-cone structure, B1-410/G3-482/R1-571/R2-556. Existence of multiple B, G, and R types is inferred from shifts in peaks with red or blue backgrounds. Cones were either high or low semisaturation types. The more sensitive, low semisaturation types included U, B1, and G1 cones [3.0-3.6 log(quanta·μm-2·s-1)]. The less sensitive, high semisaturation types were B2, G3, G4, R1, and R2 types [4.3-4.7 log(quanta·μm-2·s-1)]. In both WT and roy, U- and B- cone saturation amplitudes were greater in larvae than in adults, while G-cone saturation levels were greater in adults. R-cone saturation amplitudes were the largest (50-60% of maximal dataset amplitudes) and constant throughout development. WT and roy larvae differed in cone signal levels, with lesser UV- and greater G-cone amplitudes occurring in roy, indicating strain variation in physiological development of cone signals. These physiological measures of cone types suggest chromatic processing in zebrafish involves at least four to seven spectral signal processing pools.

There are more than 30 distinct types of mammalian retinal ganglion cells, each sensitive to different features of the visual environment. In rabbit retina, they can be grouped into four classes according to their morphology and stratification of their dendrites in the inner plexiform layer (IPL). The goal of this study was to describe the synaptic inputs to one type of Class IV ganglion cell, the third member of the sparsely branched Class IV cells (SB3). One cell of this type was partially reconstructed in a retinal connectome developed using automated transmission electron microscopy (ATEM). It had slender, relatively straight dendrites that ramify in the sublamina a of the IPL. The dendrites of the SB3 cell were always postsynaptic in the IPL, supporting its identity as a ganglion cell. It received 29% of its input from bipolar cells, a value in the middle of the range for rabbit retinal ganglion cells studied previously. The SB3 cell typically received only one synapse per bipolar cell from multiple types of presumed OFF bipolar cells; reciprocal synapses from amacrine cells at the dyad synapses were infrequent. In a few instances, the bipolar cells presynaptic to the SB3 ganglion cell also provided input to an amacrine cell presynaptic to the ganglion cell. There was apparently no crossover inhibition from narrow-field ON amacrine cells. Most of the amacrine cell inputs were from axons and dendrites of GABAergic amacrine cells, likely providing inhibitory input from outside the classical receptive field.

Considerable theoretical and experimental effort has been dedicated to understanding how neural circuits detect visual motion. In primates, much is known about the cortical circuits that contribute to motion processing, but the role of the retina in this fundamental neural computation is poorly understood. Here, we used a combination of extracellular and whole-cell recording to test for motion sensitivity in the two main classes of output neurons in the primate retina-midget (parvocellular-projecting) and parasol (magnocellular-projecting) ganglion cells. We report that parasol, but not midget, ganglion cells are motion sensitive. This motion sensitivity is present in synaptic excitation and disinhibition from presynaptic bipolar cells and amacrine cells, respectively. Moreover, electrical coupling between neighboring bipolar cells and the nonlinear nature of synaptic release contribute to the observed motion sensitivity. Our findings indicate that motion computations arise far earlier in the primate visual stream than previously thought., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Predicting species distributions has long been a valuable tool to plan and focus efforts for biodiversity conservation, particularly because such an approach allows researchers and managers to evaluate species distribution changes in response to various threats. Utilizing data from a long-term monitoring program and land cover data sets, we modeled the probability of occupancy and colonization for 38 bird Species of Greatest Conservation Need (SGCN) in the robust design occupancy modeling framework, and used results from the best models to predict occupancy and colonization on the Iowa landscape. Bird surveys were conducted at 292 properties from April to October, 2006-2014. We calculated landscape habitat characteristics at multiple spatial scales surrounding each of our surveyed properties to be used in our models and then used kriging in ArcGIS to create predictive maps of species distributions. We validated models with data from 2013 using the area under the receiver operating characteristic curve (AUC). Probability of occupancy ranged from 0.001 (SE < 0.001) to 0.995 (SE = 0.004) for all species and probability of colonization ranged from 0.001 (SE < 0.001) to 0.999 (SE < 0.001) for all species. AUC values for predictive models ranged from 0.525-0.924 for all species, with 17 species having predictive models considered useful (AUC > 0.70). The most important predictor for occupancy of grassland birds was percentage of the landscape in grassland habitat, and the most important predictor for woodland birds was percentage of the landscape in woodland habitat. This emphasizes the need for managers to restore specific habitats on the landscape. In an era during which funding continues to decrease for conservation agencies, our approach aids in determining where to focus limited resources to best conserve bird species of conservation concern.

Bioluminescent and fluorescent reporter systems have enabled the rapid and continued growth of the optical imaging field over the last two decades. Of particular interest has been noninvasive signal detection from mammalian tissues under both cell culture and whole animal settings. Here we report on the advantages and limitations of imaging using a recently introduced bacterial luciferase (lux) reporter system engineered for increased bioluminescent expression in the mammalian cellular environment. Comparison with the bioluminescent firefly luciferase (Luc) system and green fluorescent protein system under cell culture conditions demonstrated a reduced average radiance, but maintained a more constant level of bioluminescent output without the need for substrate addition or exogenous excitation to elicit the production of signal. Comparison with the Luc system following subcutaneous and intraperitoneal injection into nude mice hosts demonstrated the ability to obtain similar detection patterns with in vitro experiments at cell population sizes above 2.5 × 10(4) cells but at the cost of increasing overall image integration time.

Background: The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo., Methodology/principal Findings: Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2)) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH(2) supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background., Conclusions/significance: The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) has been shown to be a useful target for molecular assays that quantify form- or clade-specific RNA transcript concentrations as a proxy for the carbon fixation activity of marine phytoplankton. To improve the phylogenetic specificity and sensitivity of RNA probe hybridization methods, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay has been reported for diatom and pelagophyte rbcL RNA. Here we detail enhancements made to this PCR method and development of additional assays to specifically quantify rbcL expression from haptophytes, Synechococcus and high-light Prochlorococcus. In vitro RNA transcripts were tested to demonstrate specificity and quantitative accuracy. Application of these methods on seawater samples from two depth profiles in the northern Gulf of Mexico showed a fair degree of agreement between PCR and hybridization results, with results for the chromophytic or form ID rbcL-containing organisms having better agreement between the two methods. Diatoms and other heterokonts were shown to be the primary carbon fixers at these locations by PCR, in agreement with greater form ID rbcL RNA measured by hybridization.

Over the past several decades healthcare delivery systems have received increased pressure to become more efficient from both a managerial and patient perspective. Many researchers have turned to simulation to analyze the complex systems that exist within hospitals, but surprisingly few have published guidelines on how to analyze models with multiple performance measures. Moreover, the published literature has failed to address ways of analyzing performance along more than one dimension, such as performance by day of the week, patient type, facility, time period, or some combination of these attributes. Despite this void in the literature, understanding performance along these dimensions is critical to understanding the root of operational problems in almost any daily clinic operation. This paper addresses the problem of multiple responses in simulation experiments of outpatient clinics by developing a stratification framework and an evaluation construct by which managers can compare several operationally different outpatient systems across multiple performance measure dimensions. This approach is applied to a discrete-event simulation model of a real-life, large-scale oncology center to evaluate its operational performance as improvement initiatives affecting scheduling practices, process flow, and resource levels are changed. Our results show a reduction in patient wait time and resource overtime across multiple patient classes, facilities, and days of the week. This research has already proven to be successful as certain recommendations have been implemented and have improved the system-wide performance at the oncology center.

Aims: To use molecular beacon based nucleic acid sequence-based amplification (NASBA) to develop a rapid, sensitive, specific detection method for norovirus (NV) genogroupII (GII)., Methods and Results: A method to detect NV GII from environmental samples using real-time NASBA was developed. This method was routinely sensitive to 100 copies of target RNA and intermittent amplification occurred with as few as 10 copies. Quantitative estimates of viral load were possible over at least four orders of magnitude., Conclusions: The NASBA method described here is a reliable and sensitive assay for the detection of NV. This method has the potential to be linked to a handheld NASBA device that would make this real-time assay a portable and inexpensive alternative to bench-top, lab-based assays., Significance and Impact of the Study: The development of the real-time NASBA assay described here has resulted in a simple, rapid (<1 h), convenient testing format for NV. To our knowledge, this is the first example of a molecular beacon based NASBA assay for NV.

An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17beta-estradiol over the concentration range of 1.2 x 10(-8) through 5.6 x 10(-12) M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 +/- 1.1) x 10(-10) and (2.4 +/- 1.0) x 10(-10) M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 x 10(-11) to 2.8 x 10(-9) M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.

We have developed a rapid, sensitive, and specific assay for the detection and quantification of enteroviruses using nucleic acid sequence-based amplification (NASBA). The inclusion of an internal control (IC) increased the precision and accuracy of the method over a standard NASBA assay and provided a way to detect assay inhibition. The assay was sensitive to 10 viral particles with amplification and detection occurring in as little as 18 min. The assay detected a variety of different enteroviruses to the exclusion of non-target viruses. The standard NASBA method resulted in predictions of viral load to within an order of magnitude of the expected number, as compared with prediction to within less than a half order of magnitude using the IC-NASBA method. Rapid and sensitive detection of enteroviruses is important in both clinical samples to diagnose illness and in environmental samples to assess risk of wastewater contamination and potential health hazards.

Detection and quantification of low abundance target RNA has wide utility in the fields of clinical diagnostics, environmental monitoring, gene expression analysis, and biodefense. Nucleic acid based sequence amplification (NASBA) is an isothermal amplification method that provides the sensitivity needed for these applications. However, the requirement for three separate enzymes in NASBA often results in a greater variability between replicate samples than that seen in PCR-based assays. To overcome this problem, we have adapted the bioMérieux Nuclisens Basic Kit and Nuclisens EasyQ Analyzer along with the introduction of a synthetic internal control RNA (IC-RNA) for quantification of potentially any RNA sequence. Using the rbcL gene from the Florida red tide organism Karenia brevis as our target, we describe a simple method to accurately quantify the native target by computing the ratio of the time to positivity (TTP) values for both the wild-type and IC-RNA, and plotting this ratio against the starting number of target molecules or cells. By utilizing this simple method, we have significantly increased our accuracy and precision of prediction over the standard TTP calculations.

Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5x10(5) vs 2.9x10(6) relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7x10(7) RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells.

The luxA, B, C, D, and E genes from Photorhabdus luminescens were cloned and functionally expressed in Saccharomyces cerevisiae to construct a bacterial lux-based yeast bioreporter capable of autonomous bioluminescence emission. The bioreporter was engineered using a series of pBEVY yeast expression vectors that allowed for bi-directional constitutive or inducible expression of the individual luxA, B, C, and E genes. The luxD gene, encoding the acyl-ACP transferase that ultimately supplies the requisite aldehyde substrate for the bioluminescent reaction, was fused to a yeast internal ribosomal entry site (IRES) sequence to ensure high bi-cistronic expression. Although self-generation of bioluminescence was achieved by the bioreporter, the signal was relatively weak and decayed rapidly. To overcome this instability, a flavin oxidoreductase gene (frp) from Vibrio harveyi was co-expressed to provide sufficient concentrations of the FMNH(2) co-factor required for the bioluminescent reaction. Expression of frp with the lux genes not only stabilized but also enhanced bioluminescence to levels approaching 9.0x10(5) times above background.

The coauthors of this article had developed the clinical impression that whereas surgically placed amalgam fillings are successful on a short-term basis, the long-term prognosis for these cases may not be nearly as favorable. In order to test this hypothesis, as many patients as could be contacted, either directly or indirectly, who had been treated before 1981 were evaluated. Excluded from this study were cases for which failure could be attributed to any reason other than failure from the amalgam reverse filling. Therefore, all cases included had to demonstrate periapical healing prior to ultimate breakdown. On this basis, 60 of 104 teeth (57.7%) were considered to be successful and 44 teeth (42.3%) were determined to be failures.

The effect of cobalt-60 teletherapy on the pulps of restored teeth of monkeys was studied. Gold crowns and amalgam and composite resin restorations were placed in 28 teeth of two Macaca speciosa monkeys. One monkey then received cobalt-60 radiation to both maxillary and mandibular teeth and bones, with a total dose of 7,600 rads. Decalcified semiserial paraffin sections, 7 mum thick, of the teeth and supporting tissues were prepared and stained with hematoxylin and eosin. Microscopically, there was no discernible difference between the irradiated and the non-irradiated pulps.

We examined 30 male chronic hemodialysis patients and 18 male controls without known bone or renal disease to determine the utility of maxillomandibular, non-dominant hand, shoulder and pelvis films in the evaluation of renal osteodystrophy. We used panoramic periapical radiographs to examine the maxilla and mandible and sensitive rapid processing films for the hand, shoulder and pelvis. Films were evaluated by experienced personnel without knowledge of the patients. There were significant differences between patients and controls in creatinine, urea nitrogen, total protein, albumin, alkaline phosphatase and phosphorus. Twenty-three patients had abnormal hand radiographs and 22 patients had abnormal jaw radiographs (p less than 0.05 vs. controls). Four patients had changes in the hands, but not in the jaw; 4 had opposite findings. Changes in the jaw tended to be more severe than in the hands in those with involvement of both. We concluded that dental and hand radiography are good screening techniques for evaluating bone disease. They may be useful in evaluating treatment for renal osteodystrophy.