Browsing by Author "Friedlander, Michael J."

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    Antarctic teleosts with and without hemoglobin behaviorally mitigate deleterious effects of acute environmental warming
    Ismailov, Iskander I.; Scharping, Jordan B.; Andreeva, Iraida E.; Friedlander, Michael J. (PLoS, 2021-11-24)
    Recent studies forecast that many ectothermic animals, especially aquatic stenotherms, may not be able to thrive or even survive predicted climate change. These projections, however, generally do not call much attention to the role of behavior, an essential thermoregulatory mechanism of many ectotherms. Here we characterize species-specific locomotor and respiratory responses to acute ambient warming in two highly stenothermic Antarctic Notothenioid fishes, one of which (Chaenocephalus aceratus) lacks hemoglobin and appears to be less tolerant to thermal stress as compared to the other (Notothenia coriiceps), which expresses hemoglobin. At the onset of ambient warming, both species perform distinct locomotor maneuvers that appear to include avoidance reactions. In response to unavoidable progressive hyperthermia, fishes demonstrate a range of species-specific maneuvers, all of which appear to provide some mitigation of the deleterious effects of obligatory thermoconformation and to compensate for increasing metabolic demand by enhancing the efficacy of branchial respiration. As temperature continues to rise, Chaenocephalus aceratus supplements these behaviors with intensive pectoral fin fanning which may facilitate cutaneous respiration through its scaleless integument, and Notothenia coriiceps manifests respiratory-locomotor coupling during repetitive startle-like maneuvers which may further augment gill ventilation. The latter behaviors, found only in Notothenia coriiceps, have highly stereotyped appearance resembling Fixed Action Pattern sequences. Altogether, this behavioral flexibility could contribute to the reduction of the detrimental effects of acute thermal stress within a limited thermal range. In an ecologically relevant setting, this may enable efficient thermoregulation of fishes by habitat selection, thus facilitating their resilience in persistent environmental change.
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    Biology and Physics Competencies for Pre-Health and Other Life Sciences Students
    Hillborn, Robert C.; Friedlander, Michael J. (The American Society for Cell Biology, 2013-02-12)
    The recent report on the Scientific Foundations for Future Physicians (SFFP) and the revised Medical College Admissions Test (MCAT) reframe the preparation for medical school (and other health professional schools) in terms of competencies: what students should know and be able to do with that knowledge, with a strong emphasis on scientific inquiry and research skills. In this article, we will describe the thinking that went into the SFFP report and what it says about scientific and quantitative reasoning, focusing on biology and physics and the overlap between those fields. We then discuss how the SFFP report set the stage for the discussion of the recommendations for the revised MCAT, which will be implemented in 2015, again focusing the discussion on biology and physics. Based on that framework, we discuss the implications for undergraduate biology and physics education if students are to be prepared to demonstrate these competencies.
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    A comparative analysis exposes an amplification delay distinctive to SARS-CoV-2 Omicron variants of clinical and public health relevance
    Brown, Katherine L.; Ceci, Alessandro; Roby, Clinton; Briggs, Russell B.; Ziolo, D.; Korba, R.; Mejia, R.; Kelly, S. T.; Toney, D.; Friedlander, Michael J.; Finkielstein, Carla V. (Taylor & Francis, 2023)
    Mutations in the SARS-CoV-2 genome may negatively impact a diagnostic test, have no effect, or turn into an opportunity for rapid molecular screening of variants. Using an in-house Emergency Use Authorized RT-qPCR-based COVID-19 diagnostic assay, we combined sequence surveillance of viral variants and computed PCR efficiencies for mismatched templates. We found no significant mismatches for the N, E, and S set of assay primers until the Omicron variant emerged in late November 2021. We found a single mismatch between the Omicron sequence and one of our assay's primers caused a > 4 cycle delay during amplification without impacting overall assay performance. Starting in December 2021, clinical specimens received for COVID-19 diagnostic testing that generated a Cq delay greater than 4 cycles were sequenced and confirmed as Omicron. Clinical samples without a Cq delay were largely confirmed as the Delta variant. The primer-template mismatch was then used as a rapid surrogate marker for Omicron. Primers that correctly identified Omicron were designed and tested, which prepared us for the emergence of future variants with novel mismatches to our diagnostic assay's primers. Our experience demonstrates the importance of monitoring sequences, the need for predicting the impact of mismatches, their value as a surrogate marker, and the relevance of adapting one's molecular diagnostic test for evolving pathogens.
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    Determinants of Rotavirus Polymerase Localization and Activity
    McKell, Allison Overstreet (Virginia Tech, 2017-09-19)
    Rotavirus (RV) is a viral pathogen that causes severe, watery diarrhea and vomiting in the young of humans and other animals. RV infections result in over 200,000 pediatric deaths around the world each year, especially in developing nations. Within the infected host cell, RV forms inclusion bodies, called viroplasms, where many stages of viral replication occur. The RV polymerase, known as VP1, must localize to viroplasms during infection where it replicates the virus' RNA genome. The work described in this dissertation focused on identifying region(s) of VP1 essential for its viroplasmic localization and its function as a polymerase. We found that a single amino acid change in a region of the polymerase called the N-terminal domain negatively impacted its capacity to localize to viroplasms during infection as well as its enzymatic activity in a test tube. Follow up studies using VP1 proteins from divergent strains and a mutant containing only the N-terminal domain of VP1 provided more insight into polymerase localization determinants. In total, our work suggests that the VP1 N-terminal domain plays an important role in localizing the polymerase to viroplasms via interactions with other viral proteins and supporting its function as a polymerase.
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    Development and implementation of a scalable and versatile test for COVID-19 diagnostics in rural communities
    Ceci, Alessandro; Muñoz-Ballester, Carmen; Tegge, Allison N.; Brown, Katherine L.; Umans, Robyn A.; Michel, F. Marc; Patel, Dipankumar; Tewari, Bhanu P.; Martin, James E.; Alcoreza, Oscar Jr.; Maynard, Thomas M.; Martinez-Martinez, Daniel; Bordwine, Paige; Bissell, Noelle; Friedlander, Michael J.; Sontheimer, Harald; Finkielstein, Carla V. (Nature Publishing Group, 2021-07-20)
    Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand.
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    Development of MRI-based Yucatan Minipig Brain Template
    Norris, Caroline N. (Virginia Tech, 2019-04-05)
    Yucatan minipigs have become increasingly common animal models in neuroscience where recent studies, investigating blast-induced traumatic brain injury, stroke, and glioblastoma, aim to uncover brain injury mechanisms [1-3]. Magnetic Resonance Imaging (MRI) has the potential to validate and optimize unknown parameters in controlled populations. The key to group-level MRI analysis within a species is to align (or register) subject scans to the same volumetric space using a brain template. However, large animal brain templates are lacking, which limits the use of MRI as an effective research tool to study group effects. The objective of this study was to create an MRI-based Yucatan minipig brain template allowing for uniform group-level analysis of this animal model in a standard volumetric space to characterize brain mechanisms. To do this, 5-7 month old, male Yucatan minipigs were scanned using a 3 Tesla whole-body scanner (Siemens AG, Erlangen) in accordance with IACUC. T1-weighted anatomical volumes (resolution = 1×1×1 mm3; TR = 2300 ms; TE= 2.89 ms; TI = 900 ms; FOV = 256 mm2 ; FA = 8 deg) were collected with a three-dimensional magnetization prepared rapid acquisition gradient echo (MPRAGE) pulse sequence [4]. The volumes were preprocessed, co-registered, and averaged using both linear and non-linear registration algorithms in AFNI [5] to create four templates (n=58): linear brain, non-linear brain, linear head, and non-linear head. To validate the templates, tissue probability maps (TPMs) and variance maps were created, and landmark variation was measured. TPMs computed in FSL [6] and AFNI show enhanced tissue probability and contrast in the non-linear template. Additionally, variance maps showed a more uniform spatial variance in the non-linear template compared to the linear. Registration variation within the brain template was within 1.5 mm and displayed improved landmark variation in the non-linear brain template. External evaluation subjects (n=12), not included in the template, were registered to the four templates to assess functionality. The results indicate that the developed templates provide acceptable registration accuracy to enable population comparisons. With these templates, researchers will be able to use MRI as a tool to further neurological discovery and collaborate in a uniform space.
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    LTD Induction in Adult Visual Cortex: Role of Stimulus Timing and Inhibition
    Perrett, Stephen P.; Dudek, Serena M.; Eagleman, David M.; Montague, P. Read; Friedlander, Michael J. (Society for Neuroscience, 2001-04-01)
    One Hertz stimulation of afferents for 15 min with constant interstimulus intervals (regular stimulation) can induce longterm depression (LTD) of synaptic strength in the neocortex. However, it is unknown whether natural patterns of lowfrequency afferent spike activity induce LTD. Although neurons in the neocortex can fire at overall rates as low as 1 Hz, the intervals between spikes are irregular. This irregular spike activity (and thus, presumably, irregular activation of the synapses of that neuron onto postsynaptic targets) can be approximated by stimulation with Poisson-distributed interstimulus intervals (Poisson stimulation). Therefore, if low-frequency presynaptic spike activity in the intact neocortex is sufficient to induce a generalized LTD of synaptic transmission, then Poisson stimulation, which mimics this spike activity, should induce LTD in slices. We tested this hypothesis by comparing changes in the strength of synapses onto layer 2/3 pyramidal cells induced by regular and Poisson stimulation in slices from adult visual cortex. We find that regular stimulation induces LTD of excitatory synaptic transmission as assessed by field potentials and intracellular postsynaptic potentials (PSPs) with inhibition absent. However, Poisson stimulation does not induce a net LTD of excitatory synaptic transmission. When the PSP contained an inhibitory component, neither Poisson nor regular stimulation induced LTD. We propose that the short bursts of synaptic activity that occur during a Poisson train have potentiating effects that offset the induction of LTD that is favored with regular stimulation. Thus, natural (i.e., irregular) low-frequency activity in the adult neocortex in vivo should not consistently induce LTD.
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    Mechanisms underlying retinogeniculate synapse formation in mouse visual thalamus
    Monavarfeshani, Aboozar (Virginia Tech, 2018-01-22)
    Retinogeniculate (RG) synapses connect retinal ganglion cells to the thalamic relay cells of the dorsal lateral geniculate nucleus (dLGN). They are critical for regulating the flow of visual information from retina to primary visual cortex (V1). RG synapses in dLGN are uniquely larger and stronger than their counterparts in other retinorecipient regions. Moreover, in dLGN, RG synapses can be classified into two groups: simple RG synapses, which contain glia-encapsulated single RTs synapsing onto relay cell dendrites, and complex RG synapses, which contain numerous RTs that converge onto the shared regions of relay cell dendrites. To identify target-derived molecules that direct the transformation of RTs into unique RG synapses in dLGN, I used RNAseq to obtain the whole transcriptome of dLGN and its adjacent retinorecipient nucleus, vLGN, at different time points during RG synapses development. Leucine-Rich Repeat Transmembrane Neuronal 1 (LRRTM1), a synaptogenic adhesion molecule, was the candidate I selected based on its expression pattern. Here, I discovered that LRRTM1 regulates the development of complex RG synapses. Mice lacking LRRTM1 (lrrtm1-/-) not only show a significant reduction in the number of complex RG synapses but they exhibit abnormal visual behaviors. This work reveals, for the first time, a high level of retinal convergence onto dLGN relay cells in thalamus and the functional significance of this convergence for vision.
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    Role of GABAA-Mediated Inhibition and Functional Assortment of Synapses onto Individual Layer 4 Neurons in Regulating Plasticity Expression in Visual Cortex
    Sáez, Ignacio; Friedlander, Michael J. (PLOS, 2016-02-03)
    Layer 4 (L4) of primary visual cortex (V1) is the main recipient of thalamocortical fibers from the dorsal lateral geniculate nucleus (LGNd). Thus, it is considered the main entry point of visual information into the neocortex and the first anatomical opportunity for intracortical visual processing before information leaves L4 and reaches supra- and infragranular cortical layers. The strength of monosynaptic connections from individual L4 excitatory cells onto adjacent L4 cells (unitary connections) is highly malleable, demonstrating that the initial stage of intracortical synaptic transmission of thalamocortical information can be altered by previous activity. However, the inhibitory network within L4 of V1 may act as an internal gate for induction of excitatory synaptic plasticity, thus providing either high fidelity throughput to supragranular layers or transmittal of a modified signal subject to recent activity-dependent plasticity. To evaluate this possibility, we compared the induction of synaptic plasticity using classical extracellular stimulation protocols that recruit a combination of excitatory and inhibitory synapses with stimulation of a single excitatory neuron onto a L4 cell. In order to induce plasticity, we paired pre- and postsynaptic activity (with the onset of postsynaptic spiking leading the presynaptic activation by 10ms) using extracellular stimulation (ECS) in acute slices of primary visual cortex and comparing the outcomes with our previously published results in which an identical protocol was used to induce synaptic plasticity between individual pre- and postsynaptic L4 excitatory neurons. Our results indicate that pairing of ECS with spiking in a L4 neuron fails to induce plasticity in L4-L4 connections if synaptic inhibition is intact. However, application of a similar pairing protocol under GABAARs inhibition by bath application of 2μM bicuculline does induce robust synaptic plasticity, long term potentiation (LTP) or long term depression (LTD), similar to our results with pairing of pre- and postsynaptic activation between individual excitatory L4 neurons in which inhibitory connections are not activated. These results are consistent with the well established observation that inhibition limits the capacity for induction of plasticity at excitatory synapses and that pre- and postsynaptic activation at a fixed time interval can result in a variable range of plasticity outcomes. However, in the current study by virtue of having two sets of experimental data, we have provided a new insight into these processes. By randomly mixing the assorting of individual L4 neurons according to the frequency distribution of the experimentally determined plasticity outcome distribution based on the calculated convergence of multiple individual L4 neurons onto a single postsynaptic L4 neuron, we were able to compare then actual ECS plasticity outcomes to those predicted by randomly mixing individual pairs of neurons. Interestingly, the observed plasticity profiles with ECS cannot account for the random assortment of plasticity behaviors of synaptic connections between individual cell pairs. These results suggest that connections impinging onto a single postsynaptic cell may be grouped according to plasticity states.
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    The role of Lynx1, an endogenous modulator of cholinergic transmission, in NMJ development, maintenance, and repair
    Vaughan, Sydney Katherine (Virginia Tech, 2019-05-08)
    The cholinergic system drives muscle contraction and plays a central role in the formation, maintenance, and repair of mammalian neuromuscular junctions (NMJs) and skeletal muscles. Because of these essential actions, much effort has been devoted to identifying primary and auxiliary modulatory components of the cholinergic system at NMJs and throughout skeletal muscles. Here, I asked if Lynx1, a GPI-anchored protein shown to modulate nAChRs in the brain, is present and affects the activity of nAChRs at NMJs. Molecular and cellular analysis revealed that Lynx1 levels increase in skeletal muscles, specifically at NMJs, during development. Its expression pattern also closely mirrors changes in cholinergic transmission in vivo and in vitro. As expected, I found by co-immunoprecipitation that Lynx1 interacts with muscle nAChRs and using electrophysiology, I show that Lynx1 desensitizes nAChRs to ACh at NMJs. These findings demonstrate that Lynx1 regulates the cholinergic system at NMJs, suggesting roles for this gene in developing and adult NMJs. To determine the role of Lynx1 at NMJs, I examined Lynx1 knockout mice at different ages. While deletion of Lynx1 has no discernable effect on developing NMJs, its absence increases the incidence of NMJs with age-related morphological features, such as fragmentation and denervation, in young adult and middle-aged mice. Loss of Lynx1 also increases the number of slow-type muscle fibers in young and middle-aged mice, another hallmark of aging. Along with these morphological changes, deletion of Lynx1 affects expression of genes associated with NMJ stability, myogenesis, and muscle atrophy in young adult and middle-aged mice. Not surprisingly, the loss of Lynx1 reduces the density and stability of nAChRs at NMJs. Because of these findings, I surmised that loss of Lynx1 would adversely affect NMJs under other physiological stressors. However, I found the opposite as the loss of Lynx1 augments the capacity of NMJs to repair damages during exercise, following injury to motor axons, and during the initial symptomatic stage of amyotrophic lateral sclerosis (ALS). Since Lynx1 modulates the activity of nAChRs, these contrasting findings likely represent the positive and negative effects of heightened cholinergic transmission on aging compared to injury and disease-afflicted NMJs.
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    Role of retinal inputs and astrocytes for the development of visual thalamus
    Somaiya, Rachana Deven (Virginia Tech, 2022-06-01)
    Axons of retinal ganglion cells (RGCs) send visual information to a number of retinorecipient regions in the brain. In rodents, visual thalamus receives dense innervations from RGC axons and is important for both image-forming and nonimage-forming visual functions. Retinal inputs invade visual thalamus during embryonic development, before the arrival of non-retinal inputs (such as local interneurons and axonal inputs from other brain regions). In this dissertation, I explore how early innervation of RGC axons affects circuitry in visual thalamus and the role of visual experience, neural activity, and molecular cues in the development. While the development of astrocytes in cortex has been well-described, they have been largely overlooked in visual thalamus. Using immunohistochemical, functional, and ultrastructural analysis, I show that astrocytes in visual thalamus reach adult-like morphological properties and functionality at retinogeniculate synapses early in development, by eye-opening and before visual experience. These studies reveal that while experience-dependent visual activity from RGC axons is critical for many aspects of visual thalamus development, astrocytic maturation occurs independent of that information about our visual environment. As with astrocytes, little progress has been made in understanding the development of interneurons in the visual thalamus. Here, I show that retinal inputs interact with thalamic astrocytes to influence the recruitment of GABAergic interneurons into visual thalamus. I found that this interaction between RGC axons and astrocytes is not dependent on neural activity of RGCs. Using transcriptomic analysis, in situ hybridization, and reporter lines, I observed thalamus-projecting RGCs express SHH and astrocytes in visual thalamus express SHH signaling molecules. My results reveal that SHH signaling between RGC axons and astrocytes is critical for astrocytic fibroblast growth factor 15 (FGF15) expression in developing visual thalamus. Ultimately, FGF15 serves as a potent motogen that is essential for thalamic interneuron migration. These data identify a novel morphogen-dependent and activity-independent mechanism that mediates crosstalk between RGCs and astrocytes to facilitate the recruitment of interneurons into the developing visual thalamus.
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    Synthesis, Characterization and Performance of Gelatin Biopolymer based Nanoparticle Formulations for Molecule Encapsulations
    Stevenson, Andre Terrance Jr. (Virginia Tech, 2018-04-24)
    Gelatin's ability to dissolve in water while also forming a gel upon cooling, produces melt in your mouth candies and frozen desserts, along with hard and soft capsules and tablets. This protein, which is extracted from pork skin and cattle hide, is categorized by a rigidity or stiffness value and remains one of the most common materials in food and pharmaceutical formulations. Its established use and safe certification are appealing characteristics for manipulation into nanoparticles (NPs) to encapsulate therapeutic molecules as medicine. NPs are generally spherical materials, yet their abilities hold great promise to improve medical outcomes. These abilities include: protecting molecules from harsh locations like the stomach, improved therapeutic delivery through biological barriers such as the brain and controlled release for minimal side effects. NPs typically less than 200 nanometers (nm) overcome biological barriers more effectively than larger particles. For reference, 200 nm is equivalent to dividing the length of an ant (~4 millimeters) by 20,000. The potential applications of gelatin NPs to treat disease is impressive; however, an inability to consistently obtain ideal NP sizes (<200 nm average diameter) exists. Furthermore, gelatin NPs are commonly stabilized (or cross-linked) using toxic chemicals. The motivation for this research was to (1) contribute new understanding why ideal gelatin NPs are difficult to obtain and (2) form NPs using a non-toxic chemical for prospective brain injury treatment. This dissertation determined low rigid and high rigid gelatin can consistently form NPs less than 200 nm, indicating rigidity alone is not a main factor for obtaining ideal NPs. Instead, characterization approaches indicated gelatin sample composition prior to NP formation must be very uniform. As a result, filtering solutions prior to NP formation proved a new technique to prepare ideal NPs. Glyceraldehyde is a sugar and has shown to be a non-toxic gelatin NP stabilizer. For the first time, glyceraldehyde's non-toxicity was shown using various brain cell types and NPs were formed to be ~130 nm. After incorporation of a new therapeutic molecule for brain injury treatment, average particles were ~149 nm with slow therapeutic release profiles determined in simulated body fluid.
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    Vaccine Effectiveness During an Outbreak of COVID-19 Alpha Variant (B.1.1.7) in a Men’s Correctional Facility in Rural Virginia
    Silverman, Rachel A.; Ceci, Alessandro; Cohen, Alasdair; Helmick, Meagan; Short, Erica; Bordwine, Paige; Friedlander, Michael J.; Finkielstein, Carla V. (2022-07)
    In April 2021, a COVID-19 outbreak occurred at a correctional facility in rural Virginia, USA. Eighty-four infections were identified among 854 incarcerated persons by facilitywide testing with reverse transcription quantitative PCR (qRT-PCR). We used whole-genome sequencing to link all infections to 2 employees infected with the B.1.1.7α (UK) variant. The relative risk comparing unvaccinated to fully vaccinated persons (mRNA-1273 [Moderna, https:// www.moderna.com]) was 7.8 (95% CI 4.8–12.7), corresponding to a vaccine effectiveness of 87.1% (95% CI 79.0%–92.1%). Average qRT-PCR cycle threshold values were lower, suggesting higher viral loads, among unvaccinated infected than vaccinated cases for the N, E, and S genes. Vaccination was highly effective at preventing SARS-CoV-2 infection in this high-risk setting. This approach can be applied to similar settings to estimate vaccine effectiveness as variants emerge to guide public health strategies during the ongoing pandemic.
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    Vaccine Effectiveness during Outbreak of COVID-19 Alpha (B.1.1.7) Variant in Men’s Correctional Facility, United States
    Silverman, Rachel A.; Ceci, Alessandro; Cohen, Alasdair; Helmick, Meagan; Short, Erica; Bordwine, Paige; Friedlander, Michael J.; Finkielstein, Carla V. (Centers for Disease Control and Prevention, 2022-07)
    In April 2021, a COVID-19 outbreak occurred at a correctional facility in rural Virginia, USA. Eighty-four infections were identified among 854 incarcerated persons by facilitywide testing with reverse transcription quantitative PCR (qRT-PCR). We used whole-genome sequencing to link all infections to 2 employees infected with the B.1.1.7α (UK) variant. The relative risk comparing unvaccinated to fully vaccinated persons (mRNA-1273 [Moderna, https:// www.moderna.com]) was 7.8 (95% CI 4.8–12.7), corresponding to a vaccine effectiveness of 87.1% (95% CI 79.0%–92.1%). Average qRT-PCR cycle threshold values were lower, suggesting higher viral loads, among unvaccinated infected than vaccinated cases for the N, E, and S genes. Vaccination was highly effective at preventing SARS-CoV-2 infection in this high-risk setting. This approach can be applied to similar settings to estimate vaccine effectiveness as variants emerge to guide public health strategies during the ongoing pandemic.