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Biology subjects

Johnson, D. A.

Publications and source records attributed to Johnson, D. A..

10 recordsLinked to original sources

Top-down control of the descending pain modulatory system drives placebo analgesia

In placebo analgesia, prior experience and expectations lead to pain suppression by the administration of an inert substance, but causal evidence for its neural basis is lacking. To identify the underlying neural circuits, we reverse-translated a conditioned placebo protocol from humans to mice. Surprisingly, the placebo effect suppresses both nociception and unconditioned emotional-motivational pain-related behavior. Descending pain modulatory neurons in the periaqueductal gray (PAG) are critical for both morphine and placebo antinociception. The placebo effect depends on input to the PAG from the medial prefrontal and anterior cingulate cortices, but not anterior insular cortex. Conditioning enhances noxious stimulus-evoked endogenous opioid release in the PAG to produce analgesia. Our results suggest that cortical control of the descending pain modulatory system (DPMS) is gated by rapid endogenous opioid signaling in the PAG during placebo trials. This study bridges clinical and preclinical research, establishing a central role for the DPMS in placebo analgesia.

neuroscience↗

In vivo photopharmacology with light-activated opioid drugs

Traditional methods for site-specific drug delivery in the brain are slow, invasive, and difficult to interface with recordings of neural activity. Here, we demonstrate the feasibility and experimental advantages of in vivo photopharmacology using "caged" opioid drugs that are activated in the brain with light after systemic administration in an inactive form. To enable bidirectional manipulations of endogenous opioid receptors in vivo, we developed PhOX and PhNX, photoactivatable variants of the mu opioid receptor agonist oxymorphone and the antagonist naloxone. Photoactivation of PhOX in multiple brain areas produced local changes in receptor occupancy, brain metabolic activity, neuronal calcium activity, neurochemical signaling, and multiple pain- and reward-related behaviors. Combining PhOX photoactivation with optical recording of extracellular dopamine revealed adaptations in the opioid sensitivity of mesolimbic dopamine circuitry during chronic morphine administration. This work establishes a general experimental framework for using in vivo photopharmacology to study the neural basis of drug action. HighlightsA photoactivatable opioid agonist (PhOX) and antagonist (PhNX) for in vivo photopharmacology. Systemic pro-drug delivery followed by local photoactivation in the brain. In vivo photopharmacology produces behavioral changes within seconds of photostimulation. In vivo photopharmacology enables all-optical pharmacology and physiology.

neuroscience↗

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram-positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase-mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae, identifying a number of putative {beta}7-{beta}8 loop-mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1 position of the substrate motif, underlined in LPXTG, in contrast to the strict P1 Gly recognition of SrtA from Staphylococcus aureus. However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C-terminus, and three others of {beta}7-{beta}8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified {beta}7-{beta}8 loop-mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. We argue that a greater understanding of individual SrtA sequence and structural determinants of target selectivity can facilitate the design or discovery of improved sortagging tools.

biochemistry↗

The Influence of Public Health Faculty on College and University Plans during the COVID-19 Pandemic

The purpose of this study was to determine whether the institutional presence of public health faculty within colleges and universities influenced operational plans for the fall semester of 2020. Using cross-sectional data collected by the College Crisis Initiative of Davidson College, six levels of instructional modalities (ranked from least to most restrictive) were compared between Council on Education of Public Health (CEPH)-accredited and non-CEPH-accredited 4-year institutions. Institutions with CEPH-accredited schools and programs were more likely to select some restrictive teaching modalities: 63.8% more likely to use hybrid/hyflex or more restrictive and 66.9% more likely to be primarily online (with some in person) or more restrictive. However, having CEPH-accredited programs did not push institutions to the most restrictive modalities. COVID-19 cases in county, enrollment, and political affiliation of the state governor were also found to influence instructional modality selection. While any ecological study has certain limitations, this study demonstrates that college and university fall plans appear to have been influenced by the presence of CEPH-accredited schools and programs of public health, and/or the input of their faculty. The influence of relevant faculty expertise on institutional decision-making can help inform college and university responses to future crises.

scientific communication and education↗

A second specificity-determining loop in Class A sortases: Biochemical characterization of natural sequence variation in chimeric SrtA enzymes

Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice for SML reactions. However, the stringent specificity of saSrtA for the sequence motif LPXTG limits its uses. Here, we use principal component analysis to identify a structurally conserved loop with a high degree of variability in all classes of sortases. We investigate the contribution of this {beta}7-{beta}8 loop, located between the catalytic cysteine and arginine residues and immediately adjacent to the target binding cleft, by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of Class A sortases from 8 species engineered into the SrtA sequence from Streptococcus pneumoniae (spSrtA). While some of our chimeric enzymes mimic the activity and selectivity of the wild-type protein from which the loop sequence is derived (e.g., that of saSrtA), others result in chimeric spSrtA enzymes able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural, and sequence analyses, we identify three interactions facilitated by {beta}7-{beta}8 loop residues that appear to be broadly characteristic of Class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.

biochemistry↗

Stratified Random Sampling Methodology for Observing Community Mask Use within Indoor Settings: Results from Louisville, Kentucky during the COVID-19 Pandemic

Wearing a facial mask can limit COVID-19 transmission. Measurements of communities mask use behavior have mostly relied on self-report. This studys objective was to devise a method for measuring the prevalence of mask-wearing and proper mask use in indoor public areas without relying on self-report. A stratified random sample of retail trade stores (public areas) in Louisville, Kentucky, USA, was selected and targeted for observation by trained surveyors during December 14-20, 2020. The stratification allowed for investigating mask use behavior by city district, retail trade group, and public area size. The average mask use prevalence among observed visitors of the 382 visited public areas was 96%, while the average prevalence of proper use was 86%. In 17% of the public areas, at least one unmasked visitor was among the observed visitors; in 48%, at least one improperly masked visitor was observed. The average mask use among staff was 92%, but unmasked staff were observed in fewer public areas, as an unmasked staff member was observed in 11% of the visited public areas. The average prevalence of proper make use among staff was 87%, similar to the average among visitors. However, the percentage of public areas where at least one improperly masked staff was observed was 33. Significant disparities in mask use and its proper use were observed among both visitors and staff by public area size, retail trade type, and geographical area. Observing unmasked and incorrectly masked visitors was more common in small (less than 1500 square feet) public areas than larger ones, also in food and grocery stores than other retail stores. Also, the majority of the observed unmasked persons were male and middle age adults.

scientific communication and education↗

Regions of Highly Recurrent Electrogram Morphology at Sites of Low Cycle Length Accurately Reflect Arrhythmogenic Substrate for Atrial Fibrillation - Implications For a New, Mechanism Guided Therapeutic Approach for Atrial Fibrillation

BackgroundAlthough atrial electrograms (EGMs) are thought to reflect pathophysiological substrate for atrial fibrillation (AF), it is not known which electrograms are suitable targets during AF ablation. We hypothesized that electrogram morphology recurrence (EMR) better reflects arrhythmogenic AF substrate than traditional frequency and complexity measures of AF. In a canine rapid atrial pacing (RAP) model of AF, we assessed the relationship between EMR and traditional AF electrogram measures, rotational activity in the atria, fibrosis, myofiber orientation and parasympathetic innervation. MethodsPersistent AF was induced in 13 dogs by RAP for 6-8 weeks. High-density epicardial mapping (117 electrodes) was performed in six atrial sub-regions. EMR measures Recurrence percentage (Rec%) and cycle length of the most frequent electrogram morphology (CLR), Fractionated Interval (FI), Organization Index (OI), Dominant Frequency (DF) and Shannons Entropy (ShEn) were analyzed before and after atropine administration. Myocyte fiber orientation, amount of fibrosis and spatial distribution of parasympathetic nerve fibers were quantified. ResultsRec% was greatest in the appendages, and CLR was lowest in the posterior left atrium. Rec%/CLR correlated with FI, OI and the complexity measure ShEn, but not with DF. All electrogram measures were poorly correlated with fibrosis and myofiber anisotropy. Rec% correlated closely with stability of rotational activity. Unlike other measures, Rec% correlated closely with spatial heterogeneity of parasympathetic nerve fibers; this was reflected in CLR response to atropine. ConclusionEMR correlates closely with stability of rotational activity and with the pattern of atrial parasympathetic innervation. CLR may therefore be a viable therapeutic target in persistent AF.

physiology↗

Manage flowering time in Miscanthus and sugarcane to facilitate intra- and intergeneric crosses

Miscanthus is a close relative of Saccharum and a potentially valuable genetic resource for improving sugarcane. Differences in flowering time within and between Miscanthus and Saccharum hinders intra- and interspecific hybridizations. A series of greenhouse experiments were conducted over three years to determine how to synchronize flowering time of Saccharum and Miscanthus genotypes. We found that day length was an important factor influencing when Miscanthus and Saccharum flowered. Sugarcane could be induced to flower in a central Illinois greenhouse using supplemental lighting to reduce the rate at which days shortened during the autumn and winter to 1 min d-1, which allowed us to synchronize the flowering of some sugarcane genotypes with Miscanthus genotypes primarily from low latitudes. In a complementary growth chamber experiment, we evaluated 33 Miscanthus genotypes, including 28 M. sinensis, 2 M. floridulus, and 3 M. xgiganteus collected from 20.9{degrees} S to 44.9{degrees} N for response to three day lengths (10 h, 12.5 h, and 15 h). High latitude-adapted M. sinensis flowered mainly under 15 h days, but unexpectedly, short days resulted in short, stocky plants that did not flower; in some cases, flag leaves developed under short days but heading did not occur. In contrast, for M. sinensis and M. floridulus from low latitudes, shorter day lengths typically resulted in earlier flowering, and for some low latitude genotypes, 15 h days resulted in no flowering. However, the highest ratio of reproductive shoots to total number of culms was typically observed for 12.5 h or 15 h days. Latitude of origin was significantly associated with culm length, and the shorter the days, the stronger the relationship. Nearly all entries achieved maximal culm length under the 15 h treatment, but the nearer to the equator an accession originated, the less of a difference in culm length between the short-day treatments and the 15 h day treatment. Under short days, short culms for high-latitude accessions was achieved by different physiological mechanisms for M. sinensis genetic groups from the mainland in comparison to those from Japan; for mainland accessions, the mechanism was reduced internode length, whereas for Japanese accessions the phyllochron under short days was greater than under long days. Thus, for M. sinensis, short days typically hastened floral induction, consistent with the expectations for a facultative short-day plant. However, for high latitude accessions of M. sinensis, days less than 12.5 h also signaled that plants should prepare for winter by producing many short culms with limited elongation and development; moreover, this response was also epistatic to flowering. Thus, to flower M. sinensis that originates from high latitudes synchronously with sugarcane, the former needs day lengths >12.5 h (perhaps as high as 15 h), whereas that the latter needs day lengths <12.5 h.

plant biology↗

Disruption of NOX2-dependent Oxidative Injury with a Targeted Gene-Therapy Approach Prevents Atrial Fibrillation in a Canine Model

Atrial fibrillation is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal as they are not targeted to the molecular mechanisms underlying AF. In this study, we demonstrated using a novel gene-based strategy in a clinically relevant large animal of AF that oxidative injury is a key mechanism underlying the onset and perpetuation of AF. First, we demonstrated that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through induction of NADPH oxidase 2 (NOX2) and generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electrical remodeling in AF by upregulating a constitutively active form of acetylcholine-dependent K+ current (IKACh) - called IKH - by a mechanism involving frequency-dependent activation of protein kinase C epsilon (PKC{varepsilon}). To understand the mechanism by which oxidative injury promotes the genesis and/or maintenance of AF, we performed targeted injection of NOX2 shRNA in atria of normal dogs followed by rapid atrial pacing. The time to onset of non-sustained AF increased by more than 5-fold in NOX2 shRNA treated dogs. Furthermore, animals treated with NOX2 shRNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, with attenuated activation of PKC{varepsilon}, a likely molecular mechanism underlying this beneficial electrophysiological remodeling. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.\n\nOne Sentence SummaryTargeted disruption of NOX2-dependent oxidative injury with a novel gene therapy approach prevents onset as well as perpetuation of atrial fibrillation.

physiology↗

Collective cell behaviour in mechanosensing of substrate thickness

Extracellular matrix stiffness has a profound effect on the behaviour of many cell types. Adherent cells apply contractile forces to the material on which they adhere, and sense the resistance of the material to deformation - its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 {micro}m) thicknesses. Here, we hypothesised that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic ECM-functionalised polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker labelled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 m. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater halfmaximal response at 54 m. Depth-sensing was dependent on ROCK-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances.

biophysics↗