bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacologic Microglial Depletion Disrupts Breathing and Body Temperature Regulation During Torpor in 13‐lined Ground Squirrels
Authors: Sprenger Ryan J. , Radcliff Abigail B. , Meza Armand L. , Mickelson Carly R. , Watters Abby , Romero Phinea Z. , Milsom William , Carey Hannah V. , Watters Jyoti J. , Baker Tracy L.
Published: May 2022

Mammalian hibernation initiates dramatic changes in physiology that include large reductions in ventilation (V̇E), heart rate, oxygen consumption rate (V̇o2), and body temperature (Tb) upon entrance into torpor, effects that reverse upon arousal. Several physiologic processes must maintain function throughout a wide range of temperatures. Recent evidence indicates that neuronal synapses loosen and retract in regions of the brain that are silenced during a torpor bout, although little is understood yet about how these connections are regulated during hibernation. Microglia, brain resident immune cells, are potential facilitators of these essential neural homeostatic activities due to their critical roles in neuronal support and synaptic strengthening and pruning. Thus, we hypothesized that microglia are required for maintaining normal ventilatory neural control during hibernation. To test this, we treated 13‐lined ground squirrels with either vehicle or colony‐stimulating factor receptor‐1 (CSF1R) antagonist, PLX3397 (80mg/kg, po), a receptor whose activation is necessary for microglial survival in the adult nervous system, and measured V̇E, V̇o2, and Tb throughout a torpor bout. During entrance, PLX‐treated squirrels took ~1.5 hours longer to reach their minimum torpor temperature (Tb; 12°C) compared to control animals (Tb; 9.3°C) (ambient temperature; 5‐6°C). Similarly, arousal time in microglia‐depleted animals was as much as 1 hour longer relative to controls, despite starting at a higher minimum Tb. In both treatment groups, V̇o2 during entrance fell and spiked periodically. However, in PLX‐treated animals, these spikes were much greater and more frequent. Minimum V̇o2 in torpor was ~3 times greater in PLX‐treated animals compared to control animals. Upon arousal from torpor, V̇o2 remained elevated, longer, in PLX‐treated squirrels. Total V̇E tended to be elevated in PLX‐treated animals through entrance, steady state torpor, and arousal, but it was most striking during steady state torpor, where V̇E in PLX‐treated animals was 15‐fold higher than control animals. The net result in torpor was an elevated air convection requirement, thus microglia‐depleted animals hyperventilated throughout the torpor bout. Together, these results suggest that microglia play an important regulatory role in normal ventilatory and temperature control throughout torpor and influence the rate at which animals enter into and arouse from torpor. Mechanisms whereby microglia contribute to hibernation neurophysiology are currently under investigation.
Vitamin D Regulates Natriuretic Peptide Receptor‐A Gene Expression via Modulation of Histone Deacetylases and DNA Methyltransferase Activity
Authors: Kumar Prerna , Pandya Krishna , Nguyen Christian N. , Pandey Kailash N
Published: May 2022

Cardiac hormones atrial and brain natriuretic peptides (ANP and BNP) activate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and produces the second messenger cGMP. cGMP activates downstream signaling and biological consequences of ANP/NPRA such as natriuresis, diuresis, vasorelaxation, antimitogenic responses, and cardiac and renal anti‐hypertrophic effects. The activity and expression of Npr1 gene (coding for GC‐A/NPRA) assessed primarily through ANP‐stimulated cGMP production, are mediated by agents, including autoregulation involving natriuretic peptides, osmotic sensors, and other external and internal stimuli, but the hormonal and epigenetic mechanisms mediating Npr1 regulation are not well understood. The objective of present study was to study the effect of Vitamin D (vitD) in the regulation of Npr1 gene transcription and expression via modulation of epigenetic factors. Our bioinformatics study with the murine Npr1promoter has shown presence of 4 vitD response elements (VDRE) in the region ‐583 to ‐495 from the transcription start site having perfect and VDRE‐like consensus sequence. Npr1promoter activity was determined by transient transfection of Npr1 promoter deletion constructs in cultured rat thoracic aortic smooth muscle cells (RTASMCs) and mouse mesangial cells (MMCs) treated with Vitamin D₃ (1α, 25‐dihydroxy; VD3) using dual luciferase assay kit. Luciferase assay demonstrated that VD3 enhances Npr1 promoter activity by more than 5‐6‐fold in RTASMCs and MMCs in a dose‐dependent manner. Western blot and densitometry analysis showed increasing concentrations of VD3 significantly induced NPRA protein levels by 3.5‐fold in MMCs and 4.5‐fold in RTASMCs and maximum effect was observed at 100 nM. There was 45%‐50% inhibition of histone deacetylase (HDAC) activity in presence of VD3 in both cell lines as measured by HDAC activity/inhibition ELISA kit. Moreover, treatment with VD3 reduced class I HDAC enzymes, HDAC1 and HDAC3 protein levels and dose‐dependently enhanced acetylation level of histones, H3 at lysine residues 9 and 14 (H3‐K9/14 ac) and H4 at lysine residue 12 (H4‐K14ac). Treatment of RTASMCs and MMCs with VD3 attenuated DNA methyltransferase (DNMT) activity by 40%‐55% as measured by DNMT activity/inhibition ELISA kit. The results demonstrate that VD3 regulate Npr1 gene expression by modulation of histone deacetylases and DNMT activity. The identification of epigenetic targets of vitamin D signaling as a regulator of Npr1gene transcription and protein expression will have important implications in hypertension and cardiovascular regulation.
Metabolomic Kidney Input and Output Analyses in Salt‐Sensitive Hypertension
Authors: Hoffmann Brian R. , Shimada Satoshi , Greene Andrew S. , Liang Mingyu , Dash Ranjan , Cowley Allen
Published: May 2022

Rationale

Salt‐sensitive hypertension is globally widespread and associated with increased risk of cardiovascular eventsand kidney disease. Although excess dietary salt intake is a major cause of these damaging effects, many of the underlying mechanisms remain poorly defined. Specifically, it is not understood how the critical energy needs of the kidney are met in face of constantly changing environmental nutrients and how this affects metabolic homeostasis in the kidney.

Methods

We hypothesized that a high salt diet in normal salt‐insensitive Sprague Dawley rat would lead to alterations of renal tubular bioenergetics and substrate metabolism. To test this, metabolomic analysis of arterial plasma (input) and renal venous plasma (filtered output) obtained from unanesthetized instrumented rats was performed using tandem mass spectrometry for comparison on a Q‐Exactive Orbitrap mass spectrometer coupled to a dual‐channel Vanquish Ultra‐Performance Liquid Chromatography system. Plasma metabolites were extracted from 20 µL of plasma from SD rats consuming low salt (0.4% NaCl) or high salt (4% NaCl) for 7, 14, or 21 days. These samples were then analyzed with separation over a hydrophobic (C18) and hydrophilic (HILIC) column under both positive and negative polarity to maximize identification of metabolites identified. Varying amounts of quality control plasma sample pools were used to determine injection sample concentration to analyze for each sample and remaining pools were used for normalization of the runs over time.

Results

A total of 4,603 metabolite features were detected, with 1,205 being identified named compounds. After applying further stringency filters to these named metabolites, 350 were significantly different in response to the varying high salt diets (p <0.05; Benjamini‐Hochberg correction) in at least one of the group comparisons performed (versus low salt and artery versus vein). Differential metabolites include numerous lipids, amino acids, and bioenergetic compounds, among others. There appeared to be a shift in these differential compounds between 14 and 21 days of high salt diet, suggesting a potential metabolic shift between initial responses and prolonged high salt responses. Complementary studies are also underway to compare urine metabolite flux, outer medulla, and cortex in conjunction with the plasma analysis to produce a complete computational prediction model.

Conclusion

Together, implementation of this methodology will provide key insights and be valuable in further predicting the emergent properties of complex metabolic interactions in the kidney of rats during the development of salt‐induced hypertension.
Impact of O2 Availability on Convective and Diffusive O2 Transport and Skeletal Muscle Intracellular PO2 at VO2max
Authors: Broxterman Ryan M. , Wagner Peter D. , Richardson Russell S.
Published: May 2022

Cross‐sectional evidence suggests that maximal O2 uptake (VO2max) is the consequence of well‐matched O2 supply and demand in untrained skeletal muscle (symmorphosis) and in trained skeletal muscle is limited, not by mitochondrial O2 capacity, but, by convective and diffusive O2 transport (dysmorphosis). However, it is unknown if, longitudinally, aerobic exercise training transitions skeletal muscle VO2max from a sym‐ to a dys‐morphosis of O2 transport and utilization capacities. The purpose of this study was to test the hypothesis that, compared to normoxia, hyperoxia would increase skeletal muscle mean capillary PO2 (capPO2) and intracellular PO2 (iPO2) both pre‐ and post‐training, but would only increase VO2max post‐training. We tested this in 5 healthy sedentary males (mean±SD; age: 27±5 yrs, height: 175±6 cm, mass: 73±7 kg, cycle VO2max: 33±4 ml/kg/min) using the unique combination of human quadriceps single‐leg knee‐extensor exercise (KE), directly measured arterial and femoral PO2, thermodilution measured leg blood flow (LBF), and 1H nuclear magnetic resonance spectroscopy measurement of myoglobin (Mb) desaturation at 100% maximal work rate in randomized conditions of hypoxia, normoxia, and hyperoxia (12, 21, and 100 % O2, respectively). Mb desaturation was converted to iPO2 using an O2 half‐saturation pressure of 3.2 mmHg. All measurements were made pre‐ and post‐training, which consisted of eight weeks of KE (1 hour/visit, 3 visits/week). Pre‐training, increasing inspired O2 increased capPO2 (33.0±4.8, 41.4±4.1, and 49.6±8.7 mmHg, all p<0.019), Mb desaturation decreased from hypoxia to normoxia (46±4 and 31±4 %, p=0.004), but not from normoxia to hyperoxia (29±14 %, p=0.79), iPO2 increased from hypoxia to normoxia (3.8±0.6 and 7.2±1.4 mmHg, p=0.009), but not from normoxia to hyperoxia (10.0±6.6 mmHg, p=0.375), and VO2max was not altered by inspired O2 (0.47±0.10, 0.52±0.13, and 0.54±.01 l/min, all p>0.285). Post‐training, increasing inspired O2 increased capPO2 (32.8±2.6, 39.6±2.8, and 47.5±6.1 mmHg, all p<0.010), did not decrease Mb desaturation (79±20, 53±11, and 53±8 %, all p>0.050), iPO2 increased from hypoxia to normoxia (1.1±1.2 and 3.1±1.1 mmHg, p=0.019), but not from normoxia to hyperoxia (3.0±1.1 mmHg, p=0.954), and VO2max increased with greater inspired O2(0.59±0.11, 0.68±0.11, and 0.76±0.09 l/min, all p<0.044). There was a pre‐ to post‐training difference within all three inspired O2 conditions for VO2max (all p<0.010) and in hypoxia and normoxia for both Mb desaturation (both p<0.020) and iPO2 (both p<0.011). When untrained, the concomitant increase in capPO2 and iPO2, with no change in VO2max, from normoxia to hyperoxia infers a mitochondrial O2 capacity limited skeletal muscle VO2max. In trained skeletal muscle, the concomitant increase in capPO2 and VO2max, with minimal changes in iPO2, from normoxia to hyperoxia infers an O2 transport limited VO2max.
Macrophages Contribute to Sex Differences in Obesity‐dependent Microvascular Dysfunction
Authors: Domingos‐Souza Gean , Hogan James O. , White Carl
Published: May 2022

Sex differences in obesity‐induced microvascular dysfunction have been reported, however, the mechanisms are poorly understood. We previously identified macrophages as key cells that mediate microvascular dysfunction in obesity. Interestingly, sex differences in the inflammatory response to obesity have also been described. The current aim was to define the interaction between biological sex and macrophages on obesity‐dependent microvascular dysfunction. We placed male and female mice (C57BL/6) on either a high‐fat diet to induce obesity or a low‐fat control diet. After twelve weeks mice were treated with either clodronate‐containing liposomes to deplete macrophages or PBS‐containing control liposomes. Macrophage depletion had no effect on body weight, glycemia, or mean arterial pressure, which were all increased by obesity in both male and female. Obesity also caused an expansion of the macrophage population in both male and female mesenteric perivascular adipose tissue, as quantified by flow cytometry, which was similarly attenuated by clodronate treatment. We then assessed microvascular function in third order mesenteric arterioles using pressure myography. The sensitivity of vasoconstriction to norepinephrine was increased by obesity in both male and female. Macrophage depletion dramatically attenuated vasoconstriction in male but only minimally affected vessels from females. In contrast, macrophage depletion had no effect on acetylcholine‐dependent vasodilation in either male or female. Taken together, these data show that macrophages contribute to obesity‐dependent microvascular hypercontractility in males but not females.
The Role of Endothelin Receptors on Sensory Nerve Mediated Dilation in Postmenopausal Women
Authors: Del Vecchio Angelica R. , Welti Laura M. , Kuczmarski Andrew V. , McGinty Shane J. , Wong Brett J. , Wenner Megan M.
Published: May 2022

Rapid local heating of the skin elicits biphasic vasodilation. The initial peak in skin blood flow (SkBF) is mediated by sensory nerves (i.e., the axon reflex). Both endothelin (ETA and ETB) receptors are located on axon terminals on nociceptor fibers, and therefore may be involved in the sensory nerve mediated vasodilation. Although prior work shows a blunted axon reflex with aging, this has yet to be examined in women, and the role of ETA and ETB receptors in this initial sensory nerve‐mediated vasodilatory response remains unclear.

PURPOSE

We tested the hypothesis that the initial peak is blunted in postmenopausal women (POST) compared to young women (YW), and that blockade of ETA and ETB receptors would improve sensory nerve‐mediated dilation in POST.

METHODS

We retrospectively analyzed data in 25 healthy YW (27±10 yr) and 16 healthy POST (56±1 yr). Cutaneous vasodilation to local heating was measured using laser doppler flowmetry during microdialysis perfusions of lactated Ringer’s (control), ETA receptor blockade (BQ‐123, 500nM), and ETB receptor blockade (BQ‐788, 300nM). Cutaneous vascular conductance (CVC) was calculated as SkBF/MAP at baseline and during the initial peak of local heating (42°C); CVC were normalized to maximal vasodilation achieved by perfusion of sodium nitroprusside (28mM) and heating to 43°C.

RESULTS

All women were normotensive (MAP: YW 83±8 mmHg; POST 94±2 mmHg, p < 0.001) and not obese (YW 23±3 kg/m2; POST 24±1 kg/m2, p = 0.153). Sensory nerve‐mediated dilation at the control site was reduced in POST (YW 63 ±14 vs POST 49 ±17 %CVCmax, p=0.004). Blockade of ETA (YW 54 ±10 vs POST 55 ±9 %CVCmax, p=0.579) and ETB (YW 62 ±17 vs POST 59 ±13 %CVCmax, p=0.476) receptors did not impact the initial peak of local heating in either group.

CONCLUSIONS

These preliminary data suggest the sensory nerve‐mediated vasodilation during local heating is attenuated with aging in women, but endothelin receptors do not contribute to this age‐related decrease. Additional research is needed to further understand the interactions between sex hormones and aging in regulating sensory nerve mediated vasodilation.
SPARC: The structure of vagal nociceptive nerve fibers in the mouse esophagus
Authors: Patil Mayur J. , Harsanyiova Jana , Kim Seol‐Hee , Kollarik Marian , Taylor‐Clark Tom
Published: May 2022

The esophagus is innervated by nociceptive sensory afferent nerve fibers originating from the vagus nerve. The nature of the vagal nociceptive fiber innervation, specifically which esophageal layers, however, remains unknown. We hypothesized that the vagal nociceptive TRPV1+ afferent fibers innervate the esophageal mucosa. We evaluated our hypothesis by using genetic neuronal tracing in several mutant mice, including two new strains we generated. The wholemount preparations of separated esophageal mucosa and muscle were immunostained for appropriate fluorescent proteins and imaged by spinning‐disk confocal microscopy. In the TRPV1‐Flp/RC: FLTG mice(N=8) in which TRPV1‐expressing cells express tdTomato the TRPV1+ fibers with varicose appearance densely innervated both the mucosa and the muscle. In the mucosa the TRPV1+ fibers are located just beneath the epithelium, run in parallel registers of two or more fibers, have craniocaudal orientation and branch repeatedly. In the muscle the TRPV1+ fibers form a dense network in the myenteric plexus innervating virtually all myenteric ganglia forming intraganglionic varicose endings (IGVEs). Many TRPV1+ fibers connecting IGVEs run in parallel to the fibers of the outer and inner striated muscle. In the TRPV1‐Flp/Tac1‐Cre/RC:FLTG mice(N=4) in which GFP is selectively expressed in cells positive for both TRPV1 and Tac1 (the marker of esophageal nociceptors derived from neural crest) the esophageal innervation by TRPV1+/Tac1+ fibers mimicked the dense innervation by TRPV1+ fibers. In contrast, in the TRPV1‐Flp/P2X2‐Cre/RC:FLTG mice(N=4) in which GFP is selectively expressed in cells positive for both TRPV1 and P2X2 (the marker of esophageal nociceptors derived from placodes) the TRPV1+/P2X2+ fibers were almost absent in the esophagus. Consistent with this finding, in the P2X2‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP virus into vagal ganglia(N=2) in which GFP is selectively expressed in vagal P2X2+ fibers, the nodose TRPV1‐negative tension mechanosensor nerve terminals (intraganglionic laminar endings) were visualized in the esophageal muscle. Finally, in the TRPV1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP virus into vagal ganglia(N=3), in which only the vagal TRPV1+ fibers express GFP, the vagal TRPV1+ fibers had the same pattern as TRPV1+/Tac1+ fibers, but with lower density and patchy distribution. Our data indicate that vagal nociceptive TRPV1+ fibers innervate both the esophageal mucosa and myenteric ganglia and originate from neural crest‐derived jugular portion of the vagal jugular‐nodose ganglia complex.
Cochlear Duct Abnormalities in Mice With Osteogenesis Imperfecta
Authors: Huston Lila A. , Menegaz Rachel , Handler Emma , Organ Jason , Gonzales Lauren
Published: May 2022

Osteogenesis imperfecta (OI), a developmental disorder of type I collagen, is known to cause hearing loss in ~ 60% of the diseased population. Identified forms include conductive hearing loss (17.4% of OI patients), involving loss of function within the ossicular chain, and sensorineural hearing loss (25.8%), resulting from damage to the cochlea, with the most predominant form being mixed hearing loss (56.8%), involving damage to both the cochlea and ossicles. While OI‐related pathologies have largely focused on the middle ear, the pathological appearance of the cochlea (the organ most often compromised in OI‐related hearing loss) has gained little focus. In this study, we examine OI‐related pathologies on the cochlea in a mouse model for the severe type III OI, to document 1) any visible variation between WT and OI variants, and 2) assess the encroachment of the otic capsule onto the cochlea by analyzing differences in duct volumes. We hypothesize that cochlea in mice with OI will have less consistent morphology overall than their WT counterparts due to abnormal growth of the bony capsule.

16 week old OIM mice (B6C3Fe a/a‐Col1a2oim/J) (n=6) were compared to unaffected wildtype (WT) littermates (n=6) with no known hearing defects. High‐resolution micro‐CT scans were created for all specimens and 3D models and volumes of the cochlea were generated using 3D Slicer software. Two‐tailed Mann‐Whitney U‐tests were used to investigate differences between 1) right and left ears of the same mouse to examine intraindividual symmetry and 2) differences in volumes between WT and OI cochlea.

No major morphologic differences between OI and WT were observed, except for minor areas of higher ossification at the base of the cochlea, mostly within the OI sample. Within WT specimens, we observed little intraindividual difference in the cochlear volume (0‐3%). Within OI specimens, significant differences were observed in cochlear volume between right and left ears in the same animal (4‐15%; p<0.05), indicating potential unilateral effects. When average WT and OI volumes were compared, there was much overlap between the two samples although the OI volumes had a significantly larger range than the WT range (Mann‐Whitney U, p<0.05).

Overall, our results indicate that mice with OI are much more likely to have evidence of unilateral cochlear volume losses, despite very little difference in overall shape appearance, possibly due to bony capsule encroachment. This find indicates an extremely high potential for sensorineural and mixed hearing loss in OI‐bred mice and elucidates at least one mechanism behind how this type of hearing loss might be occurring.

Little is known about the pathological appearance of the cochlea in OI, leading to difficulty in managing hearing loss. Further investigation of the etiology and progression of cochlear pathologies will allow for better outcomes in hearing for those patients afflicted with OI‐related hearing loss.
Discovery of a Bilateral Fibulocalcaneus Externum Invertus Muscle: A Novel Fibularis Quartus Variant
Authors: Hess Allison J. , Martignetti Reagan E. , Gillis B. Diane , Zdilla Matthew J. , Davis Jillian S. , Lambert H. Wayne
Published: May 2022

The fibularis (peroneus) quartus is a variant muscle found within the lateral leg compartment in ~11.5% of cadaveric specimens, though recent studies have shown a higher prevalence of ~20.9 – 21.9%. The proximal muscular attachments can originate from the fibula, fibularis brevis, fibularis longus, and/or the intermuscular septi that form the boundaries of the lateral leg compartment, but it then descends to reside posterior to the lateral malleolus of the fibula. The distal attachment of the fibularis quartus varies greatly, and fibularis quartus muscular variants are often named according to location of its distal attachment. The most common distal insertion site of a fibularis quartus muscle is the lateral surface of the calcaneus into either the fibular trochlea or the retrotrochlear eminence. Therefore, this variant of the fibularis quartus inserting into the lateral surface of the calcaneus is called the fibulocalcaneus externum. During routine cadaveric dissection of a 79‐year‐old Caucasian male, a rare and novel variation of the fibularis quartus muscle was discovered. Although this variant is seen arising from the fibula, fibularis brevis, and the posterior intermuscular septum, descending posterior to the lateral malleolus, and attaching distally to the lateral surface of the calcaneus, it is unique in that the muscle is inverted, with the tendonous part being more proximal and the muscular portion arising distally. This novel muscle was found unilaterally in the left leg via dissection initially with its distal head inserting on the fibular trochlea of the calcaneus, but ultrasonographic imaging verified its bilateral presence. The subsequent dissection of the right leg revealed a similar inverted muscle, but this variation split into two distal muscle bellies inserting into the retrotrochlear eminence posteriorly and the fibular trochlear anteriorly. Given its anatomy, we have named this rare fibularis quartus variant the fibulocalcaneus externum invertus muscle. Like most fibularis quartus variants, the fibulocalcaneus externum invertus muscle can cause ankle instability, ankle locking, chronic lateral ankle pain, and peroneal compartment syndrome, especially due to acting as a space‐occupying lesion within the superior peroneal tunnel due to its distal muscle belly/bellies. While it is unknown if this person had any clinical signs and/or symptoms, clinicians, surgeons, and/or radiologists should be aware of the fibulocalcaneus externum invertus when assessing radiological imaging or preparing for ankle surgeries in and around the lateral leg compartment.
Mechanism of Phosgene Oxime Induced Skin Toxicity in C57BL/6 Mouse Model
Authors: Goswami Dinesh G. , Singh Satyendra K. , Madadgar Omid , Croutch Claire R. , Noland Erica L. , Brown Jared , Agarwal Rajesh , Tewari‐Singh Neera
Published: May 2022

Phosgene Oxime (CX) is a potent chemical warfare agent. It is grouped with vesicants; however, its effect resembles of an urticant or nettle agent. CX has faster penetration compared to other vesicating chemical warfare agents and causes erythema, urticaria, blanching, itching hives, necrosis, and systemic toxic effects upon exposure. The mechanism of CX induced toxicity is not known and there are no approved therapies. We have shown that dermal CX exposure in SKH‐1 hairless mice causes clinical lesions, epidermal cell death, mast cell degranulation, infiltration of immune cells, and increase in the level of DNA damage and inflammatory markers. In this study we examined the mechanism of CX‐induced inflammatory response in C57BL/6 mice. Dorsal skin of mice was exposed to neat CX for 0.5 or 1.0 min using two 12 mm vapor caps at MRI Global. CX exposure caused decrease in heart rate and breath rate with significant mortality (50%) after 1.0 min CX exposure. CX exposure caused erythema, and edema at the exposure site and led to increase in epidermal thickness. Significant mast cell degranulation was observed within 2h of CX exposure in male and female mice at both the exposure durations. The increase in mast cell degranulation was associated with increased tryptase and chymase levels in the mouse skin. CX exposure also led to increased expression of inflammatory mediators, cyclooxygenase‐2 (COX‐2), matrix metallopeptidase‐9 (MMP‐9), myeloperoxidase (MPO), and MAP kinases JNK. Inflammatory cytokines and chemokines were analyzed in the skin tissue lysates using cytokine array. CX exposure caused increases in several pro‐inflammatory cytokines and chemokines including IL‐6 and TNF‐α, and a decrease in anti‐inflammatory cytokines IL‐4 and IL‐10. Together, these results suggest mast cells as important contributors of CX induced dermal inflammation and toxicity. Further studies are being carried out using WT (C57BL/6) and mast cell deficient mice to evaluate the role of mast cells in CX‐induced toxicity.
Time‐dependent Characterization of Respiratory Parameters post Bleomycin‐induced Lung Injury
Authors: Karpuk Nikolay , Kamra Kajal , Lisco Steven J. , Zucker Irving H. , Wang Hanjun
Published: May 2022

Background‐

Acute lung injury (ALI) induces inflammation that disrupts the normal alveolar‐capillary endothelial barrier which impairs gas exchange to induces hypoxemia that reflexively increases respiration. Increased respiratory rate (RR) usually develops within a few hours to a few days after ALI. The bleomycin (Bleo)‐induced lung injury rat model has been one of the most widely used animal models to study the mechanisms of respiratory failure post ALI. However, systemic and time‐dependent characterization of respiratory parameters in this model has not been done. In the current study, we aimed to use the plethysmography (DSI system) to characterize the changes in variable respiratory parameters at different stages of lung injury in the bleomycin‐induced ALI rat model.

Methods and Results‐

ALI was induced in male SD rats (200‐250g) using a single intra‐tracheal injection of Bleo (day 0) and respiratory variables‐ Respiratory rate (RR), Tidal Volume (TV), Minute Ventilation (MV), enhanced pause (Penh), pause (PAU), ratio rate of achieving peak expiatory flow (Rpef), peak inspiratory flow (box) (PIFb), peak expiratory flow box (PEFb), inspiratory time (Ti), expiratory time (Te), expiratory flow at the point of 50%TV (EF50), end inspiratory pause (EIP), end expiratory pause (EEP), relaxation time (Tr) and time of brake (TB) were measured at room air for one hour weekly from W1 (acute phase of ALI) to W4 (recovery phase of ALI) using whole‐ body plethysmography (WBP). Our data showed that MV and RR were significantly elevated from baseline (W0) to W1‐W4 in Bleo‐treated rats whereas TV was decreased from baseline to W1‐W3 post Bleo (p≤0.01 compared to W0). Associated with increased RR, Ti, Te and Tr were significantly shorter at W1‐W4 post Bleo compared to baseline (W0). Penh and Rpef, measures of airway obstruction, were significantly increased at W2 and W1‐W4 post Bleo, respectively. PIFb and PEFb were significantly increased whereas EIP and EEP were significantly decreased in W1‐W4 post Bleo. EF50, a measurement of exhalation flow rate, was significantly increased at W1‐W4 post Bleo.

Conclusions‐

These data suggest that a single intratracheal instillation of Bleo induced long‐lasting changes in respiratory function from acute to recovery phase of lung injury. Although blood gas returned to the normal level at W4 post Bleo, many respiratory parameters remained abnormal, indicating that pulmonary function might require a longer time to recover in this model.
Seeing and Understanding the Light: Student‐Built Fluorescence Microscopes for Dynamic Calcium Imaging in the Undergraduate Physiology Lab Class
Authors: Johnson Bruce R. , Ryan James , Li Felisha , Deitcher David
Published: May 2022

Students often treat laboratory equipment as a black box with little knowledge of how scientific instrumentation works to gather data. We are creating interdisciplinary classrooms where students gain confidence to build and understand tools to solve biological questions as future scientists. We describe student construction, use and assessment of our Do‐It‐Yourself (DIY) fluorescence imaging microscope. Students were first introduced to principles of fluorescence microscopy and Drosophila neurogenetics through lecture material, readings and online resources. Mixed teams of 2‐3 Biology and Engineering students assembled their own imaging microscopes with the following parts: 20X objective lens, excitation and emission filters and dichroic mirror, CMOS camera, 3‐D printed holders and housings for these parts, and a student‐built LED light source (total parts/microscope cost ~$1000). Construction was guided by pre‐recorded build videos and assistance from course instructors and teaching assistants. Student teams tested their microscope by collecting images from prepared slides with GFP fluorescence in fly synaptic boutons. Teams also acquired images from living fly larval preparations in two simple experiments. These compared fluorescence intensity before and during application of a high potassium saline that: 1) released GFP‐labelled neuropeptide from fly synaptic boutons, and 2) activated glutamate neurons expressing GCaMP. They analyzed images for fluorescence intensity and adjusted and colorized images with ImageJ. Assessment of the student learning experience was conducted for two Spring 2021 undergraduate laboratory courses at Cornell University (BioNB/BME/ECE 4910, Principles of Neurophysiology and BioNB 4300, Experimental Molecular Biology) and a Fall 2021 undergraduate Neurobiology laboratory course (Neurobiology 340) at Hobart and William Smith colleges. Students rated statements on all the following topics highly positive: knowledge of fluorescence imaging principles, affective experience of building and understanding the instrumentation, working in teams and clarity of guidance during the microscope build and use sessions. We continue our DIY efforts to expand the teaching, learning and research toolbox for physiology students and faculty.
Elucidating the Role of the NRF2 pathway in Nitrogen Mustard Induced Toxicity in Mouse Epidermal Skin Cells
Authors: Kerin Ryan , Wright Holly , Fuertes Fabiola , Monson Anna , Sharma Swati , Singh Satyendra , Goswami Dinesh , Madadgar Omid , Lundback Steve , Rockwell Cheryl , Liby Karen , Tewari‐Singh Neera
Published: May 2022

Nitrogen mustard (NM), a structural analog of warfare agent sulfur mustard (SM), is a chemical vesicant that causes blistering of skin and damage to mucus membranes. NM is a strong alkylating and DNA damaging agent. Currently, we lack effective therapies to rescue skin injuries by NM and SM. The insight into the mechanisms of NM and SM‐induced skin injuries is crucial for the development of effective targeted therapies. Our lab’s previous studies suggest that skin toxicity from NM exposure can cause macromolecular damage and activation of signaling pathways, especially those related to inflammation, oxidative stress, and DNA damage. Therefore, targeting anti‐oxidative stress and anti‐inflammatory pathways such as the nuclear factor erythroid 2‐related (Nrf2) pathway can be essential to treat epidermal injuries from this vesicant exposure. Nrf2 is a key transcription factor that controls the basal and induced expression of an array of antioxidant response element–dependent genes to provide anti‐inflammatory and cytoprotective effects to cells. Thus, activating the Nrf2 pathway can be a targeted approach for the treatment of NM skin toxicity. To study the role of the Nrf2 pathway in NM‐induced toxicity, we used mouse skin epidermal JB6 cells. The JB6 cells were treated with 75µM of NM for 2, 6, 12 or 24 hours where approximately 50% of cell viability was achieved (MTT assay and trypan blue cell counting). Using qPCR analysis, we further studied the RNA expression of the Nrf2 pathway and its targeted genes including heme oxygenase‐1 (HO‐1), NADPH Quinone oxidoreductase enzyme (NQO‐1), Glutamate‐Cysteine Ligase Modifier Subunit (GCLM), Glutamate‐Cysteine Ligase Catalytic Subunit (GCLC) and Catalase. Our results showed an upregulation in RNA expression for HO‐1 at 2 and 6 hours post NM exposure, and a downregulation in Nrf2, NQO‐1, Catalase, GCLM, and GCLC. There was also an upregulation for HO‐1 and NQO‐1 at 12 hours post NM exposure. However, there was a downregulation in RNA expression for all these markers at 24 hours post NM exposure. To induce the activation of Nrf2 pathway and its targeted genes, we want to treat the cells with synthetic oleanane triterpenoid derivatives of 2‐cyano‐3,12‐dioxooleana‐1,9(11)‐dien‐28‐oic acid (CDDO), which are among the most potent Nrf2 activators. These results will help us further investigate in in vivo studies if triterpenoids via cytoprotective and antioxidant effects could be used for the treatment of acute as well as long‐term skin toxicity from NM exposure.
Multiple roles of β‐Arrestin2 in initiating and sustaining D3R‐mediated ERK1/2 phosphorylation by G protein‐biased and unbiased D3R agonists
Authors: Xu Wei , Bearoff Frank , Kortagere Sandhya
Published: May 2022

β‐arrestins play key roles in both terminating G protein signaling and activating G protein‐independent signaling across many G protein coupled receptors (GPCRs); however, the roles of β‐arrestins in dopamine D3 receptor (D3R)‐mediated ERK phosphorylation/signaling are poorly characterized. Our previous studies showed that novel compounds SK609 and its analog SK608 are selective D3R agonists and display Gi/o biased signaling without significant recruitment of β‐arrestin2 (βarr2) compared with dopamine which can potently activate G proteins and recruit β‐arrestins. We hypothesize that βarr2 plays multiple roles in initiating and sustaining D3R‐mediated ERK1/2 phosphorylation by G protein‐biased and unbiased D3R agonists. We tested this hypothesis using neuron‐like SH‐SY5Y cells stably expressing D3R (SH‐SY5Y‐D3R cells) with βarr2 knockdown (βarr2KD) in vitro. Our results show: SK609 and SK608 or pramipexole (PRX) time‐dependently elicited both the early and late phases of mono phosphorylation of ERK1/2; SK608 or SK609 only elicited the early phase of dual phosphorylation, whereas PRX induced both the early and late phases and sustained for 1‐4 h; βarr2KD partially reduced mono ERK1/2 phosphorylation by PRX but not SK608 or SK609; βarr2KD significantly enhanced both the early and late phases of dual ERK1/2 phosphorylation by SK608 or SK609 whereas it partially reduced the early phase by PRX and completely abolished the late phase; Gi/o inhibitor PTX abolished PRX‐ or SK608 and SK609‐induced mono ERK1/2 phosphorylation and SK608‐ and SK609‐induced the early phase of dual ERK1/2 phosphorylation, whereas the early phase of dual phosphorylation by PRX was abolished but the late phase was not affected; Gbg inhibitor Galllein had no effect on PRX‐ induced dual phosphorylation of ERK1/2 but inhibited SK609‐ or SK608‐indued dual phosphorylation; PKC inhibitor Go6983 greatly reduced or completely abolished PRX‐ or SK608‐ and SK609‐induced the mono phosphorylation, and blocked SK608‐ or SK609‐induced the dual phosphorylation in SH‐SY5Y‐D3R cells. In contrast, Go6983 abolished PRX‐induced late phase of dual phosphorylation, it only partially reduced PRX‐induced early phase; PTX plus Go6983 completely abolished PRX‐ or SK608‐ and SK609‐induced mono or dual ERK1/2 phosphorylation in SH‐SY5Y‐D3R/βarr2KD cells. In summary, βarr2 not only participates in inhibiting G protein‐biased D3R agonist‐induced early and late phases of dual ERK1/2 phosphorylation and activating unbiased D3R agonists‐induced the early and late phases of the dual phosphorylation, but also is required for Gi/o‐ or PKCs‐dependent the early phase of the mono and dual ERK1/2 phosphorylation. These findings support multiple roles in which βarr2 acts as both signal terminator for G protein biased agonist‐induced ERK1/2 signaling and signal transducer for unbiased D3R agonist‐induced signaling interactions among βarr2 and Gi/o and PKCs which collaboratively regulate ERK1/2 signaling.
Effects of Cannabinoid Agonists and Antagonists in Rats Discriminating Fentanyl
Authors: Alkhelb Dalal A. , Kirunda Andre , Papanastasiou Ioannis , Jiang Shan , Iliopoulos‐Tsoutsouvas Christos , Zvonok Alexander , Nikas Spyridon P. , Vemuri Kiran , Makriyannis Alexandros , Desai Rajeev I.
Published: May 2022

Extensive evidence suggests the existence of a functional interaction between endogenous cannabinoids and opioid systems. Targeting cannabinoid CB1 receptors might be a promising approach for developing new medications for opioid addiction. The present studies were undertaken to evaluate the effects of CB1 agonists and antagonists in rats (n=8) trained to discriminate, i.p., injections of 0.032 mg/kg fentanyl from saline on a 10‐response fixed‐ratio (FR‐10) schedule of food reinforcement. Results show that the µ‐opioid agonists (fentanyl, morphine, and oxycodone) substituted fully for fentanyl, whereas the muscarinic antagonist atropine did not substitute for fentanyl. The CB1 agonists Δ9‐THC, AM2201, and AM8936 all partially substituted for fentanyl’s discriminative‐stimulus effects suggesting some overlap in the discriminative‐stimulus effects of cannabinoid agonists and fentanyl. Pretreatment studies with a µ‐opioid receptor antagonist show that naltrexone antagonized fentanyl’s effects. Interestingly, pretreatment studies with CB1 antagonists show that rimonabant (inverse agonist) produced some attenuation of fentanyl’s discriminative‐stimulus effects at doses that significantly decreased response rates, whereas the neutral CB1 antagonist AM4113 blocked fentanyl discrimination at doses that did not modify rates of responding. Our findings are consistent with our recent work showing that AM4113 attenuates heroin self‐administration in rats, without producing depressive‐like effects. Collectively, these data suggests that CB1 neutral antagonists that block CB1 receptors with rimonabant‐like potency, devoid of unwanted side‐effects, may be therapeutically advantageous for countering the abuse‐related behavioral effects of opioids.
Investigating Zmym2 and Zmym4 as Putative Six1 Co‐Factors
Authors: Jourdeuil Karyn , Tavares Andre L. , Moody Sally A.
Published: May 2022

Branchio‐oto‐renal syndrome (BOR) is a condition that presents with variable craniofacial, otic, and/or renal malformations. Half of the BOR patients have a mutation in SIX1or its co‐factor EYA1. As the other 50% of cases are of unknown cause, it is important to identify other putative co‐factors of SIX1 that may contribute to BOR. A screen of the fly interactome identified zinc‐finger MYM‐containing proteins, Zmym2 and Zmym4, as putative co‐factors of Six1. Recent studies have established that Zmym2 is a transcriptional repressor that interacts with Six4 during renal development. The function of Zmym4 has not yet been well characterized. In situ hybridization revealed that they are co‐expressed with six1and eya1 in the pre‐placodal ectoderm (PPE) and are later expressed in the otic vesicle (OV) and neural crest (NC) of Xenopus. Luciferase assays indicate that Zmym4 enhances Six1‐Eya1 transcriptional activity whereas Zmym2 has a mild repressive effect. Loss of either Zmym2 or Zmym4 affects neural border, NC, and PPE marker genes, suggesting that both genes are important for craniofacial development. At later stages, loss of either Zmym2 or Zmym4 results in a reduction in the size of the OV as well as the branchial arch NC; the later results in altered craniofacial cartilages. Current experiments aim to decipher whether Zmym2 and Zmym4 interact with Six1 and/or Eya1 through co‐immunoprecipitation experiments. Collectively, these data indicate that Zmym4 modulates Six1 transcriptional activity, Zmym2 and Zmym4 are required for craniofacial development, and suggests they may contribute to BOR phenotypes.
Impact of Genetics and Age on Muscle‐type Creatine Kinase
Authors: Mosher Eric P. , Eberhard Colten D. , Bumpus Namandje N.
Published: May 2022

Muscle‐type creatine kinase (CKM) plays an important role in physiology by maintaining ATP concentrations. Cells use phosphocreatine as a means to store high energy phosphate. As ATP is rapidly depleted during events such as muscle contraction, creatine kinase enzymes replenish ATP by transferring the phosphate from phosphocreatine to ADP. Additionally, it has been found that CKM has pharmacologically‐relevant activity, as it is involved in the activation of tenofovir (TFV), a nucleotide analog reverse transcriptase inhibitor used for HIV pre‐exposure prophylaxis (PrEP). TFV is a prodrug that requires phosphorylation by intracellular kinases to produce the pharmacologically active tenofovir diphosphate (TFV‐DP) metabolite. In colon tissue, a site of HIV infection, CKM is responsible for the second phosphorylation reaction, converting TFV‐monophosphate (TFV‐MP) to TFV‐DP. While TFV‐based PrEP has been largely successful, some people on PrEP still become infected with HIV. Due to the importance of CKM in TFV activation, we hypothesize that person‐to‐person differences in CKM activity in colon tissue may impact TFV‐DP formation and thus protection from infection. To this extent, we have used a mix of biochemical and mass spectrometry‐based tools to study the impact of both genetics and age on muscle‐type creatine kinase. Wild‐type (WT) human CKM and 15 naturally‐occurring mutants were recombinantly expressed and purified and the impact of these mutations on the activities of the enzyme were determined using in vitro assays. It was found that ten of these mutations led to reductions in TFV‐MP phosphorylation to less than 25% of that of WT. Additionally, only four of these ten mutations resulted in decreases in both the canonical ADP phosphorylation and ATP dephosphorylation reactions: R130H, R132C, W211R, and N286I. Positions 130, 132, and 286 are implicated by protein crystal structures to be involved in substrate binding and phosphoryl transfer. Further biochemical studies using a thermal shift assay and hydrogen‐deuterium exchange mass spectrometry revealed the W211R mutation reduces enzyme activity by impacting the protein structure in a destabilizing manner. In pursuit of understanding the impact of age on CKM, colon tissue from 12‐ and 85‐week‐old C57BL/6J mice (n=5) were analyzed using nano‐liquid chromatography mass spectrometry proteomics. It was found that the abundance of CKM in colon tissue increased by 20% in the older mice compared to the younger comparison group. Additionally, phosphorylation at serine 372 was found in three 12‐week‐old mice and just one of the 85‐week‐old mice. These data suggest that the abundance and post‐translational modification of CKM in colon tissue may change as a function of age, which could have an effect on TFV activation. Together, these data demonstrate that two factors, age and genetics, may lead to interindividual differences in TFV activation, suggesting a potential role for a more personalized approach to PrEP.
Role of Neuregulin 3‐ErbB4 Signaling in a Murine Model of Co‐Morbid Nicotine Dependence and Schizophrenia
Authors: Prantzalos Emily , Turner Jill
Published: May 2022

According to the NIH, rates of nicotine dependence among patients with schizophrenia are at nearly 90%, compared to smoking rates of approximately 12% in the general population. Current research shows that variations in the gene for Neuregulin 3 (NRG3), a member of the EGF superfamily and the cognate ligand for ErbB4 receptors, are considered some of the strongest genetic links contributing to schizophrenia risk. Additionally, NRG3 protein and mRNA have been found to be highly enriched following chronic nicotine exposure and withdrawal. However, specific mechanisms underpinning NRG3’s association with both increased schizophrenia risk and increased nicotine withdrawal symptomology are unknown. In this ongoing experiment, we are testing the overall hypothesis that NRG3‐ErbB4 signaling effects on NMDA receptor function may provide a discrete, mechanistic link between nicotine dependence and schizophrenia.

We plan to utilize a novel genetic mouse model of both nicotine dependence and schizophrenia in a multidimensional approach to study how NRG3 mediates nicotine withdrawal‐induced alterations on behavioral and molecular levels and how the alterations relate to schizophrenic‐like dysfunction. To first begin these investigations, we examined the behavioral and molecular baseline effects of nicotine and aripiprazole, a long‐acting atypical antipsychotic used to treat schizophrenia, in wildtype littermates. Animals treated chronically for nicotine and then undergoing spontaneous nicotine withdrawal were examined in the alternating Y‐maze, social interaction test, and open field test for efficacy in reducing behavioral endophenotypes common to both nicotine withdrawal and schizophrenia in our model. Following behavioral testing, PFC tissues were analyzed to determine mRNA and protein expression of factors in the NRG3 signaling pathway. Preliminary results show differential changes in mRNA expression associated with glutamatergic and serotonergic transmission in the mPFC, OFC, and hippocampus. Ongoing experiments will extend these studies to include time‐ and site‐specific genetic deletion of NRG3 in the PFC and compare these results to those derived in wildtype littermates. Our findings will provide insight into the underlying mechanisms linking the NRG3 signaling pathway and nicotine dependence, and may provide a novel link between nicotine use and its highly co‐morbid psychiatric disorder, schizophrenia. Findings from these highly translational studies may lead to novel therapeutic development for both nicotine dependence and schizophrenia.