bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular NAD+, fibroblast senescence and pulmonary fibrosis
Authors: Liu Tianju , Rinke Andrew E. , Wang Jing , Phan Sem H.
Published: April 2020

Aging is a risk factor in fibrotic interstitial lung diseases, including idiopathic pulmonary fibrosis (IPF). Cellular senescence is observed in IPF and animal models of lung fibrosis along with telomere dysfunction, although the potential differential pathogenic significance of their cell type specific localization is unknown. Removal of senescent cells ameliorates fibrosis in animal models and are considered as potential therapeutic approaches. However the pathogenic role of aging and/or cellular senescence in the development of lung fibrosis are not fully understood. Declining cellular nicotinamide adenine dinucleotide (NAD+) levels as seen in aging, may impair the activity of senescence suppressor Sirt1, an NAD+‐dependent deacetylase, hence boosting the levels of NAD+ have been proposed to delay or inhibit aging related illnesses and improved longevity. To elucidate the role of NAD+ in cellular senescence in pathogenesis of pulmonary fibrosis, we investigated the impact of increasing or reducing NAD+ levels on bleomycin (BLM)‐induced mouse pulmonary fibrosis by supplementation with nicotinamide mononucleotide (NMN) by i.p injection, or nicotinamide phosphoribosyltransferase (Nampt) deficiency, respectively. The latter was induced by treating transgenic mice bearing the floxed Nampt and Col1a2‐CreERT transgenes with tamoxifen, resulting in mesenchymal cell specific Nampt deletion. The results showed that pre‐treatment of NMN injection before BLM injury significantly reduced lung myofibroblast differentiation as manifested by decreased Acta2 mRNA, which was accompanied by decreased numbers of senescent type II alveolar epithelial cells (SPC+/p16+), and BM‐derived monocytic myeloid cells. In contrast, deletion of Nampt in mesenchymal cells with consequent reduction of NAD+ levels caused increased myofibroblast differentiation, and more severe lung fibrosis measured by lung hydroxyproline and histology after BLM injury when compared to the response in wild type animals. In contrast to wild type cells, Nampt‐deficient lung fibroblasts exhibited elevated expression of p16, and senescence‐associated secretory phenotype (SASP) cytokines, including TNFα, IL‐1β, and IL‐6. Notably enhanced recruitment of BM‐derived myeloid cells into BLM‐injured lung was also observed. These findings suggest that NAD+ may play a significant role in prevention of cellular senescence and protection from pulmonary injury/fibrosis.

Support or Funding Information

This study is supported by NIH grants HL112880 and HL 138417.
Identification of Allosteric Modifiers of Mosquito Odorant Receptor Function and Odor‐Triggered Behaviors
Authors: Iatrou Kostas , Kythreoti G , Sdralia N , Tsitoura P , Papachristos D P. , Michaelakis A , Schulz S
Published: April 2020

Insect odorant receptors (ORs) are heteromeric ion channels each consisting of a common subunit, ORco, and one of multiple variable subunits, ORx, to which odorants acting as specific receptor agonists or antagonists bind. In cells expressing the ORco subunit alone, functional homomeric channels are formed that can be activated by specific ORco agonists. We have previously shown that mosquito repellents that inhibit odor‐specific responses of multiple Anopheles gambiae heteromeric receptors are ORco‐specific antagonists. Additionally, the responses of heteromeric receptors induced by ORx‐agonists were significantly enhanced in the presence of an ORco‐agonist (OA), a finding suggesting that OA binding causes structural rearrangements in the liganded ORx heteromers.

We are now reporting on the extension of our studies for discovery of A. gambiae ORco agonists and antagonists using an insect cell‐based ORco expression platform that allows fast screening of compound collections. Screening of a collection of volatile organic compounds (VOCs) of natural origin for relevant bioactive ligands resulted in the identification of several hits acting as ORco antagonists. The functional relevance of the identified hits has been established by in vivo repellence assays against Aedes albopictus mosquitoes, which are capable of vectoring a number of infectious agents. Nine newly identified ORco antagonists displayed significant mosquito repellent activities, with three of them causing mosquito anosmia‐like effects similar to those of equivalent doses of the widely used insect repellent DEET. Moreover, binary mixtures of the most active compounds caused stronger repellence effects relative to the single compounds, a finding suggesting the possible existence of different antagonist binding sites on ORco and functional synergies caused by allosteric interactions within the homomer and the heteromers. These results provide proof of concept that natural ORco antagonists may modify olfaction‐based mosquito behaviors in a predicted fashion. Moreover, the screening platform employed in this study yields enhanced possibilities for personal protection against mosquito‐borne infectious diseases.

Support or Funding Information

Supported by OPENSCREEN‐GR “An Open‐Access Research Infrastructure of Chemical Biology and Target‐Based Screening Technologies for Human and Animal Health, Agriculture and the Environment” (MIS 5002691) funded under the Action “Reinforcement of the Research and Innovation Infrastructure” of the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020), and LIFE CONOPS (LIFE12 ENV/GR/000466) funded by the European Commission under the program LIFE + Environment Policy and Governance. We thank Asteria Karadima for the repellence test schematic.
The role of myeloid cells in lung injury‐induced muscle atrophy
Authors: Bivona Joseph J. , Crymble Hanna M. , Stapleton Renee D. , Files D. Clark , Toth Michael , Suratt Benjamin T.
Published: April 2020

Rationale

Intensive care unit acquired weakness (ICUAW) occurs in nearly 50% of patients admitted to the ICU. Patients exhibiting ICUAW have increased in‐hospital and post‐ICU mortality. ICUAW, and the associated muscle atrophy specifically, cannot be completely explained by disuse and may involve a secondary trigger to enhance the breakdown of muscle fibers. Recent work has suggested that excessive systemic inflammation may contribute to this process. We hypothesized that muscle atrophy in ICUAW may, in part, be induced by myeloid cell recruitment to muscle.

Methods

C57Bl6/J mice received intratracheal (IT) instillation of lipopolysaccharide (LPS, 3 μg/g) or saline and were euthanized 3, 6, 12, 24, 72 hours thereafter. For antibody‐mediated myeloid cell depletion, 200 μg of αGR‐1, αLy6G, or isotype control antibodies were administered IP 24 hours and 1 hour prior to injury and mice were examined 24 hours post‐injury. Whole gastrocnemius muscle was analyzed using RT‐qPCR. For in vitro studies, C2C12 myoblasts were differentiated for 7 days and stimulated with LPS (1 μg/ml) to elicit an inflammatory response. Bone marrow derived macrophages (BMDMs) were differentiated with 10 ng/ml of macrophage‐colony stimulation factor for 7 days and seeded at a density of 2600 cells/cm2 (4940 cells/well) onto C2C12 myotubes after 6 days of differentiation. On day 7 of C2C12 differentiation, cocultures were stimulated as previously described. Supernatants and cell lysates were analyzed by ELISA and RT‐qPCR. Myotube diameter was measured using ImageJ.

Results

IT LPS upregulates the atrogene muscle RING‐finger 1 (MuRF1) and induces atrophy in vivo. However, this effect of LPS is not seen in cultured C2C12 myotubes alone. Depletion of neutrophils and monocytes with αGR‐1 antibody reduced MuRF1 expression in vivo. Both C2C12 myotubes and whole muscle show rapid, transient expression of myeloid chemoattractants MCP‐1 and KC (CXCL1). Cocultures of BMDMs and C2C12 myotubes enhance the expression of IL‐6 and MCP‐1 synergistically and induce C2C12 myotubes to produce TNFα.

Conclusions

Our data indicate that IT LPS induces MuRF1 in vivo and our in vitro data in myotubes suggests that this effect may require myeloid cells. Further supporting this conclusion, we found that antibody depletion of myeloid cells reduced IT LPS‐induced MuRF1 expression. Moreover, expression of key myeloid cell chemoattractants are induced in muscle both in vitro and in vivo in response to LPS and addition of BMDMs to C2C12 myotubes with LPS treatment reveals synergistic increases of IL‐6 and MCP‐1 and induces C2C12 myotubes to produce TNFα. The current study implicates macrophages and neutrophils as modulators of the muscle atrophy response to following lung injury.

Support or Funding Information

NIH 5R01HL143452‐02NIH 2T32HL076122‐16
Dantrolene Improves Cerebral Perfusion More than Nimodipine in a Rat Model of Cerebral‐induced Vasospasm
Authors: Roman Marie , Tirado Guillermo , Martinez Solianne , Martins Antonio , Ferrer Yancy , Morales Myrna , Crespo Maria J.
Published: April 2020

A cerebral vasospasm (CVSP) is a potent vasoconstriction of the cerebral vasculature, and the primary cause of morbidity and mortality following a hemorrhagic stroke. The middle cerebral artery (MCA) is one of the most common locations affected by CVSPs. Experimental evidence shows that the ryanodine receptor inhibitor dantrolene may be as effective as the Ca+2 channel blocker nimodipine in reducing vasospasms in the systemic vasculature. To determine if this effect also extends to the cerebral vasculature, we used a rat model of cerebral‐induced vasospasms to investigate the effects of intravenously administered dantrolene (2.5 mg/kg) and nimodipine (1 mg/kg) on cerebral blood flow velocity in the MCA. Blood flow velocity was measured in the MCA seven days after vasospasm‐induction, using a PeriFlux 5000 Laser Doppler System. Measurements were performed 30 minutes before and one hour after either dantrolene or nimodipine administration. In addition, hemodynamic parameters (i.e., systolic, diastolic, and mean blood pressures; heart rate) were measured before and after administration of the drugs. Dantrolene decreased MCA blood flow velocity by 35% (from 364.7± 60 to 231.0 ± 34 perfusion units), whereas nimodipine reduced this parameter by only 14% (N=6, P<0.05). For both dantrolene and nimodipine, the hemodynamic parameters remained unchanged (N=6, P>0.05, when compared to results before drug administration). Our results suggest that 2.5 mg/kg dantrolene is more effective than 1 mg/kg nimodipine in reducing vascular reactivity and improving cerebral blood perfusion in the MCA, without altering systemic hemodynamic parameters. Therefore, dantrolene may provide a promising therapy for the management of CVSP and its complications.

Support or Funding Information

This work was supported by Grants from the National Institute of Health (MBRS‐RISE Grant R25GM061838), the NIMHD‐CCRHD‐RCMI Program (U54‐MD007600), and the Anesthesiology Department of the University of Puerto Rico‐School of Medicine.
Post‐exposure treatment with esomeprazole protects pregnant mice from mortality and from compromised fetal growth after exposure to chlorine gas.
Authors: Molyvdas Adam , Ren Changchun , Dunigan Katelyn , Tipple Trent , Matalon Sadis , Jilling Tamas
Published: April 2020

Exposure to Chlorine (Cl2) and Bromine (Br2) causes significant cardiopulmonary injury to mice, rats, rabbits and humans triggering extensive systemic injury. After exposure to Br2 pregnant mice develop preeclampsia‐like symptoms and increased mortality as compared to identically exposed male or non‐pregnant female mice (Lambert et al Hypertension 2018). Heme oxygenase (HO‐1), which has been shown recently to be induced by proton pump inhibitors such as esomeprazole (ESO), has been implicated as a protective molecule against halogen inhalation induced injury in non‐pregnant animals and, additionally, against the development of symptoms in models of preeclampsia in pregnant mice.

Timed pregnant C57BL/6 mice were exposed to 400ppm Cl2 for 10min at E14.5, then treated with vehicle or with 30mg/kg ESO by gavage 6h post exposure, then at every 24h thereafter. Survival and body weights were monitored until E18.5. Fetal weights were recorded at E18.5 after euthanasia. Finally, using nuclear factor (erythroid‐derived 2)–like 2 (Nrf2)‐knock out and Nrf2 competent murine transformed club cells (mtCC) we tested whether HO‐1 production was induced via Nrf2.

Survival was significantly higher (P=0.0056) in Cl2 exposed pregnant mice that received ESO (88.9% survival, n=9) compared to the vehicle treated animals (29.4% survival, n=17). Maternal exposure to Cl2 severely compromises fetal growth (0.6366g±0.0666 (S.E.M.), n=5) when compared to normal fetuses (1.167g±0.0399 (S.E.M.), n=5) (P<0.0001). However, the weight of fetuses collected from Cl2 exposed pregnant mice that received ESO (0.9264g±0.0542 (S.E.M.), n=8) compared to weights of fetuses from vehicle treated animals (0.6366g±0.0666 (S.E.M.), n=5) was significantly higher (P=0.0058). Finally, maternal weight, as normalized to weight recorded preexposure, in Cl2 exposed ESO treated pregnant mice (93.64%±4.038% (S.E.M.), n=8) compared with the vehicle treated animals (81.31%±5.717% (S.E.M.), n=5) was not significantly different (P=0.1772). Additionally, we found that HO‐1 gene expression is induced in a dose dependent manner by ESO only in Nrf2‐competent cells (p<0.001).

Our results suggest that ESO may be an effective countermeasure to increase maternal survival and improve fetal growth post halogen inhalation in pregnancy. Further mechanistic studies will have to explore organ/tissue specific role of HO‐1 induction in the protective effect.

Support or Funding Information

This work was supported by: 5U01ES027697 (to Tamas Jilling) and 5U01ES026458 (to Sadis Matalon)
DHHC21‐mediated α1 Adrenergic Receptor Palmitoylation Contributes to Renal Blood Flow Regulation During Sepsis
Authors: Yang Xiaoyuan (Sherry) , Zheng Ethan , Chatterjee Victor , Creasy Alexandria , Ma Yonggang , Overstreet Jonathan , Yuan Sarah Y.
Published: April 2020

Palmitoylation is a reaction of 16‐carbon palmitic acid attachment to a cysteine residue of a protein via thioester linkage, catalyzed by a family of palmitoyl acyltransferases containing the DHHC motif. This post‐translational modification plays a critical role in regulating protein function, subcellular localization and interactions with other cellular molecules. A recent study shows that vascular α1 adrenergic receptor (α1AR) requires DHHC21‐mediated palmitoylation for its signaling activity to induce vasoconstriction. In the kidney, arterial vasoconstriction has been implicated in the development of renal hypoperfusion following sepsis. In this study, we sought to examine whether DHHC21 contributes to kidney dysfunction by affecting renal blood flow through α1AR palmitoylation. Cecal ligation puncture (CLP) was performed to induce polymicrobial sepsis in wild type and DHHC21 functional deficient (Zdhhc21dep/dep) mice. Their renal function was dynamically assessed based on tracer (IRdye 800CW) clearance measured by multispectral optoacoustic tomography (MSOT). The results showed that in wild‐type mice, IRdye 800CW movement from kidney cortex to the medulla/pelvis region was significantly slower after CLP, compared to sham control. The sepsis‐induced impairment in renal clearance was alleviated in Zdhhc21dep/dep mice. Consistently, histological analysis (Periodic acid‐Schiff staining) revealed that CLP caused kidney structural damages, evidenced by tubular vacuolation, brush border loss, tubular cell detachment and leukocyte infiltration; these pathologies were greatly ameliorated in DHHC21 loss‐of‐function mice. Moreover, compared with wild‐type mice, Zdhhc21dep/dep mice exhibited reduced CLP‐induced increases in circulating creatinine and urea nitrogen. Mechanistically, a decreased ratio of oxygenated to deoxygenated hemoglobin was detected in kidneys of wild‐type mice following CLP, suggesting renal tissue hypoxia in response to sepsis. We then asked whether this lack of oxygenation is associated with reduced renal blood flow due to arterial constriction and whether α1AR palmitoylation is involved in this process. Indeed, we found a significant increase in α1AR palmitoylation, along with activation of the α1AR downstream effector ERK, in renal arteries of wild‐type mice following CLP. These molecular responses occurred concomitantly with a reduced renal blood flow, measured by a transonic flowmeter. In contrast, sepsis‐induced α1AR palmitoylation/activation in renal arteries was greatly attenuated in Zdhhc21dep/dep mice, consistent with a better‐preserved renal blood flow in these mice. Taken together, our findings indicate that DHHC21‐mediated α1AR palmitoylation contributes to renal blood flow reduction and kidney injury during sepsis.

Support or Funding Information

This work was supported by the National Institutes of Health Grant HL070752 (to SYY)
Akt‐PI3K signaling pathway is a crucial player in dopamine D2 receptor‐mediated emesis in least shrews
Authors: Belkacemi Louiza , Zhong Weixia , Darmani Nissar Ahmad
Published: April 2020

Dopamine is a member of the catecholamine family involved in multiple physiological functions. Together with its five dopaminergic receptors (DRs), DA is associated with several neurological diseases including restless leg syndrome, schizophrenia, Parkinson’s disease, and depression. Unfortunately, most DR‐based agonists used to treat some of these diseases evoke nausea and vomiting as impending side‐effects. Coincidently, activation of the kinase phosphoinositide 3‐kinase (PI3K)/protein kinase B (Akt) pathway not only plays a significant role in the onset and the progression of neurodegenerative disorders but may also evoke emesis caused by stimulation of dopamine D2 receptor (DR2). Thus, the aim of this study was to decipher the role of Akt in DR2‐induced emesis using the least shrew model of vomiting (Cryptotis parva). Shrews were injected intraperitoneally (i.p.) with a fully effective emetic dose of the DR2 preferring agonist quinpirole (2 mg/kg) in the presence and the absence of either: i) a corresponding DR2 antagonist sulpiride (subcutaneous (s.c.), 8 mg/kg), or ii) incremental doses of the PI3K inhibitor LY294002 (0, 5, 10 mg/kg, i.p.). The treated shrews were observed for 30 mins for vomiting behavior following quinpirole injection. Another set of shrews were treated with 2 mg/kg quinpirole alone (N = 4 per group) and sacrificed at 5, 15, and 30 mins to determine the time‐dependent profile of Akt phosphorylation. The animals were then perfused, and brains collected for immunohistochemistry studies or brainstems harvested for Western blot analysis on protein extracts. For antagonist studies, different groups of shrews were given sulpiride (8 mg/kg, s.c) or LY294002 (10 mg/kg, i.p.) (N = 4–6 per group) for 30 mins prior to injection of quinpirole (2 mg/kg) and brainstems were collected after 15 mins for Western blot analysis. Behavioral results showed that quinpirole induced vomiting was significantly blocked by sulpiride (8 mg/kg) and LY296004 (10 mg/kg). Akt phosphorylation at Ser473 was significantly increased at 15 and 30 mins in the brainstem with quinpirole treatment alone. The increase of phospho‐Akt protein in brainstem emetic nuclei [area postrema (AP), nucleus of the solitary tract (NTS), and dorsal motor nucleus of the vagus (DMNX)] was further confirmed through immunostaining brainstem sections of quinpirole‐treated least shrews. Sulpiride on the other hand significantly inhibited quinpirole‐induced Akt and PI3K protein phosphorylation at 15 mins. Likewise, LY29004 prevented Akt activation at 15 mins. Our findings uncovered a crucial role for DR2‐mediated Akt activation in emesis and suggest targeting signals up upstream of Akt enzyme may offer new avenues for potential potent antiemetics.

Support or Funding Information

This work was supported in part by NIH‐NCI grant RO1CA207287 and the WesternU startup fund (1395) to ND.
The Origins & Evolution of Stress Research: From Distress to Eustress
Authors: Szabo Sandor
Published: April 2020

Hans Selye (1907, Vienna ‐ 1982, Montreal) first published his seminal papers in 1936 (Nature) & 1937 (Science) that described the three stages of stress reaction (i.e., alarm reaction, resistance & exhaustion) in rats exposed to cold, immobilization or toxic agents. He identified the adrenals & the pituitary as the main endocrine glands involved in this nonspecific reaction. He also knew & documented that although the sudden release of epinephrine (adrenaline) from the adrenal medulla, as described earlier by Walter Canon, plays a critical role in the initial alarm reaction, but the long‐term effects of stress reaction are due to the enhanced synthesis & release of glucocorticoids from the adrenal cortex. Based on this mechanistic reasoning he described in his first publications what has later become known the “morphologic triad of stress”: adrenal hypertrophy, gastric erosions/ulcers & thymo‐lymphatic atrophy. Furthermore, what we call today ‘stress‐related diseases’ in man, he described these in the 1940s & 1950s as ‘diseases of adaption’ in rats. In the last stages of his productive life, Selye realized that virtually all the stressors (i.e., stress‐causing agents) he used in experimental animals were unpleasant, negative factors that caused ‘distress’. But around the early 1970s, mostly based on the work of Swedish psychosocial scientist Lennart Levi who distinguished “negative stress” from “positive stress”, Selye in his last scientific books (1974 & 1976) “Stress without distress” & “Stress in Health & Disease”, he called
distress the stress reaction caused by negative, unpleasant factors &
eustress (from ‘euphoria’) the similar reaction triggered by positive, pleasant factors like joy over major rewards, receiving surprising good news. Namely, we learned during the last about 40 years that our pituitary gland cannot distinguish between positive & negative stimuli to release ACTH (only our brain cortex, amygdala & hippocampus may perceive the difference). Because of these stress‐related creative discoveries Hans Selye is known as ‘father of biologic stress’, but he also made numerous other major discoveries, e.g., he also introduced the first physiologically sound, structure‐activity classification of steroids that was also based on the chemical structure of steroids in 1943. He not only introduced the names of glucocorticoids & mineralocorticoids but discovered their anti‐ & pro‐inflammatory properties, respectively, of these steroids in animal models. Furthermore, he not only described the first stress‐induced gastric ulcers in rats (1936 & 1937), but he also recognized the first human ‘stress ulcers’ during the air‐raids in London during World War II (Lancet, 1943). Thus, Hans Selye not only introduced the concept & bodily reaction what we call today “stress”, but he also creatively synthesized the contributions of Walter Canon & Lennart Levi in a mechanistic molecular & cellular pathway.
Those Who Can’t Do, Teach&[hellip]Anatomy? The Presence of Stereotype Threat in Anatomical Sciences Educators
Authors: Cale Andrew S. , Lee Lisa M. J.
Published: April 2020

Background

Anatomical sciences educators play a critical role in training future health professionals and researchers. However, in academic culture, teaching is often negatively stereotyped and less respected. As a result, educators are vulnerable to stereotype threat, a situation in which an individual underperforms due to their fear of confirming a negative stereotype associated with their group.

Methods

This IRB‐Exempt study (COMIRB #19‐1572) was designed to identify the associations of stereotype awareness and concern in anatomical sciences educators. Educators from post‐secondary and health professional institutions were invited to complete a survey consisting of demographic and 5‐point Likert scale questions regarding six domains: educator identification, institutional identification, stereotype awareness and concern, imposter tendencies, anxiety, and confidence. Spearman’s correlation was applied to survey responses from all educators as well as subgroups based on gender, ethnicity, highest degree, and academic appointment to identify statistically significant correlations.

Results

A total of 100 anatomical sciences educators completed the survey. In these individuals, stereotype awareness and concern was positively correlated with anxiety (r=0.293, p<0.003), imposter tendencies (r=0.348, p<0.001), and teaching load (r=0.275, p=0.006) as well as negatively correlated with identification with institution (r=−0.404, p<0.001) and confidence (r=−0.340, p=0.001). Ethnic minorities, non‐doctoral degree holders, and early career educators also exhibited statistically significant, but more pronounced correlations.

Conclusion

This study demonstrates that awareness and concerns regarding negative teaching faculty stereotypes is linked to varying degrees with institutional disidentification, imposter tendencies, anxiety, and diminished confidence in anatomical sciences educators.
Heat stress defense in dental pulp stem cells
Authors: Xiao Nan Tori , Dakafay Harmony Matshik , Thor Der
Published: April 2020

Many dental treatment procedures can generate excessive heat within a short period of time. The heat on the tooth surface can be transduced to the dental pulp chamber and lead to alteration of dental pulp stem cells, vascular structures and sensory neurons within the chamber. It has been reported that elevation of temperature by 5.5°C in the pulp chamber caused 15% irreversible pulpal damage. However, the molecular changes of dental pulp cells post heat stress has not been characterized. In this study, we explored the changes in human dental pulp Stem Cells from the Exfoliated Deciduous teeth (SHED) after exposure to heat stress.

Lysate of SHED cells were collected after exposure to heat stress at 42°C or normal culture temperature at 37°C. Western blots were used to examine the expression of Heat Shock Proteins (HSP). Human cytokine arrays (AAH‐CYT‐5, RayBiotech) were used to evaluate the changes in the expression of 80 human cytokines in SHED cells after heat stress.

Short term exposure to heat stress induced phosphorylation of HSP27. Prolonged exposure further increased expression of other heat shock proteins, which have been reported to play an important role in cell protection under stress. In addition, transient exposure to heat stress caused differential expression of cytokines in SHED cells, suppressing expression of cytokines as IL‐1a and IL‐10, while increasing expression of glial cell derived neurotrophic factor (GDNF) and insulin‐like growth factor ‐1 (IGF‐1).

The results showed HSPs and multiple cytokines play important role in detecting and responding to heat stress on the SHED cells. These factors could potentially protect SHED cells from thermal damage and promote stem cell regeneration post thermal damage.

Support or Funding Information

Faculty Scholarly/Artistic Activities Award DDNA12‐013 University of the Pacific.
Maternal sleep disordered breathing during pregnancy as a cause of autism relevant neuronal and behavioral aberrations in the offspring.
Authors: Vanderplow Amanda M. , Kermath Bailey A. , Ewald Andrea C. , Gums Kimberly T. , Seablom Erin N. , Baker Tracy L. , Watters Jyoti J. , Cahill Michael E.
Published: April 2020

Sleep disordered breathing (SDB) is characterized by recurring breathing cessations during sleep, causing intermittent hypoxia (oxygen deprivation) often several hundred times per night. SDB is present in ~15% of pregnant women as compared to ~2–4% of non‐pregnant women of childbearing age. Risk factors for gestational SDB development bear striking similarities to the collection of maternal risk factors for autism spectrum disorders (ASD), including advanced maternal age and maternal obesity. Maternal immune activation/inflammation is both a consequence of SDB, and an independent risk factor for, ASD. We posit that an overlooked, yet increasingly common inflammatory stimulus, maternal SDB, contributes to ASD etiology. We recently developed a rat model in which to study the consequences of maternal SDB on the offspring by exposing pregnant rats to nightly intermittent hypoxia from gestational days 10–21. Our findings indicate that offspring of mothers subjected to intermittent hypoxia during pregnancy exhibit deficits in several autism‐relevant behaviors, including a constellation of cognitive impairments, reduced interest in social interaction, and heightened anxiety. In most instances, the magnitude of behavioral disruption was more severe in male than female offspring. To begin to understand the mechanisms contributing to these behavioral aberrations, we examined the density of dendritic spines in the prefrontal cortex (PFC) and CA3 region of the hippocampus. Our data suggest that the offspring of mothers subjected to nightly intermittent hypoxia exhibited a striking increase in dendritic spine density in the PFC when compared to control offspring. However, the dendritic spine density in the CA3 region showed no significant differences between groups. Similar to our behavioral observations, dendritic spine aberrations were more prominent in male vs. female offspring. Additionally, these male offspring showed significant differences in spine morphology when compared to control offspring. Taken together, these findings indicate that maternal sleep disordered breathing not only induces autism‐relevant behavioral impairments, but also mimics the heightened cortical synaptic connectivity phenotypes recently uncovered in autism. Future studies are aimed at identifying a role for microglial inflammation and concomitant reduction in microglial‐mediated synaptic pruning in the behavioral and synaptic structural alterations that we have revealed in this model.

Support or Funding Information

R25 Team‐ScienceUW2020 Pilot Award
Ectopically Expressed Human Olfactory Receptor OR51E1: a Search for Novel Molecular Probes
Authors: Pronin Alexey , Janczura Karolina , Volmar Claude-Henry , Brothers Shawn , Slepak Vladlen
Published: April 2020

Olfactory receptors comprise the largest family of G protein‐couples receptors (GPCRs) ‐ ~1400 functional genes in the mouse (Olfrs) and ~400 in the human (ORs). Most genes were assigned to it based solely on the sequence similarity. Recent studies demonstrated that many Olfrs are expressed outside of the nasal cavity and possibly involved in physiological processes such as muscle regeneration and controlling blood pressure. These ectopically expressed olfactory receptors might recognize cues other than odorants and be involved in processes independent of the sense of smell. Ligands were identified for fewer than 100 of ORs and Olfrs.

Using transcriptomics analysis followed by RT‐PCR and in situ RNA hybridization we previously showed expression of several Olfrs and other olfactory signaling genes expressed in the mouse ocular tissues. We focused our attention on Olfr558 and demonstrated its expression in blood vessels of the eye. The human ortholog of Olfr558 (OR51E1 or PSGR2) was shown to be expressed in the prostate and dramatically up‐regulated in prostate cancer and is an established biomarker of malignancy. The human and mouse genes encoding this receptor are almost identical (94%), which is unique for GPCRs in general, and particularly for sensory chemoreceptors. This unusual evolutionary conservation points to the importance of its physiological function. While the function of this receptor or its native ligand is unknown, in vitro it responds to some short‐ and medium‐chain fatty acids (e.g., butyrate and valerate) by activating adenylyl cyclase. We found that growth of human prostate cancer cells that express OR51E1 is inhibited by valerate. The knockdown of OR51E1 gene with shRNA eliminated this effect of valerate.

While butyrate and valerate can be used in such experiments, their low affinity and selectivity complicates interpretation of the data. Additionally, they are quickly metabolized in the body. Thus, there is a clear need for novel probes that can be used to dissect OR51E1 function. To address this need, we established a stable cell line overexpressing OR51E1 and demonstrated that in response to known OR51E1 agonists this cell line increases intracellular cAMP level. We then used this cell line for screening of a diverse chemical library of 9600 compounds, showing its suitability for high throughput screening. To demonstrate that our expression system can be used for expression of other ORs, we generated stable cell lines overexpressing two other receptors – OR51E2 and OR2AT4. We showed that both receptors are expressed as proteins of the expected molecular weight and localize on the cell surface. Since these ORs represents a large class of previously unexplored yet druggable targets, this project facilitates investigation of the entire family of ectopic olfactory receptors.
Measuring Effects of Treatment with a Novel Metalloprotease Inhibitor, Extracellular Matrix Protection Factor‐2, on Total Protein Production by Human Gingival Fibroblast Cultures
Authors: Maloney Trevor J. , Mattioli Patrisia , Laird Patrick , Musiol Joseph , Popper Hannah , Otto Ashley , Nguyen Stephanie , Graham Morgan , Green Kevan , Shamseddin Seyed , Borghaei Ruth , D’Angelo Marina
Published: April 2020

Collagen, produced by fibroblasts, serves an integral role in the structural integrity of the extracellular matrix of the human gingiva, but during periodontal disease, matrix metalloproteases (MMPs) are upregulated and contribute significantly to the degradation of collagen. In this study, we test the effects of the novel MMP inhibitor, Extracellular Matrix Protection Factor‐2 (ECPF‐2) on total protein production by human gingival fibroblasts (HGVFs) reared in serum‐free media. Cells are enzymatically released from gingival tissue isolated during oral surgery and cultures produced from patient samples: normal, non‐inflamed (NTN); inflamed non‐smoker (IFN); inflamed previous smoker (IFP); inflamed current smoker (IFC). Subconfluent cultures were switched to 0.1% fetal bovine serum containing DMEM media overnight and then treated for 24 hours with complete serum‐free DMEM (Control); 5ug ECPF‐2; or 50ug ECPF‐2. Conditioned media was collected and concentrated and the cell layer was extracted with 0.5% CHAPS buffer. Total protein of the samples was quantified using the Pierce Modified Lowry Protein Assay. Regardless of treatment or patient pathology, all cultures tested produced approximately 1.3–1.5 ug/ml/culture of total protein. There was no statistically significant differences between patient samples or within treatment conditions per patient. These data suggest that our culture system allows for viable metabolism in serum‐free medium and treatment with the novel MMP inhibitor, ECPF‐2, does not affect the metabolic activity of HGVF cultures. Therefore, this culture system can be used to measure the therapeutic effects of ECPF‐2 on collagen metabolism associated with periodontal disease.

Support or Funding Information

Support from the PCOM Division of Research
Cardiomyocyte SNAP29 Insulates Desmosomes from Targeted Protein Degradation by Selective Autophagy to Restrict Arrhythmias
Authors: Wang Jie , Pellman Jason , Liang Yan , Lyon Robert , Mezzano Valeria , Zanella Fabian , Zhang Jing , Nigam Vishal , Gu Yusu , Dalton Nancy , Peterson Kirk L. , Sheikh Farah
Published: April 2020

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a fatal and untreatable inherited cardiomyopathy associated with ventricular dysfunction and cardiac arrhythmias leading to sudden cardiac death in the young. Early detection is hindered as people present with complex disease patterns and harbors a “concealed phase”, where lethal arrhythmias are observed in the absence of overt structural changes to the heart. Mutations and loss of components of the desmosome, a cardiac mechanical cell‐cell junction, have been linked to the development of ARVC through human and mouse genetic studies. However, limited information exists on how desmosomal protein dysregulation/loss can trigger the complex features of ARVC, especially the asymptomatic “concealed phase”. Using a yeast two‐hybrid screen we identified Synaptoso‐malassociated protein 29 (SNAP29) as a novel desmosomal (desmoplakin, DSP)‐interacting protein. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy in non‐cardiomyocytes; however, its role at the desmosome and/or heart is undefined. We hypothesize that SNAP29 may play a critical role in maintaining desmosomal protein homeostasis and have direct relevance to ARVC disease features, as it is a disease associated with desmosomal protein dissolution. We show that SNAP29 co‐localizes with DSP in both the adult mouse and human heart. Cardiac‐specific SNAP29‐deficient mice (SNAP29‐cKO) displayed baseline and pacing‐ induced ventricular arrhythmias in an age‐dependent manner in the absence of cardiac structural and functional deficits. Furthermore, molecular analyses revealed that the arrhythmias found in SNAP29‐cKO hearts and cardiomyocytes were associated with a molecular loss of a subset of desmosomal proteins (DSP and plakophilin‐2) and the gap junction protein, connexin43. Furthermore, an accumulation of autophagic markers (p62 and LC3BII), as well as machinery, was observed at the cardiac cell‐cell junction in SNAP29 deficient cardiomyocytes. Acute blockade of autophagy using specific inhibitors (chloroquine and bafilomycin A1) was sufficient to rescue levels of desmosomal and gap junction proteins as well as arrhythmias in SNAP29 deficient cardiomyocytes. Our data suggest that SNAP29 insulates a subset of desmosomal proteins from selective autophagy‐mediated degradation to restrict cardiac arrhythmias. Thus, SNAP29 loss may directly contribute to the “concealed phase” of ARVC. Our results further identify autophagy defects as an underlying mechanism in desmosomal disease pathogenesis and highlight new potential therapeutic targets for ARVC.

Support or Funding Information

NIH/NHLBI (HL09780)
Assessing Paracoccus denitrificans zinc metallochaperone AztD as a potential novel antibiotic drug target
Authors: Hendricks Grace , Meni Anusha , Yukl Erik
Published: April 2020

Zinc is an important cofactor required for the catalytic activity and structural stability of enzymes. Additionally, zinc plays critical roles in host‐pathogen interactions during bacterial infection. As such, zinc homeostasis and its regulation are vital for every organism. Bacteria use ATP Binding Cassette (ABC) transporters to acquire zinc from their extracellular surroundings. In Paracoccus denitrificans, the ABC transporter consists of a cytoplasmic ATPase (AztA), a transmembrane permease (AztB), a periplasmic solute binding protein (AztC), and a metallochaperone (AztD). The P. denitrificans zinc import system is closely related to that of carbapenem resistant Enterobacteriaceae (CRE), a group of pathogenic organisms considered an urgent threat to human health by the Centers for Disease Control. These zinc import system proteins are necessary for bacterial virulence, have no known homologs in eukaryotes, and are conserved in human pathogens. Thus, this study aims to assess AztD as a potential novel antibiotic drug target. Current work involves using site‐directed mutagenesis to delete the N‐terminal domain of AztD in order to further understand its role in zinc binding and transfer to AztC. Future experiments include screening for pore blocking molecules that could act as zinc transfer inhibitors.

Support or Funding Information

National Institutes of Health 1R01GM122819‐01A1
Beta‐cell Prohormone Convertase 1/3 Plays a Larger Role in Glucose Homeostasis Than Beta‐cell Prohormone Convertase 2 in Mice
Authors: Taylor Austin James , Chen Yi-Chun , Verchere C. Bruce
Published: April 2020

Insulin is synthesized in pancreatic beta cells from its precursor proinsulin, which is cleaved by the prohormone convertases 1/3 (PCSK1) and 2 (PCSK2) to generate mature insulin. In patients with type 1 or type 2 diabetes, proinsulin processing to mature insulin is impaired and elevated circulating proinsulin:insulin ratios are found in both conditions. It is unknown whether the beta‐cell prohormone processing impairments observed in diabetes contribute to or are secondary consequences of beta‐cell failure. Given the key role of PCSK1 and PCSK2 in insulin synthesis and beta‐cell function, we hypothesized that beta‐cell PCSK1 or PCSK2 deficiency would result in beta‐cell dysfunction and glucose intolerance.

Beta‐cell specific knockout mouse models of Pcsk1 (Pcsk1betaKO) and Pcsk2 (Pcsk2betaKO) were generated using cre‐lox recombination and monitored for changes in glycemia on chow and high‐fat (HF) diets. Neither Pcsk1betaKO or Pcsk2betaKO chow‐fed animals displayed hyperglycemia or impaired glucose tolerance in the first 14 weeks of life. By 26 weeks of age Pcsk1betaKO, but not Pcsk2betaKO, chow‐fed male mice displayed fasting hyperglycemia and impaired glucose tolerance. After 19 weeks of HF diet, 3 of 5 Pcsk1betaKO male mice developed diabetes (fasting glycemia > 16mM), while no hyperglycemia was observed in Pcsk2betaKO mice up to 30 weeks of diet duration. Pcsk1betaKO mice displayed elevated islet and plasma (pro)insulin levels, decreased islet mature insulin levels, translucent insulin granules, and trended toward increased beta‐cell area in the pancreas. Despite altered insulin biosynthesis and immature granules, Pcsk1betaKO islets displayed similar insulin secretion kinetics to Pcsk1 wild‐type islets ex vivo.

Our data suggest that Pcsk1 plays a larger role in proinsulin processing and beta‐cell function in mice than Pcsk2. However, despite severe impairments in insulin biosynthesis, Pcsk1‐deficient beta cells compensate remarkably well at early ages and hyperglycemia is only observed in states of increased beta‐cell secretory demand (mild insulin resistance associated with aging or HF diet in mice). Thus, impaired Pcsk1‐mediated prohormone processing, but not Pcsk2‐mediated processing, in beta cells increases diabetes susceptibility in mice.

Support or Funding Information

Funding was provided by the Canadian Institutes of Health Research and Canucks for Kids Fund Childhood Diabetes Laboratories
Effects of nicotine + morphine on reward‐related behavior and nicotinic acetylcholine receptor regulation in mouse midbrain.
Authors: Avelar Alicia Jo , Akers Austin T. , Cooper Skylar Y. , Kasireddy Vishnupriya , Henderson Brandon J.
Published: April 2020

In the United States about 480,000 deaths/year are caused by cigarette smoking and about 50,000 due to opioid abuse. Co‐use of more than one drug is common, therefore, it is vital to study the effects of drug combinations to understand how co‐use alters the brain. Especially given that in America, > 90% of opioid dependents are heavy smokers. The objective for this study is to determine how combined nicotine + morphine alters reward‐related behavior and nicotinic acetylcholine receptor (nAChR) expression in the midbrain. Our hypothesis is that co‐use of nicotine + morphine leads to enhanced GABA neuron disinhibition of VTA dopamine neurons. Female and male (3–5 months old) C57BL/6J background strain mice with or without α4‐mCherryα6‐GFP nAChRs were used in conditioned place (CPP) preference assays to examine reward‐related behavior with nicotine + morphine. We used confocal microscopy to analyze the density of α4‐containing (α4*), α6*, and α4α6* nAChRs in ventral tegmental area (VTA), substantia nigra pars compacta (SNc), and substantia nigra pars reticulata (SNr). In our CPP assays, a Two‐way ANOVA (factors: treatment and sex) showed a significant effect of sex (F (1, 42) = 5.75; p = 0.02) on reward‐related behavior, suggesting a sex‐dependent dose effect with morphine. In our microscopy assays, nicotine + morphine treatment downregulated α4* nAChRs on SNr GABA cells. Our CPP data supports that sex is an important variable in the effects of drugs on reward‐related behavior. Our receptor regulation data suggests that downregulation of α4* nAChRs on SNr GABA cells is important to the combined effects of nicotine + morphine.

Support or Funding Information

Support: Funding was provided through start‐up funds through the Marshall University Research Corporation.
Interaction between Ras and the two Ras‐Binding Domains of Raf kinase
Authors: Cookis Trinity , Volmar Alicia , Merritt Kathleen , Mattos Carla
Published: April 2020

The interaction between Ras and Raf kinase at the membrane promotes cell proliferation through the mitogen activated protein kinase (MAPK) pathway. Ras mutations drive 20% of all human cancers and despite great efforts, there are currently no drugs targeting Ras. Raf interacts with Ras via its two N‐terminal Ras‐binding domains: the Ras‐binding domain (RBD) and the cysteine‐rich domain. Binding of both the Raf‐RBD and CRD are required for Ras‐mediated activation of Raf kinase, however, the mechanism that results in the activation of the C‐terminal Raf kinase domain remains unknown. Here we present the 2.8 Å crystal structure of Ras in complex with a Raf construct containing both the RBD and CRD, revealing the interface for Raf‐CRD binding. In combination with molecular dynamic simulations, we identify allosteric effects induced by Raf‐CRD binding that stabilize the Ras active site to promote a Ras conformation poised for intrinsic hydrolysis.

Support or Funding Information

NSF, MCB‐1517295. Northeastern Office of Undergraduate Research and Fellowships
Novel Anti‐Caner Nucleoside Analogs: Synthesis and Activity
Authors: Abdullah Rola Hassan , Hamzeh Khalil , Bouhadir Kamal , Eid Ali
Published: April 2020

Cancer continues to be the second leading cause of death worldwide, with colorectal cancer (CRC) being the second most common type. Despite significant advances in cancer therapies, current treatment of CRC remains inefficient. In addition, effectiveness of currently available chemotherapeutic drugs such as 5‐FlouroUracil (5FU) is limited owing to developed resistance in CRC. Here, we provide novel schemes for synthesis of four novel nucleoside analogs, as well as describe their effects on proliferation, migration, aggregation, adhesion and de‐adhesion of human colorectal cancer cells, both wild‐type and 5‐FU‐resistant HCT116. In either cell type, our synthesized novel analogs significantly inhibited cell viability in a concentration and time‐dependent manner. Importantly, all the four analogues inhibited proliferation at a much lower concentration than that of 5FU, indicating higher potency of these analogs. In addition, these compounds inhibited cell migration in a time‐dependent manner. They also inhibited adhesion and de‐adhesion of both cell types to collagen, as well as promoted homotypic cell‐cell interaction. ERK1/2 and iNOS were both inhibited by these analogs. This was also concomitant with a decrease in the production of nitric oxide (NO) and vascular endothelial growth factor (VEGF), as well as a decrease in MMP‐2 and MMP‐9 release. Taken together, by inhibiting these hallmarks of malignancy, our data highlight that the four analogs could act as potent chemotherapeutic drugs against CRC, including the resistance form.