bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of ATG9A‐dependent basal autophagy causes an accumulation of ubiquitin‐rich condensates which act as a platform for inflammatory signaling
Authors: Jayatunge Dissanayake Arachchilage Dasun N. , Andersen Joshua L. , Poole Daniel M. , McEwan Colten M.
Published: May 2022

Excessive inflammation underlies many human diseases, such as neurodegeneration, autoimmune disorders, cancer, and COVID‐19. Thus, cellular mechanisms that control inflammation are of high therapeutic interest. Autophagy has been implicated in the suppression of inflammation, yet mechanistic links between autophagy and inflammation are not completely understood. Previous work demonstrated that loss of ATG9A, but not ATG5 or ATG7, increased inflammatory signaling through the STING‐IRF3 cascade, suggesting that perhaps an autophagy‐independent function of ATG9A regulates inflammation.1 Here, we show that loss of the essential basal autophagy regulators ATG9A and ATG101 sensitizes cells to dsDNA‐induced IRF3 activation. Importantly, this effect was not observed with the loss of ATG5 or ATG7, suggesting that the canonical LC3‐lipidation autophagy machinery is not required for suppression of IRF3 and inflammatory signaling.2 In an effort to understand the role of ATG9A and ATG101 in suppressing inflammatory signaling, we found that loss of ATG9A and ATG101, but not ATG5 or ATG7, caused an accumulation of ubiquitin‐rich condensates. We also found that the accumulation of ubiquitin‐rich condensates coincided with an overactivation of the ubiquitin‐sensing, IRF3‐targeted kinase TBK1. Importantly, we found that inhibiting the accumulation of ubiquitin‐rich condensates via knock‐out of p62/SQSTM1 abrogated the increased inflammatory signaling caused by loss of ATG9A. Together, our data suggest a model in which the loss of basal autophagy machinery causes an accumulation of ubiquitin‐rich condensates, which, in turn, act as a platform for the aberrant activation of TBK1‐mediated inflammatory signaling. We propose that these data have important implications for diseases of protein aggregation wherein similar ubiquitin‐rich condensates form and aberrant inflammatory signaling plays a pathological role.

References

(1) Saitoh, T.; Fujita, N.; Hayashi, T.; Takahara, K.; Satoh, T.; Lee, H.; Matsunaga, K.; Kageyama, S.; Omori, H.; Noda, T.; Yamamoto, N.; Kawai, T.; Ishii, K.; Takeuchi, O.; Yoshimori, T.; Akira, S. Atg9a Controls DsDNA‐Driven Dynamic Translocation of STING and the Innate Immune Response. PNAS 2009, 106 (49). https://doi.org/10.1073/pnas.0911267106.

(2) Kannangara, A. R.; Poole, D. M.; McEwan, C. M.; Youngs, J. C.; Weerasekara, V. K.; Thornock, A. M.; Lazaro, M. T.; Balasooriya, E. R.; Oh, L. M.; Soderblom, E. J.; Lee, J. J.; Simmons, D. L.; Andersen, J. L. BioID Reveals an ATG9A Interaction with ATG13‐ATG101 in the Degradation of P62/SQSTM1‐ubiquitin Clusters. EMBO reports 2021, 22 (10). https://doi.org/10.15252/embr.202051136.
Type 1 Diabetes: A Promising Dialogue between Promiscuous T Cell Receptor and H‐2Db Peptide Complex
Authors: Sharma Ruby , Ghosh Agnidipta , Garforth Scott , Almo Steven C. , DiLorenzo Teresa P.
Published: May 2022

Obliteration of the beta cells mediated by CD8 T cells in the islets of Langerhans leads to the development of autoimmune disease, type 1 diabetes (T1D). During the kickoff of T1D, CD8 T cells play a pivotal role in the destruction of these pancreatic beta cells. The CD8 T cell clone AI4, previously isolated from the islets of a young diabetes‐prone NOD mouse, is found to be the most pathogenic among the population of CD8 T cell clones. Multiple natural and synthetic ligands for AI4 have been identified. These ligands include an unusually short seven‐residue peptide derived from the insulin A chain (InsA14‐20) that is recognized by AI4 in the context of H‐2Db but lacks a C‐terminal anchor residue. High diabetogenic activity of AI4 is accredited to the promiscuity, or cross reactivity, of its T cell receptor (TCR). We adopted the structural biology studies for understanding the mechanism of cross‐reactivity of the AI4 TCR with its multiple ligands. Unraveling the three‐dimensional structure of H‐2Db with AI4 ligands will help us to unveil the distinct mechanisms involved in the recognition and the cross‐reactivity of the AI4 TCR. In previous studies, our group has already reported two structures of the H‐2Db peptide complexes recognized by AI4. In our present work we have solved two additional structures of H‐2Db with AI4 ligands and several others are in progress. In addition to these studies, we have cloned, expressed and purified the AI4 TCR in a soluble form, which should allow us to obtain its structure alone and in complex with its multiple ligands. Our approach to study the interaction of promiscuous TCRs with their multiple ligands will allow us to uncover the likely multiple mechanisms that underlie TCR cross‐reactivity in autoimmunity.
Autophagy is Disrupted in the Livers of Obese Mice Exposed to Asparaginase
Authors: Zalma Brian A. , Mirek Emily T. , Anthony Tracy G.
Published: May 2022

Amino acid insufficiency activates general control nonderepressible 2 (GCN2), resulting in preferential synthesis of Activating Transcription Factor 4 (ATF4) to promote cellular protection and recovery. Asparaginase is an enzyme which breaks down asparagine and glutamine and is used to treat acute lymphoblastic leukemia in children and adults. Patients who are overweight or obese carry greater risk of developing hepatotoxicity during treatment and we reported that diet‐induced obesity predisposes mice to liver steatosis and dysfunction during asparaginase exposure. To understand the mechanism behind these risks, we subjected liver‐specific ATF4 knock‐out mice (ls‐Atf4KO) and their wild type controls, to a high‐fat diet (60% fat by kcal) or low fat chow (~18% fat by kcal) for 12 weeks before exposing them to 8 daily intraperitoneal injections of asparaginase (3 international units per gram body weight) or saline vehicle. Untargeted metabolomics were conducted on the livers of mice (n=6 per group; 3 male and 3 female). Asparaginase exposure decreased liver asparagine independent of diet or genotype. The liver metabolomes in lean mice were largely unaffected by asparaginase or loss of ATF4. In contrast, the liver metabolomes of obese mice exposed to asparaginase showed striking changes in sphingolipids that correlated with elevations in endoplasmic reticulum (ER) stress as indicated by the phosphorylation of protein kinase R‐like ER‐resident kinase (PERK). Increased synthesis of sphingolipids occurs during the induction of autophagy. Consistent with a sphingolipid signature of increased autophagosome formation, microtubule‐associated protein 1 light chain 3 beta‐II (LC3‐II) was elevated in obese, ls‐Atf4KO mice exposed to asparaginase. Additionally, ubiquitin‐binding protein 62 (p62), a cargo protein which binds to toxic waste and is then incorporated into the autophagosome for degradation, was elevated in obese mice exposed to asparaginase. This increase in p62 levels indicates a block in fusion of the autophagosome with the lysosome. Levels of p62 were highest in obese ls‐Atf4KO mice administered asparaginase. Overall, these data suggest that asparaginase minimally affects the liver metabolome in lean, wild‐type mice. In contrast, asparaginase causes marked shifts in the liver metabolome of obese mice which coincides with ER stress and autophagy dysfunction. Loss of ATF4 exacerbates autophagy disruption in obese mice exposed to asparaginase, highlighting its role in remediating ER stress.
Hippocampal Physiological and Behavioral Deficits Related to Alcohol‐induced Disruption of Local Blood Perfusion
Authors: North Kelsey C. , Slayden Alexandria V. , Dopico Alex M.
Published: May 2022

Despite preventative education, moderate‐heavy episodic alcohol (ethanol; EtOH) intake, such as in “binge” drinking, remains a major public health problem. Binge drinking, can lead to blood ethanol levels above legal intoxication and cause alcohol‐induced blackouts (AIB), a form of amnesia linked to dysfunction of the hippocampal CA1 region. The neurobiological bases of AIB, however, remain largely unknown. It is noteworthy that proper function of neurons in the brain, including the CA1 region, critically depends on aerobic metabolism and optimal blood flow (BF). Using rodent models, we previously showed that EtOH at intoxicating concentrations constricted cerebral arteries independently of circulatory, metabolic, and endothelial factors, but due to drug inhibition of large conductance Ca2+‐ and voltage‐gated K+ (BK) channels present in vascular smooth muscle. Using behavioral modeling of contextual fear conditioning with miniscope technology, the present study tests the hypothesis that EtOH‐induced anterograde memory dysfunction is coincidental to EtOH‐induced reduction in local blood flow and reduced neuronal activity in the CA1 region. Thus, we used Ca2+‐release imaging to map single neuron activity in combination with dynamic local BF recordings in awake and freely moving mice to simultaneously determine vascular, neurophysiological and behavioral effects of EtOH in a mouse model of AIB. Ca2+ imaging was conducted using GCaMP6, which provided a blue fluorescent light when neurons released Ca2+, indicating action potentials, while rhodamine dextran dye provided a red label for the vasculature within the CA1. AIB was modeled via contextual fear conditioning, in which mice were exposed to electric shocks after receiving intraperitoneal injections (IP) of saline or EtOH. Twenty‐four hours later, animals were placed into the same boxes, and their freezing behavior was recorded. Data revealed that IP injection of 2 mg/kg EtOH evoked a robust (>50%) decrease in Ca2+ fluorescence which was coincidental to an 18% decrease in cerebral artery diameter. Reductions in neuronal activity and cerebral artery diameter were, indeed, accompanied by a 42.3% reduction in freezing behavior (P>0.05). Thus, this study identifies both vascular and neurophysiological components of EtOH action on the hippocampus CA1 region in a behavioral model of AIB. More generally, our study is the first to investigate the simultaneous effects of EtOH on behavior, including anterograde memory, neuronal activity, and local BF dynamics in the brain.
Genomic Rearrangement in Non‐coding Region of Chromosome 1 Leads to Spontaneous Obesity and Heart Failure with Preserved Ejection Fraction
Authors: White Donnell D. , Wu Jian , Lauterboeck Lothar , Kesterson Robert , Yang Qinglin
Published: May 2022

Background

Obesity is a global epidemic leading to shorter life expectancies for the first time in recent U.S. history, and epidemiological studies predict that half of the U.S. adult population will be obese by 2030. Obesity is the most common comorbidity and risk factor for heart failure (HF) in patients with preserved ejection fraction (HFpEF), a subclass containing more than 50% of HF cases. However, effective treatment of HFpEF remains scarce due to the incomplete understanding of the mechanisms. Our lab discovered a spontaneously obese mouse line, with a genomic rearrangement in a non‐coding region of chromosome 1. Most monogenic obesity animal models exploit the hypothalamic leptin–melanocortin feeding pathway. However, monogenic models are not representative of most human metabolic disease. This obesity animal model may better represent the numerous contributing mechanisms of obesity in humans, allowing us to find better solutions to help prevent further decline of health outcomes in the US.

Methods

A mouse line (C57/B6J) of Floxed‐stop Overexpression of IF1 (FEI) was generated and maintained for Cre‐mediated conditional IF1 overexpression. We observed that severe obesity became a fixed phenotype in inbred FEI mice after inbreeding for 5‐6 generations. Targeted‐Locus‐Amplification (TLA) analyses revealed that FEI inbreeding triggered a genomic rearrangement in a non‐coding region of chromosome 1. In the present study, we focus on assessing the metabolic and cardiac phenotype of FEI mice using echocardiography, histology, glucose and insulin tolerance assays, and exercise tolerance tests.

Results

Early onset obesity was consistent in FEI mice with increased peripheral fats and circulating tissue lipids, as well as reduced systemic metabolic rates. FEI mice also exhibited increased food intake, fasting blood glucose, insulin, and leptin with impaired glucose and insulin tolerance as early as 6 weeks of age. Echocardiography revealed that the FEI mice developed concentric left ventricular hypertrophy with preserved ejection fraction. However, E/A ratio results from Doppler flow measurement at mitral valve suggest diastolic dysfunction in the FEI mice. Heart weight:tibial length was increased in FEI compared to WT. H&E and Trichrome blue staining on heart sections exhibited increased cardiomyocyte hypertrophy and cardiac fibrosis. H&E of adipose tissue and liver revealed increased adipocyte size, and increased fat deposition, respectively. FEI exhibited significantly decreased exercise tolerance compared to WT.

Conclusions

We conclude that the metabolic and cardiac phenotypes in the FEI mice closely recapitulate obese patients who suffer from HFpEF. Further studies on these mice should yield novel and translational insight into the genetic and metabolic mechanisms underlying the development of obesity and HFpEF, improving patient treatments.
Diesel exhaust particles alter cAMP dynamics in human bronchial epithelial cells
Authors: Cattani‐Cavalieri Isabella , Baarsma Hoeke , Oun Asmaa , van der Veen Melissa , Oosterhout Emily , Dolga Amalia , Ostrom Rennolds , dos Santos Valença Samuel , Schmidt Martina
Published: May 2022

Diesel exhaust particles (DEP) are one of the main causes of air pollution and are associated with lung disorders. DEP exposure may promote several pathological processes, including epithelial‐to‐mesenchymal transition (EMT), inflammation and oxidative stress, which contribute to a decline in lung function. Cyclic AMP (cAMP) has been reported to alleviate lung dysfunction. The impact of DEP on cAMP signaling is largely unknown. Human bronchial epithelial (BEAS‐2B) cells were exposed to different concentrations (≤ 300 µg/ml) for up to 72 h. Markers of oxidative stress, inflammation, EMT, components of mitochondrial bioenergetics and cAMP signaling were evaluated. DEP increased gene expression of Nrf1, thioredoxin, HO‐1 and SOD‐2 (oxidative stress markers), and interleukin (IL)‐6 and IL‐8 (inflammatory markers), as well as the EMT marker transforming growth factor‐β1 (both mRNA and protein). Epithelial cell markers (E‐cadherin, ZO‐1) were reduced by DEP, whereas mesenchymal markers (β‐catenin, collagen‐1) were increased. DEP reduced both basal and ATP‐linked respiration as well as the glycolytic capacity of mitochondria. Concomitantly, DEP profoundly altered mitochondrial morphology and increased the gene expression of A‐kinase anchoring protein (AKAP) 1 (a known mitochondrial scaffold protein). Furthermore, DEP significantly affected Epac1 mRNA levels and appeared to increase Epac1 protein, but did not alter the expression of phospho‐PKA substrates. Interestingly, DEP increased gene expression levels of several AC family members (AC3, AC6 and AC7), the β2‐adrenoceptor (β2‐AR) and prostanoid E receptor subtype 4 (EP4). Importantly, real‐time measurements of cAMP demonstrated that DEP strongly reduced cAMP induced by forskolin (AC activator), fenoterol (β2‐AR agonist) and a stable EP4 agonist, implicating that DEP might limit the therapeutic effects of β2‐AR agonists used to treat lung disorders. Our findings suggest that DEP promotes oxidative stress, inflammation, and EMT. These profound alterations in cellular behavior were accompanied by changes in mitochondrial dysfunction and real‐time cAMP dynamics, which may contribute to lung dysfunction induced by air pollution exposure.
Multisite Clinical Utilization of Antibiotics among Hospitalized Patients with COVID‐19
Authors: Potter Pamela , Lam Martin , Wright Nathan , Meyer Emily , Rondon‐Clavo Carlos , Merrill Stevie , Samano Dannielle , Hammode Emad , Ihms Benjamin , Santarelli Anthony , Ashurst John , Huynh Megan
Published: May 2022

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has infected over 212,000,000 individuals worldwide with a mortality rate of 2.1% as of August 2021. A concerning trend in the treatment of COVID‐19 has emerged wherein hospitalized patients are administered broad spectrum antibiotics to stave off secondary bacterial infection. However, the clinical justification for the use of broad‐spectrum antibiotics for viral respiratory infection remains tenuous. Over administration of these therapies contributes to the growing frequency of antimicrobial resistance, which prior to the pandemic contributed to nearly 700,000 death per year.

Objective

To describe the clinical utilization and efficacy of broad‐spectrum antibiotics among hospitalized patients with SARS‐CoV‐2 within 3 hospital systems in the American southwest.

Methods

A multisite retrospective chart review of patients admitted to inpatient care due SARS‐CoV‐2 infection from March 1st, 2020 through January 31st, 2021 was conducted. Abstracted data included patient demographics, laboratory culture results, administered antibiotics, and patient outcomes. Analysis was conducted using descriptive statistics, the Pearson chi‐square for categorical data, and the Mann‐Whitney U test for continuous data.

Results

A total of 489 patients with confirmed SARS‐CoV‐2 infection via nasopharyngeal sample were included in the analysis with 19.2% (94/489) diagnosed with a concurrent bacterial infection. Patients with concurrent bacterial infections were more likely to require critical care (50.0% vs 20.7%; p<0.001) and expire (28.7% vs 14.4%; p<0.001) while hospitalized. Though the rates of secondary bacterial infection were low, 83.4% of all patients were administered at least one broad spectrum antibiotic, with the most common being Azithromycin (60%; 297/489) followed by Ceftriaxone (46.6%; 228/489). Antibiotics were not preferentially administered to patients with concurrent bacterial infection (p=0.1619), nor did it show clinical efficacy for reducing mortality among the entire sample (p=0.190) or those with secondary infection (p=0.191).

Conclusion

The COVID‐19 pandemic has contributed to non‐specific over administration of broad‐spectrum antibiotics, which failed to improve the rate of patient mortality even when afflicted with secondary infection. Over‐administration of these drugs will continue to exacerbate the growing rates of antimicrobial resistance. Clinicians are recommended to apply early empirical approaches when selecting to employ antibiotic therapies.
Reduced microvascular expression of ADAM17 contributes to cognitive impairment in Alzheimer's disease model, APP/PS1 mice
Authors: Tian Yanna , Buncha Vadym , Fopiano Katie Anne , Balogh Marta , Martinez Carlos G. , Lang Liwei , Bagi Zsolt
Published: May 2022

The disintegrin and metalloproteinase 17 (ADAM17), abundantly expressed in cerebrovascular endothelial cells, is one of the important α‐secretases involved in the cleavage amyloid precursor protein‐α (APP). We hypothesized that abnormal function of vascular ADAM17 contributes to the cognitive impairment in Alzheimer’s disease (AD). We used 8‐10 months old male and female wild type and APP/PS1 mice containing human transgenes for both APP bearing the Swedish mutation and PSEN1 containing an L166P mutation. We found that the ADAM17 expression in brain microvessels is significantly reduced in the APP/PS1 mice, compared to wild type controls. Using the Morris Water Maze (MWM) behavior test the APP/PS1 mice showed longer latency to reach the hidden platform during the learning phase and spent significantly less time in the target quadrant during the memory probe test. ADAM17 was then overexpressed in APP/PS1 mice via tail vein systemic delivery of ADAM17‐AAV9. After 4 weeks, the ADAM17 overexpressed APP/PS1 mice spend less time to reach the platform and spend more time in the target quadrant during the MWM test compared to the control, eGFP overexpressed APP/PS1 mice. Thus, we propose that down‐regulation of cerebrovascular ADAM17 contributes to the development of cognitive dysfunction in APP/PS1 mice, which can be improved by AAV9 delivery of ADAM17.
Older Mice Show Decreased Regeneration of Neuromuscular Junctions Following Lengthening‐Contraction Injury
Authors: Paul Thomas , Machpherson Peter , Janetzke Tara , Davis Carol , Brooks Susan
Published: May 2022

Progressive muscle atrophy and loss of muscle strength associated with old age have been well documented. Although impairments in the ability of old mice to regenerate skeletal muscle following injury have been demonstrated, less is known about the way age modulates the regenerative response of motor neurons and the neuromuscular junctions (NMJ) of mice following contraction‐induced injury. The inability for NMJs to effectively regenerate following an injury could lead to the death of the denervated muscle fibers and therefore play a contributing role to age‐related sarcopenia. To investigate the relationship between age and NMJ regeneration following injury, extensor digitorum longus (EDL) muscles of adult (8‐9 months), middle‐aged (18‐19 months), and old mice (27‐28 months) were subjected to a protocol of repeated lengthening‐contractions that resulted in an acute force deficit of ~70%. After 28 days, muscles were evaluated for maximum force generating capacity, muscle mass, NMJ innervation, and endplate fragmentation. Cross sections of muscles taken at three days post injury showed extensive infiltration of mononuclear cells and widespread tissue disruption consistent with the induction of severe injury to the muscles. At 28 days, maximum isometric force recovered completely in adult mice, but was significantly reduced in middle aged and old mice when compared to muscles of adult mice. In each case, approximately 29% of fibers in the cross sections displayed containing central nuclei, whereas controls showed only 2.9%, indicating that the injured muscle fibers had undergone degeneration and regeneration. Analysis of the innervation of the NMJs in the regenerated muscles showed a moderate decrease in the number of fully innervated endplates in old mice as compared to adult and middle‐aged mice. When the architecture of the endplates in regenerated muscles was examined, the relative number of NMJs with intact postsynaptic structure varied little between the age groups, although control muscles of old mice showed an increase in the number of fragmented endplates compared with the adult and middle aged mice. Thus, the diminished ability of the skeletal muscle of old mice to recover following injury may be, in part, due to an age‐related decrease in the ability to adequately regenerate motor neurons and perhaps form NMJs in the injured muscle.
Characteristics and Outcomes of Patients Presenting to Rural or Urban Emergency Departments with COVID‐19
Authors: Potter Pamela , Sidhu Kamaldeep , Hailu Elon , Akramoff Ashley , Shah Bhairav , Merrill Stevie , Samano Dannielle , Colling Lindsey , Ihms Benjamin , Hammode Emad , Ashurst John , Santarelli Anthony
Published: May 2022

Background

The Coronavirus disease 2019 pandemic placed unprecedented strain on healthcare workers and hospital systems. While healthcare institutions across the nation all faced high census numbers, hospitals in rural settings are often burdened with less resources than their urban counterparts. Therefore, we hypothesized that receiving care in rural settings would be associated with poorer clinical outcomes.

Objective

To compare the characteristics and outcomes of patients with SARS‐CoV‐2 presenting to urban and rural emergency departments in the American southwest.

Methods

A multisite retrospective chart review of patients admitted to inpatient care due SARS‐CoV‐2 infection from March 1st, 2020 through January 31st, 2021 was conducted at three participating hospitals. Abstracted data included patient demographics, intake laboratory values, and patient outcomes. Analysis was conducted using descriptive statistics, the Pearson chi‐square for categorical data, the Mann‐Whitney U test for continuous data, and Kaplan‐Myer for disease progression.

Results

A total of 489 patients with confirmed SARS‐CoV‐2 infection via nasopharyngeal sample were included in the analysis with 57.1% (279/489) presenting to an urban emergency department. Patients admitted in the rural and urban setting showed similar demographics in regard to age (p=0.710), sex (p=0.312), and majority/minority status (p=0.062). Upon presentation, patients in rural settings were more likely to have critically low white blood cell counts (p<0.001), abnormally high hematocrit (p=0.001), and abnormally high aspartate aminotransferase levels (p<0.001) than their urban counterparts. Following admission, urban patients were 2.53 times more likely than rural patients to be transferred to critical care (p<0.001) with the transfer contributing to a reduced hospitalization length of 2.7 days in the urban setting (p=0.002). Rural patients unable to transfer to critical care were 4.95 times more likely to expire than urban patients (p=0.014).

Conclusion

Patients receiving care for SARS‐CoV‐2 infection in the rural setting often present with more severe clinical profiles and are more likely to experience negative outcomes than their urban counterparts. Rural hospitals should attempt to reduce this discrepancy by developing robust prognostic procedures to minimize critical care bed utilization.
Brain endothelial cell barrier function is compromised by autophagy depletion
Authors: Park Seul‐Ki , Cho Jae Min , Mookherjee Sohom , Brown Revi , Jacob Jaimee , Zhu Weiquan , Sun Zhonglou , Symons J. David
Published: May 2022

Age‐associated cerebral hypoperfusion and heightened blood brain barrier (BBB) permeability contribute importantly to vascular cognitive impairment. Earlier we reported that mRNA and protein expression of autophagy markers is repressed (p<0.05) in endothelial cells (ECs) from older (24‐month) mice and older (> 68 y) male subjects that exhibit concurrent peripheral arterial dysfunction. Of note, adult mice with inducible EC specific depletion of autophagy related gene 3 (Atg3) display endothelium‐dependent dysfunction (p<0.05) of femoral and middle cerebral arteries, whereas smooth muscle function is intact in both vascular beds. While these findings indicate repressed EC autophagy associates strongly with arterial dysfunction, the impact of EC autophagy on BBB permeability is unknown. Because EC autophagy repression mimics an aging phenotype, we hypothesized that BBB permeability is heightened in mice with EC specific Atg3 depletion (Atg3EC‐/‐) vs. wild type littermates (WT). In support of this, Evans blue dye was more abundant (p<0.05) in brains from Atg3EC‐/‐ vs. WT littermates (n=6 per group). Further, fluorescence assisted cell sorting results indicated a greater number (p<0.05) of white blood cells, microglia, and neutrophils in brains from Atg3EC‐/‐ vs. WT mice (n=4 per group). Using a reductionist approach we tested whether human brain microvascular ECs (HBMVECs) with autophagy depletion exhibit heightened permeability. Results from electric cell‐substrate impedance sensing procedures indicate that permeability was greater (p<0.05) in HBMVECs after : (i) IL‐1β (positive control) vs. vehicle‐treatment; (ii) 3‐methyladenine (autophagy initiation inhibitor) vs. vehicle‐treatment; and (iii) CRISPR‐cas9 mediated Atg3 knock‐down (Atg3 KD) vs. transfection with negative sgRNA (control) (p<0.05, 24 wells per treatment x three repetitions). In addition, Atg3 KD HBMVECs displayed reduced protein expression of: (i) Atg3; (ii) zonula occludens‐1; (iii) platelet endothelial cell adhesion molecule‐1; (iv) vascular endothelial cadherin; and (v) glucose transporter 1 (p<0.05, n=6). These data provide evidence that Atg3 depletion jeopardizes BBB integrity.
Imaging of Corticosteroids in Mouse Kidney Using Mass Spectrometry
Authors: Stasinopoulos Ioannis , Khan Shazia , MacKay C. Logan , Brown Roger W. , Andrew Ruth , Bailey Matthew A.
Published: May 2022

Aldosterone regulates renal sodium reabsorption which is important for blood pressure homeostasis. Glucocorticoids may also contribute to this process although this is less well understood. Analysis of steroid hormones and the metabolites in the plasma and urine gives insights into corticosteroid biology but steroid concentrations at a cellular level are largely unknown, and the kidney remains a “black box”. Mass spectrometry imaging (MSI) permits localisation of steroids in histological zones based on regional markers. This approach has been previously applied to localise steroids in brain and testes, and here is applied to kidney. Our aim was to map and quantify glucocorticoids and aldosterone in different histological zones (cortex, medulla) of kidneys from mice receiving different dietary salt, to provide new information relevant to the physiological actions of these hormones.

Cryosections kidney from male C57BL6 mouse (age 12 weeks, n=6: dietary salt low 0.03% vs normal 0.3% vs high 3%) were subject to MSI analysis following Girard T reagent derivatisation and α‐cyano‐4‐hydroxycinnamic acid matrix application. Matrix assisted laser desorption/ionisation (MALDI) was used as a sampling method, coupled to Fourier Transform Ion cyclotron mass spectrometry.

Ions with m/z 458.3010 (Δppm=0.65), 460.3166 (Δppm=0.65), and 474.2957 (Δppm=1.05) were detected, using MALDI, in renal sections for derivatives of 11‐dehydrocorticosterone, corticosterone and aldosterone respectively. Untargeted evaluation of ions was conducted to find regional markers that would allow definition of kidney histological zones. Heat maps indicated that corticosterone intensity was higher in the inner cortex than the rest of the kidney. In contrast 11‐dehydrocorticosterone, a metabolite produced by the enzymic activity of 11β‐hydroxysteroid dehydrogenase type II, was detected at highest levels in the medulla. The aldosterone signal was equally strong in medulla and outer cortex. Steroid localisation after treatment with different salt intakes indicated that the intensity of the corticosterone signal increases in the outer cortex in kidneys from mice receiving low salt diet, while 11‐dehydrocorticosterone was not affected. The intensity of the aldosterone signal within the kidney was not changed by different salt intakes. Our approach shows that it is feasible to image corticosteroids at 75μm spatial resolution within the kidney. This technology provides new insights into the physiological control of sodium transport by steroids and opens doors to understanding changes in disorders of blood pressure.
Acute and Chronic Effects of Seizures on Cardiorespiratory Control in the SSKcnj16‐/‐Rat
Authors: Eilbes Melissa A. , Manis Anna D. , Osmani Wasif , Levchenko Vladislav , Forster Hubert V. , Palygin Oleg , Staruschenko Alexander , Hodges Matthew
Published: May 2022

Epilepsy is a common neurological disorder in which 1/3rd of patients experiences repeated seizures, which puts them at greater risk for Sudden Unexpected Death in Epilepsy (SUDEP). The pathophysiological consequences of repeated seizures resulting in SUDEP, including the effects of repeated seizures on cardiorespiratory function, remain unclear. We previously established a rat model (SSKcnj16‐/‐rats) in which 10 days of repeated daily audiogenic generalized tonic‐clonic seizures (GTCSs) lead to ictal apnea, post‐ictal respiratory suppression, blunted chemoreflexes and high spontaneous mortality (PMID: 33232300). Here we tested the hypotheses that a single GTCS is sufficient to blunt ventilatory chemoreflexes and that repeated seizures lead to progressively greater post‐ictal heart rate (HR) suppression and/or increases in blood pressure (BP). In our pilot experiment, a male SSKcnj16‐/‐rat was implanted with a pressure telemeter and placed in a chronic monitoring plethysmograph to measure breathing and BP/HR continuously for 10 days. The rat was exposed to a 10 kHz sound (2 mins) to elicit a seizure once/day for 10 days. We found that audiogenic seizures led to ictal apneas of variable lengths (4‐25 sec) and ictal asystole (~1‐2 sec), followed by a 31% increase in BP and 30% decrease in HR on days 1‐4. On days 9‐10, there was a 45% increase in BP and 50% decrease in HR, suggesting a progressive effect on HR and BP with repeated seizures. Ongoing studies aim to determine if the ventilatory responses to hypoxia (12% O2) or hypercapnia (7% CO2) are affected 1 hr after recovery from a single GTCS. Naïve SSKcnj16‐/‐rats (n=4, female) were subjected to pre‐ and post‐seizure hypercapnic chemoreflex testing. Relative to room air breathing, minute ventilation (expressed as % of control) increased during hypercapnia before (262% ± 12%) and 1 hr post‐seizure (240% ± 21%). However, breathing frequency responses to hypercapnia 1 hr post‐seizure were reduced (126% ± 6%) compared to before (164% ± 14%), and tidal volume increased to a greater amount post‐seizure (192% ± 13%) compared to before (168% ± 7%), suggesting a significant shift away from a frequency‐mediated to a tidal volume‐mediated chemoreflex response in the post‐ictal period. Our preliminary data suggest that like breathing, repeated seizures may have a progressively negative and acute effect on BP and HR, and that solitary seizures are sufficient to fundamentally alter the hypercapnic ventilatory responses in this rat model, which may have high relevance to human SUDEP.
Does clozapine affect the discriminative stimulus effects of food deprivation in rats trained to discriminate between 2 and 22 hours food deprivation?
Authors: Cedarbloom Erika , Petit Sam , Grosland Regina , Norman‐Klatt Keelin , Vigil Abigail , Detert Dane , Passint Courtney , Sweeney Tatum , Quandt Haley , Jewett David
Published: May 2022

In humans, weight gain is a side effect of several atypical antipsychotics. Previous research with rodents indicates clozapine may increase food consumption for the first 2‐4 hours after injection (Antelman et al. 1977; Benvenga & Leander 1997; Lee & Clifton 2002) as well as increase motivation to acquire food under a progressive ratio schedule (Abela et al. 2020; Loh et al. 1992; Mobini et al. 2000). In other studies, however, clozapine has been shown to reduce response rates in lever pressing operant procedures (Ford et al., 1997; Kaempf & Porter, 1997; Varvel et al., 2002; Wiley et al., 1994; Abela 2020) and decrease food intake in rodents (Baptista et al., 1993). The current study investigated if clozapine would affect the discriminative stimulus effects of food deprivation. Male, Sprague‐Dawley rats (n = 11) were trained to discriminate 22 hrs of food deprivation from 2 hrs of food deprivation in a two‐lever, operant choice task. Rats were trained to respond on the right lever after 2 hrs of food deprivation to earn a 45 mg food pellet under an FR 15 schedule. Left lever presses were punished with 8 seconds of darkness. Under 22 hr deprivation conditions, the contingencies were reversed (left lever presses were reinforced with food delivery and right lever presses resulted in 8 seconds of darkness). Training sessions lasted until subjects earned 10 reinforcers or 15 minutes elapsed. Acquisition of discrimination was defined as 80% or greater condition appropriate responding both before the first reinforcer was earned and for the entire session for 8 out of 10 consecutive days. After acquiring the discrimination, subjects were food deprived for 2 or 22 hrs and administered clozapine (1.0 ‐ 5.6 mg/kg, s.c.). or vehicle (30% DMSO; 1.0 ml/kg, s.c.). During test sessions, responses toward either lever were reinforced under the FR 15. The session lasted until subjects earned 5 reinforcers or 5 minutes elapsed, whichever occurred first. The discriminative stimulus effects of food deprivation and response rates were assessed every 15 min for the succeeding 2 hrs. After test sessions, each subjects’ food intake was recorded for 1 hour. Clozapine had no effect on the discriminative stimulus effects of 2 hr deprivation, however, clozapine (1.0 ‐ 5.6 mg/kg) significantly decreased response rates. After 22 hrs food deprivation, clozapine (1.0 ‐ 5.6 mg/kg) significantly decreased % 22 hr deprivation‐appropriate responding. Response rates were significantly increased by 1.0 mg/kg clozapine, whereas response rates were decreased by 5.6 mg/kg clozapine compared to rates of responding following 30% DMSO. Clozapine (3.2 ‐ 5.6 mg/kg) decreased food intake in rats under both food deprivation conditions. These data provide further support for the notion that clozapine decreases feeding‐related behaviors in rats.
Loss of protein stability and function caused by a single point mutation (P228L) in the Cytochrome P450 Oxidoreductase
Authors: Rojas Velazquez Maria Natalia , Noebauer Mathias , Pandey Amit V.
Published: May 2022

Cytochrome P450 oxidoreductase (POR) is the obligatory redox partner of steroid and drug metabolizing cytochrome P450s located in the endoplasmic reticulum. Mutations in POR cause a broad range of disorders like congenital adrenal hyperplasia. Genome sequencing studies have revealed the existence of a POR missense variant P228L which was linked with reduced function of some P450 enzymes. We aimed to expand the enzymatic studies of POR variant P228L for its role in human metabolism.

We expressed human wild type and the P228L variant POR in bacteria and purified the proteins by Affinity Chromatography. We tested the stability of the proteins using fast proteolysis and performed kinetics assays of POR activities using small molecules substrates. POR (WT or P228L) were mixed with purified cytochrome P450 proteins and activities of cytochrome P450 proteins were assayed.

The single point mutation P228L had reduced thermal stability indicated by a lower melting point comparing to the WT. In the kinetics studies, the rates of the reactions with P228L were considerably lower than the WT but the Km did not show substantial changes. We observed a decrease in the enzymatic activities of CYP3A5 and CYP2C9 of more than 40% with P228L form of POR comparing to WT.

A single change in the amino acid sequence can affect the protein stability and cause a severe reduction in POR activity. Molecular characterization of POR mutations is crucial to have a better understanding of the impact on the functionality of its redox Partners.
Protein/protein interactions in the human cytochrome P450 system
Authors: Burris‐Hiday Sarah D. , Scott Emily E.
Published: May 2022

Human cytochrome P450 enzymes are heme‐containing monooxygenases that can act on diverse substrates including drugs, steroids, fatty acids, and vitamins. Most of these are microsomal enzymes that require electrons from NADPH‐cytochrome P450 reductase for catalysis. Specifically, the reductase FMN transfers electrons to the P450 heme, but little is known about the details of this transient interaction. Herein the reductase FMN domain was fused to various human P450 enzymes to promote their interaction, and the effects were measured on P450 ligand binding and catalysis occurring in the P450 active site some 15‐20 Å away from the FMN domain/P450 interaction. These FMN domain‐P450 fusion proteins were generated for both steroidogenic (CYP17A1 and CYP21A2) and drug‐metabolizing (CYP3A4, CYP2A6, and CYP2D6) P450 enzymes and compared with the respective isolated P450 enzyme. Fusion of the FMN domain does not appear to change the mode in which ligands bind in the P450 active site. Compounds that bound in the P450 active site either by displacing the heme water (type I interactions) or by directly binding the heme iron (type II interactions) in the isolated P450 enzyme, also did so in the corresponding FMN domain‐P450 fusion enzyme. However, among drug‐metabolizing enzymes fusion of the FMN domain can 1) alter the percentage of the P450 population that binds a particular ligand and 2) increase or decrease the ligand binding affinity, in a P450 and ligand‐specific way. The FMN domain appeared to have significantly less effects on steroidogenic enzymes, regardless of the ligand. Similar catalytic studies are being performed to relate these observed ligand binding trends to substrate metabolism. These current results demonstrate that the effect of the FMN domain is both isoform and ligand dependent, revealing an intricate communication between the P450 surface where the FMN domain binds and the buried P450 active site where substrates and inhibitors bind. This variability suggests that different P450 enzymes interact with the same reductase FMN domain in distinct ways. Since electron delivery is required for catalysis, this knowledge could potentially be exploited to selectively modulate the function of individual P450 enzymes to treat disease.
Quantifying Thermal Patterns Across Canine Body and Limbs
Authors: Brundage Cord M. , Kwon Claire
Published: May 2022

Abnormal surface patterns revealed in thermal images may indicate the presence of injury, disease, or inflammation in canines. Despite the use of infrared thermography in a laboratory setting, clinical application cannot be fully utilized without understanding isothermic patterns of the canine body. The objective of this study was to identify the thermal pattern of the canine body (Canis lupus familiaris). Rectangular regions of interest (ROIs) were drawn to represent the lateral surface of the torso (ROI 1) and hindquarter (ROI 2). Circular ROIs were analyzed along the limbs to portray joints and its diaphyses. All ROIs were detected through mean surface temperatures (STs). To identify the effect of the coat type, mean STs were categorized by each dog's hair coat type: short coat (SC), curly coat (CC), long coat (LC), and double coat (DC). The interaction between the ST of the ROIs across the body and limbs and were then analyzed using two‐way analysis of variance repeated measures (two‐way RM ANOVA) and multiple pairwise comparisons. The warmest regions of the body were found ventrally and distally in the abdominal regions of the torso (30.46 °C). Higher ST were found in areas of the hindquarter caudally and distally at the sacral end (30.53 °C). Significant differences in median ST using one‐way RM ANOVA on Ranks were observed between body percentage divisions (P < 0.001). In addition, significant interactions between location of the horizontal/vertical divisions and coat types were present (P < 0.001). In general, all dogs exhibited a decrease in temperature distally from the appendicular skeleton. No significant differences were found between joint and diaphyses (P > 0.05). Both SC and CC dogs showed that the decrease in mean ST from the shoulder becomes significant after the shoulder. DC dogs exhibited significant ST lower on the limbs whereas LC dogs did not exhibit a significant trend.
The effects of temperature changes on the mammalian spinal locomotor CPG network and the role of TRP channels in functional compensation
Authors: Wu Howard , Diaz‐Rios Manuel
Published: May 2022

Preliminary data suggests that spinal cord neural networks that contain intact sensory pathways, which include dorsal root ganglia (DRG) and thermal transient receptor potential (TRP) channels are more resilient to gradual temperature changes than isolated preparations. Moreover, how acute temperature changes affect mammalian CPG‐driven motor activity remains uncertain. We studied how acute and gradual temperature changes impact the stability of motor networks with or without sensory pathways. More specifically, we aim to investigate: 1. the difference between how acute temperature changes versus gradual temperature changes can have an impact on the stability and robustness of motor networks; 2. how sensory inputs and TRP channels affect the adaptation of spinal central pattern generator (CPG) networks to changes in temperature. We compared changes in motor neuron bursting activity between ventral L2 and L5 roots of neonatal mouse spinal cord preparations under acute (from 20℃ directly to 29℃) and gradual (gradually from 20℃, to 23℃, to 26℃, and to 29℃) temperature change conditions. Spinal cord preparations both with and without DRGs were compared. We found that the locomotor activities were increased as temperature increase under both chronic and acute changes; however, spinal cord preparations without intact DRG showed activities with less stable phasing and more overall motor bursting variability compared to those preparations with intact DRGs (on L3, L4, and L5 spinal levels). Our results suggest that the lumbar spinal locomotor CPG network activity shows more susceptibility and less adaptation to temperature changes in the absence of sensory feedback provided by DRG sensory afferents. Further studies will be conducted in the presence of TRP channel blockers to preparations with DRGs to confirm the potential compensatory role that sensory feedback afferents convey to spinal CPG networks.
Pharmacology and pharmacokinetics of fentanyl toxicity reversal by anti‐fentanyl monoclonal antibodies in combination with naloxone
Authors: Baehr Carly A. , Pravetoni Marco
Published: May 2022

AIM

Widespread access to illicit fentanyl has contributed to an ongoing epidemic of fatal drug overdoses in the United States. The incidence of fatal overdoses in the United States accelerated during the COVID‐19 pandemic, with over 100,000 deaths recorded by April 2021. Fentanyl‐specific monoclonal antibodies (mAb) offer a prophylactic and therapeutic intervention against opioid overdose by binding and sequestering fentanyl in serum, and reducing distribution to the brain. Here, the kinetics of overdose reversal by anti‐fentanyl mAb alone or in combination with naloxone were explored, and the dose‐response relationship between naloxone and high doses of fentanyl in the presence of mAb was evaluated.

METHODS

To evaluate the kinetics of fentanyl reversal, fentanyl 0.1 mg/kg was administered to male Sprague‐Dawley rats, and mAb or naloxone was administered fifteen minutes after exposure. Oxygen saturation was monitored constantly for ten minutes following administration of reversal agent. To evaluate duration and extent of protection from further fentanyl exposures, rats were pre‐treated with mAb, naloxone, or a combination, and challenged with increasing doses of fentanyl up to 0.5 mg/kg. Finally, to determine whether the presence of mAb would increase efficacy of naloxone against a lethal dose of fentanyl, 2.25 mg/kg fentanyl was administered, followed by naloxone 0.01 to 1.0 mg/kg.

RESULTS

The most rapid reversal of fentanyl toxicity was achieved by intravenous administration of a combination of anti‐fentanyl mAb and naloxone (<1 min). Intravenous naloxone reversed fentanyl toxicity more quickly than subcutaneous naloxone, and intravenous mAb was effective against fentanyl within 4 minutes, similar to subcutaneous NLX. Against further challenges of fentanyl, both mAb and 0.1 mg/kg naloxone were effective against 0.1‐0.2 mg/kg fentanyl, and the combination of mAb and naloxone was effective against doses of 0.5 mg/kg fentanyl or higher. Prophylactic administration of mAb enhanced efficacy of naloxone in reversing a potentially lethal challenge of 2.25 mg/kg fentanyl. Finally, mAb sequestered high concentrations of both fentanyl and its metabolite norfentanyl in serum, and reduced brain concentrations of fentanyl in pre‐exposure scenarios.

CONCLUSIONS

These results support translation of mAb as medical countermeasures to prevent and reverse opioid overdose related to fentanyl and its analogs.