bluuebunny/crossref_metadata_2025 huggingface dataset:
Deinococcus radiodurans RecOR Proteins Stimulate RecA Filament Formation on SSB‐Bound Single‐stranded DNA
Authors: Jones Kiel , Uranga Lee A. , Lusetti Shelley L.
Published: May 2022

Deinococcus radioduransis a radiation resistant organism with the ability to survive hundreds of DNA double‐strand breaks. The compacity to withstand such a detrimental type of DNA break stems from the efficiency of the DNA repair machinery of this organism. D. radioduransrecombination mediator proteins RecO and RecR have been shown to be required for radiation resistance in this organism. In E. colithe RecOR proteins have been reported to stimulate RecA filament nucleation onto SSB‐bound single‐stranded DNA (ssDNA) through displacement of the SSB protein. However, how D. radioduransRecO and RecR proteins work to mediate RecA filament formation is not well understand. In this study, we further elucidate the biochemical mechanism of RecOR function; the kinetic evidence demonstrates that D. radiodurans RecOR alter the rate of ATP hydrolysis by the RecA protein when bound to ssDNA regardless of the presence of SSB protein. This effect has not been previously described for other homologous recombination systems. We believe that D. radioduransRecOR functions to stimulate RecA nucleation to ssDNA directly and independently of SSB protein. Potential mechanisms for this kinetic stimulation will be discussed.
Elucidating the role of NUDT19 in renal lipid metabolism
Authors: King Rachel D. , Vickers Schuyler , King Judy , Leonardi Roberta
Published: May 2022

Coenzyme A (CoA) is an essential cofactor that plays a key role in fuel metabolism, as it is required for glucose oxidation and the synthesis and oxidation of fatty acids, amino acids, and ketone bodies. Three Nudix hydrolases, NUDT7, NUDT8, and NUDT19, hydrolyze CoA species at the phosphodiester bond, potentially contributing to the regulation of specific metabolic processes in the subcellular compartments where they reside. NUDT19 is found in the peroxisomes and is highly expressed in kidney proximal tubule cells (PTC). In vitro, NUDT19 readily hydrolyzes short and medium chain acyl‐CoAs, malonyl‐ and succinyl‐CoA, as well as free CoA. This substrate preference, combined with the peroxisomal localization of NUDT19, suggests a potential role in the regulation of peroxisomal lipid metabolism. To investigate the physiological function of NUDT19, we obtained global Nudt19‐/‐ mice and fed them a high fat diet (HFD) or a low fat control diet (CD) for 15 weeks. On either diet, Nudt19‐/‐ mice exhibit higher total CoA levels compared to wild type mice, indicating that NUDT19 contributes to the regulation of this cofactor. HFD feeding led to a higher albumin‐to‐creatinine ratio in the Nudt19‐/‐ mice compared to wild type mice, indicating a decrease in kidney function. Nudt19‐/‐ mice also exhibited elevated serum triglycerides and a higher body weight, not associated with changes in bone density or adiposity. In mice fed the HFD, deletion of Nudt19 caused only a few changes to the kidney cortex proteome, with only 8 proteins significantly altered, including 3 involved in peroxisomal and mitochondria fatty acid oxidation, which were increased in the kidneys of the Nudt19‐/‐ mice. Measurement of mitochondrial and peroxisomal fatty acid oxidation using 14C‐palmitate in kidney cortex slides did not reveal any differences between genotypes. Conversely, untargeted metabolomics analysis on whole kidneys revealed a general decrease in multiple lipid classes including lysophospholipids, monoacylglycerols, and long chain free fatty acids, without changes in triglycerides levels. Combined, the data support the conclusion that deletion of Nudt19 alters lipid metabolism.
Molecular insights into regulation of PI3Kα
Authors: Prasad Harish R.
Published: May 2022

PIK3CA, the gene for the lipid kinase p110α is one of the most frequently mutated oncogenes across all types of cancer. p110α is an enzyme that catalyzes the formation of phosphatidylinositol 3, 4, 5 Triphosphate (PIP3). PIP3 recruits effector proteins which regulate growth, proliferation and motility. Due to this role as a master cell regulator, the activity of p110α is maintained in an inactive confirmation and is only activated downstream of Receptor Tyrosine Kinases (RTKs) and RAS family of GTPases. p110α is maintained in an inactive confirmation through interactions with its regulatory subunit as well as inhibitory contacts with the C‐terminus. However, oncogenic mutations in p110α breaks these inhibitory interactions and drives hyperactivity without the need for activation signals leading to uncontrolled cell growth. We provide molecular insights into the regulation of oncogenic mutants of p110α using Hydrogen‐Deuterium Exchange Mass Spectrometry (HDX‐MS). HDX‐MS reveals the dynamic changes between the natural cytosolic state and fully‐active membrane bound states of the enzyme. We find unique molecular mechanisms regulating how mutants at the c‐terminus activate lipid kinase activity (H1047R, M1043L, G1049R and N1068KLKR). Using extensive biophysical tools, biochemical assays, and MD simulations, we have tested the oncogenic potential of these mutations. Our results elucidates a unifying theory towards the regulation of PI3Kα and how oncogenic mutations drive hyperactivity. This will aid in understanding the regulation of PI3Kα and in developing isoform specific inhibitors.
Purification and functional analysis of the ferrous iron transport protein B (FeoB) incorporated into SMA‐copolymer nanodiscs
Authors: Lee Mark , Smith Aaron T.
Published: May 2022

The acquisition and transport of ferrous iron (Fe2+) is essential for the survival and the virulence of many infectious prokaryotes. While bacteria possess several methods to acquire Fe2+, the ferrous iron transport (Feo) system is the most important Fe2+ transport complex with strong ties to bacterial virulence. Despite this significance, an understanding of the structure‐function relationship of the transporter FeoB is limited due to its intrinsically insoluble nature that renders it generally intractable outside of a lipid bilayer. Traditionally, detergents have been used to solubilize membrane proteins such as FeoB; however, detergent micelles can disrupt protein folding, lead to protein instability, and even inhibit important and essential protein‐protein interactions. In lieu of traditional amphipathic detergents, styrene‐maleic acid (SMA) copolymers have been demonstrated to solubilize membrane proteins into a nanodisc while encapsulating native lipids, thereby preventing protein misfolding and preserving protein‐protein interaction sites. In this work, we describe the first successful solubilization and purification of Escherichia coli FeoB (EcFeoB) in SMA nanodiscs. This approach has afforded our ability to obtain highly pure, monomeric EcFeoB‐SMA nanodiscs. In addition, we compare the NTPase activity of detergent‐solubilized EcFeoB to that of SMA‐solubilized EcFeoB nanodiscs to probe nucleotide‐hydrolysis rates in the presence of an artificial micelle compared to a lipid bilayer. This approach represents a promising first step in understanding the structure and the mechanism of the FeoB transporter in its native environment.
Cellular redox regulation by the neuronal glutamate and cysteine transporter, EAAT3
Authors: Radhakrishnan Saranya , Amara Susan G.
Published: May 2022

Glutamate transporters are essential for clearing glutamate from the extracellular space and thus limit the actions of glutamate within the brain. The neuronal glutamate transporter EAAT3 has also been shown to transport cysteine, which serves as a rate‐limiting substrate for the synthesis of neuronal glutathione, an essential regulator of oxidative stress in cells. Hence, EAAT3 can contribute to regulating cellular redox by acting as an essential cysteine transporter in neurons. EAAT3 mutations are also associated with several neuropsychiatric disorders such as schizophrenia and obsessive‐compulsive disorders.

The activity of plasma membrane transporters can be altered by changes in trafficking, expression levels and recycling of the transporters. EAAT3 can rapidly cycle to and from the cell surface and through intracellular compartments within a few minutes and this can be regulated through several different intracellular signaling pathways. Previous studies have demonstrated that activation of protein kinase C (PKC) can regulate EAAT3 trafficking and increase the localization of EAAT3 at the membrane surface. There is evidence for some redox‐triggered mechanisms that can directly or indirectly influence signaling by altering the catalytic properties or influencing subcellular compartmentation of different PKC isoforms. This suggests a mechanism by which EAAT3 trafficking could be modulated by PKC as a response to cellular redox stress to increase cysteine import and facilitate the synthesis of the antioxidant glutathione. To study this, we monitored if oxidizing agents such as hydrogen peroxide can stimulate a redox response in the cell and lead to increased membrane localization of EAAT3. We found that application of hydrogen peroxide leads to an increase in EAAT3 membrane localization in Neuro2A cells expressing EAAT3. We used TIRF and biotinylation assays to confirm that EAAT3 localization can be altered by PKC activation and further examined whether this surface expression can be additionally modulated during redox stress.

While studying downstream redox changes upon cysteine and antioxidant N‐acetyl cysteine treatments, we noticed a transient oxidative response in the mitochondria resulting from the generation of sulfane sulfur species and hydrogen sulfide in the mitochondria. To understand these downstream effects of cysteine and N‐acetyl cysteine on cytosolic and mitochondrial stress, we monitored the redox changes in the cytosol and mitochondria with live‐cell imaging using genetically encoded fluorescent redox sensors targeted to specific subcellular compartments. Using this approach, we were able to conduct live‐cell imaging with spatiotemporal accuracy and to dissect sub‐cellular changes in different redox couples caused by cysteine treatments. We also investigated cysteine transport and N‐acetyl cysteine transport through EAAT3, both of which appear crucial to cellular redox regulation in neuronal cells that have high mitochondrial activity and metabolic burden.
High‐throughput Assay and In Vivo Screen Identify α‐2,3‐sialylation of CD98 by ST3GAL1 and ST3GAL2 as Essential to Melanoma Survival
Authors: Chen Shuhui , Agrawal Praveen , Hernando‐Monge Eva , Mahal Lara
Published: May 2022

Melanoma is an aggressive type of skin cancer that accounts for most skin cancer deaths, and the incidence of melanoma has increased rapidly over the past decades. Early‐staged disease can be cured by surgery, however, a lack of curative treatments for patients with established melanoma metastasis results in significantly low survival rates. Thus, identification of key drivers in melanoma progression is essential to our understanding of melanoma biology. Glycosylation is a hallmark of cancer biology and altered glycosylation influences multiple facets of both tumor growth and progression. Herein, we utilized lectin microarrays to compare the glycomes of early transformation and melanoma progression. We found common glycan signatures, including an increase of α‐2,3‐sialosides, in both biological processes, and revealed glycans associated with site‐specific metastasis. Tandem analysis of using an innovative functional in vivo growth screening of essential glycogenes identified the underlying sialyltransferases ST3GAL1 and ST3GAL2 as essential for melanoma growth. We confirmed upregulation of ST3GAL1 and ST3GAL2 in melanoma via examination of transcriptomic datasets and human tissue microarrays. Proteomic analysis identified CD98 as a candidate glycoprotein responsible for promoting melanoma proliferation. Our studies reveal glycans may act as molecular drivers that functionally contribute to melanoma biology and opens up novel paths to develop glycan‐based therapeutics to treat melanoma patients.
Age‐related changes in the control mechanisms for vasomotion in the microcirculation
Authors: Ramnanansingh Timmy , Singh Shamjeet , Johnson Jenelle , Carrington Christine , Addae Jonas
Published: May 2022

Ageing causes deterioration in function and structure of the skin microcirculation which has been reported to reflect similar deterioration in the microcirculation of other organs. The deterioration in function has been shown to reduce the total microvascular blood flow. The objective of our study was to determine the extent to which the three main control mechanisms of cutaneous microcirculation i.e., myogenic, neurogenic, and metabolic (endothelium dependent and non‐endothelium dependent) are affected by ageing. We used young (approximately 2 weeks old, n=10) and older (approximately 12 weeks old, n=10) Sprague‐Dawley rats. The animals were sedated, their body temperature maintained at 35 – 37oC with a heating blanket and Laser‐Doppler Flowmetry recorded from the palmar surface of the forepaw. Perfusion analysis was used to estimate the microvascular blood flow within a selected region of the palm, whilst the spectral composition of flowmotion was analyzed with Continuous Wavelet Transform using the Morlet mother wavelet. We used the following published frequency bands for rats for the spectral analysis of the microcirculation: myogenic (0.20 – 0.70 Hz), neurogenic (0.08 – 0.20 Hz), and metabolic control (0.01 – 0.08 Hz). Statistical significance was examined with one‐tailed, two‐sample t‐Test. The weights (in grams) of the young and older rats were 114±6 and 542±11 (mean±SEM). The skin perfusion for the young and older rats were 377±31 and 189±18 (perfusion units) respectively; the difference was significant with p<0.01. The mean amplitudes of the wavelet components for myogenic, neurogenic, and metabolic microvascular control for the young rats were 56.8, 71.6 and, 105.4 respectively, whilst those for the older rats were 30.4, 48.2, and 63.3; the decreases in the values for the older rats were significant (p<0.05). The study demonstrates that aging causes significant impairment of all the three major control mechanisms that influence cutaneous microcirculation. The results provide an approach to investigate interventions that may affect specific control mechanisms of the microcirculation.
Comparison of the behavior of hybrids and domestic lambs in the early postnatal period
Authors: Plemyashov Kirill , Fedorov Valeriy , Vladimirov Leonid , Machakhtyrov Grigoriy , Machakhtyrova Varvara , Shadrina Yana , Sleptsov Evgeniy
Published: May 2022

Using hybridization, breeds of merino sheep and archaromerinos were bred. At the same time, researchers note the possibility of transferring the characteristics of behavior and habits by inheritance from parents. The research aims to study the behavioral responses of first‐generation hybrid lambs in the early postnatal period in comparison with the behavior of ordinary domestic lambs. The object of the study was 4 hybrids of the first generation (ovchubuk) obtained from domestic sheep inseminated with the epididymal sperm of a bighorn sheep (chubuku), and 4 domestic lambs. Animals were kept in a pen of 625 m2 for close and constant contact with people. The observation was carried out from 9 a.m. to 5 p.m. In the early postnatal period ‐ from birth to 4.5 months and the post‐weaning period ‐ from 4.5 to 6 months. The manifestation of reflexes or instincts was recorded on a letter, photo, video. Attempts to stand up newborn hybrids showed in the first 8‐10 minutes, 12‐15 minutes after birth they rose to all limbs, the lambs were able to stand up for 25‐30 minutes. The hybrids received the first portions of colostrum for 18‐20 minutes domestic ones for 35‐38 minutes. A characteristic feature of the hybrids was the diversity and duration of the sucking reflex. It lasted only 15‐17sec with breaks every 10‐15 minutes. In the lambs of the control group, the sucking reflex was calm and prolonged ‐ 1.5‐2 minutes. All newborn hybrids jumped on the backs of their mothers. The hybrids were distinguished by increased activity while maintaining caution good orientation in space, which was not observed in the lambs of the control group. The instinct of self‐preservation in hybrids was “silence.” When weighing and collecting biomaterial, the hybrids did not make any extra sounds, which cannot be said about the control group. In the “man‐animal” system, hybrids were quickly tamed in a smaller containment area. In the mother‐offspring system, the lambs of both groups followed their mothers everywhere. The hybrids showed: curiosity, keeping alert, jumping from a place, jumped with a 180‐degree turn in flight. In general, the ethological features of hybrid lambs differ significantly from the behavior of domestic lambs. After the birth of hybrid offspring, they use the instinct of cubs to imitate the behavior of an adult animal from a very early age. In addition, it is necessary to organize work so that pregnant sheep are in constant contact with humans in compact pens.
The N‐terminal Domain of Twinkle, the Replicative Helicase in Human Mitochondria, Binds DNA and is Essential in Supporting Processive DNA Synthesis by the Mitochondrial DNA Polymerase
Authors: Johnson Laura C. , Singh Anupam , Patel Smita
Published: May 2022

Twinkle is the replicative helicase in human mitochondria, and is relatively poorly studied compared to its nuclear counterpart. Evolutionarily related helicases in the SF4 family, including T7 gp4 and Twinkle orthologs have an N‐terminal domain that serve as the primase for replication while Human Twinkle has been demonstrated to have lost this function, with several key metal binding residues being lost evolutionarily. Without the primase activity, the utility of the NTD is in question as Human Twinkle has been demonstrated to retain its helicase functionality without the NTD, with somewhat reduced efficiency. We generated and purified a novel Twinkle construct containing only the C‐terminal domain and linker region (CTD, amino acids 360‐684) alongside a full length (aa 43‐684) and a previously studied N‐terminal domain construct (NTD, aa 43‐372). The NTD does not oligomerize in gel filtration analysis or have any of the enzymatic activities of Twinkle. Interestingly, NTD displays a notable nonspecific binding affinity for nucleic acids longer than 16 nt. The CTD retains much of the activities of full‐length Twinkle including comparable DNA binding and, annealing, and somewhat reduced ATPase and helicase activities. CTD supports strand displacement synthesis by Polymerase γ on short replication forks but does not promote formation of multi‐kilobase length products in rolling circle DNA synthesis. Strikingly, CTD is able to support long stretch DNA replication when it is part of the reconstituted mitochondrial replisome with Polymerase γ and mtSSB, albeit with a lower efficiency than Human Twinkle. Thus, the C‐terminal domain retains the majority of the lower order component abilities of full length Twinkle but struggles to implement them for processive DNA synthesis. The results suggest that the role of the N‐terminal domain of Twinkle is in optimizing the motor activity of the C‐terminal domain for synthesizing biologically relevant lengths of DNA. The implications are foundational for understanding the mechanism of action for a subsection of sometimes debilitating mitochondrial genetic diseases which result from several single point mutations in the N‐terminal region which have previously been cryptic.
Kinetics of Plant‐derived Heteropolysaccharide Bioabsorption Characterized by Fluorescence‐based Microfluidics System
Authors: Liao Matthew W. , Liu Betty S. , Wagner Willi L. , Khalil Hassan A. , Chen Zi , Ackermann Maximilian , Mentzer Steven J.
Published: May 2022

Plant‐derived structural polymer, pectin, is the “glue” between plant cells, a biomaterial with promising performance as a sealant on visceral organs. However, the nature of bioabsorption and biodurability of pectin remains unclear. Here, we investigate the feasibility of a quantitative microfluidic perfusion assay based on photochemical quenching, to define the kinetics of pectin bioabsorption.

We hypothesize that fluorescent tracers embedded into pectin biopolymers will reliably reflect predictable kinetics patterns of bioabsorption including predictable trends when modifying the chemical species used in fluorescence quenching experiments.

A fluorescent tracer, Fluorescein Sodium, was embedded into high methoxyl citrus pectin. Dried Fluorescein pectin films were placed into 24‐well plates, to monitor degradation via parallel microfluidics perfusion. This perfusion was performed via micro‐perfused exogenous 0.4% Trypan Blue, an anionic hydrophilic azo dye, which was delivered into porous matrix interfacing on pectin in each well. To assess biodurability, the fluorescence of perfused pectin under quenching by dye was monitored over time using a multi‐well plate fluorescence reader (Cytofluor 4000, PerSeptive Biosystems).

Our data show that consistent trends in fluorescence result during quenching experiments. When un‐perfused, Fluorescein‐embedded pectin exhibited statistically insignificant change in quantum yield over 1 week (p=0.457), suggesting that results were not impacted by substantial photobleaching over the course of experimentation. To minimize interfacial flow artifact, 6µm porous matrix provided an effective interface for observable perfusion of azo dye into pectin. During micro‐perfusion, serial measurements of fluorescence in Fluorescein pectin demonstrated exponential decline in fluorescence intensity. This quenching of fluorescent signal from Fluorescein correlated with the progressive bioabsorption of the pectin polymer. Absorption was feasibly modulated by adjusting dye volumes used in perfusion.

In this in vitro assay, plant‐derived pectin exhibits predictable decay characteristics and bioabsorption kinetics when incorporated with fluorescent tracers. These tracers not only provide a quantitative measure of biodurability, but also an opportunity for guided biochemical tuning of the complex biomaterial, to improve its performance as a potential therapeutic sealant.
Compound 48/80 increases bladder wall compliance independent of mast cells
Authors: Saxena Pragya , Roccabianca Sara , Tykocki Nathan
Published: May 2022

Changes in wall compliance are seen in many bladder pathologies, such as fibrosis and diabetes. Some of these pathologies can be caused by basic secretagogues like Compound 48/80 and its endogenous analogue, Substance P. Traditionally it is believed that basic secretagogues rapidly degranulate mast cells and release inflammatory mediators by acting on an orphan receptor, Mas‐related G protein‐coupled receptor B2 (Mrgprb2). While Compound 48/80 does increase wall compliance, the exact location and function of Mrgprb2 in the urinary bladder are still unknown. We hypothesize that changes in bladder wall compliance caused by Compound 48/80 are mediated through Mrgprb2 receptors but independent of mast cell degranulation.

Whole mouse bladders from C57Bl/6 (wild‐type) and C‐kitW‐sh (mast cell‐deficient) mice were dissected and mounted in a custom designed pentaplanar reflected image macroscopy (PRIM) system for simultaneous measurement of transient pressure events, intravesical pressure, bladder volume, wall stress, and wall stretch during ex vivo bladder filling. Wall compliance was derived as a measure of the stress versus stretch relationship using the pressure‐volume curves and associated imaging data. To test our hypothesis, bladders were exposed to either vehicle or Compound 48/80 (10 µg/ml) during ex vivo filling. Compound 48/80 significantly increased the leading slope of transient pressure events in both wild‐type and mast cell‐deficient mice. Compliance was also increased, as shown by decreased stress per change in stretch. We also collected urothelium and detrusor layers from C57Bl/6 and C‐kitW‐sh mouse bladders. Layers were separated using blunt dissection, and RNA was isolated for measurement of Mrgprb2 expression using qRT‐PCR. No significant difference in the expression of Mrgprb2 mRNA was observed between the two mouse models in either the urothelium or detrusor layers. These findings suggest that Mrgprb2 receptors are also expressed outside of mast cells in the urinary bladder wall and, when activated by Compound 48/80, increase wall compliance and the leading slope of transient pressure events.
Genetic and molecular basis of Adaptive and Mal‐adaptive excessive erythropoietic responses in the Andean region
Authors: Azad Priti , Zhou Dan , Haddad Gabriel
Published: May 2022

Rationale

Excessive Erythrocytosis (EE) is a predominant trait in some high‐altitude dwellers (Chronic Mountain Sickness, CMS, or Monge’s disease) but not others living at the same altitude in the Andes, we took advantage of this human “experiment in nature” and studied both populations (with CMS and without, non‐CMS). This population provides a unique opportunity to study both adaptive (non‐CMS) and mal‐adaptive (CMS) mechanism(s) and understand the molecular basis of regulating erythropoiesis at high altitude.

Methods

In order to understand the molecular basis for polycythemia of high altitude, we generated a disease in‐the dish‐model by re‐programming fibroblasts from CMS and non‐CMS subjects. In the past, we have discovered that there are selective sweeps that seem to be statistically different between CMS and non‐CMS and hence contain candidate genes. In this study, by manipulating expression of these candidate genes (Eg. SENP1, ARID1B) in our in‐vitro model system, we delineate the molecular mechanism(s) of the CMS polycythemia as well adaptive response to suppress the excessive response in non‐CMS subjects.

Results

As compared to sea level controls who responded to hypoxia by increasing their RBCs modestly, CMS cells increased theirs remarkably (up to 60 fold) with a dose‐dependent response to graded hypoxia (1.5, 5, 10% O2). Non‐CMS cells showed a blunted increase in RBC production under hypoxia. In order to understand the genetic basis of these phenotypes, we knocked down SENP1 (a desumoylase) in CMS iPS cells using lentiviral constructs and observed a striking reduction (>90%) of the CMS excessive erythropoietic response to low O2. Similarly, by knocking down ARID1B (subunit of a SWI/SNF chromatin remodeling complex) in non‐CMS we observed that RBC formation increased significantly under hypoxia. Our recent results suggest a critical role of ARID1B at the chromatin level in regulating erythroid transcriptional factors such as GATA1.

Conclusion

. Our results demonstrate the presence of a regulatory system (SENP1 and ARID1B) that is critical in erythropoiesis and that is out of balance in the CMS subjects and tightly regulated in adapted non‐CMS subjects. Our results indicate that SENP1 and ARID1B both regulate GATA1 levels albeit through unique mechanism(s) in CMS patients as well as adapted non‐CMS subjects. Therefore, studying both populations is essential in understanding the erythropoietic response under hypoxia at high altitude.
Localization and Functional Characterization of the Cannabinoid Receptor Type 1 (CB1R) in the Kidney
Authors: Rein Joshua L. , Mackie Ken , Kleyman Thomas R. , Satlin Lisa M.
Published: May 2022

Introduction

Cannabis and synthetic cannabinoid consumption is increasing worldwide. The impact of their consumption on human health including the risk of kidney disease and its associated complications is uncertain. The kidney, among other organs, produces endogenous cannabinoids (endocannabinoids, ECs) that act on the G‐protein‐coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R). ECs and CB1R agonists acutely act as diuretics with variable effects on Na+, K+, and HCO3– excretion. As the aldosterone sensitive distal nephron (ASDN) is responsible for the final renal regulation of total body electrolyte, acid/base, and water balance that is mediated by principal cells (PCs) and intercalated cells (ICs), we sought to examine the cell specific expression of components of the EC system in the mouse collecting duct and the acute changes in urine composition after administration of the cannabinoid receptor agonist WIN55,212‐2 (WIN).

Methods

CB1R and AQP2 localization were examined by immunofluorescence microscopy in cryosections of kidneys harvested from non‐manipulated male C57BL/6 mice (7‐12 weeks of age) using protein‐specific Abs. A subset of whole animal metabolic studies were performed in mice that were acid‐loaded by provision of 20 mM NH4Cl and 1% sucrose added to the drinking water x 7d. On the day of experimentation, mice were allowed to spontaneously void, then injected SC with either 1.5 mg/kg WIN or vehicle control (10% Tween‐80 and 10% DMSO in 1x PBS) in a volume <1% BW. Urine was collected for up to 4hr for measurement of volume, pH, osmolality, Na+ and K+.

Results

In the ASDN, CB1R was localized to AQP2(‐) cells, and thus presumably ICs. Acid‐loaded mice treated with WIN (n=5) had similar UpH, UNaV, and UKV to untreated mice (n= 4) but had a higher urine output (UOP) (24.1 ± 6.5 vs. 13.6 ±1.2 µL/gBW/4hr; (P=0.02) and a lower UOsm (822 ± 335 vs. 1912 ± 293 mmol/kg H2O; P=0.001). WIN‐treated non‐acid loaded mice (n=7) urinated more frequently (3.6 ± 2.2 vs. 0.4 ± 0.5 micturitions/2 hr; P=0.05), had a lower UOsm (315 ± 82 vs. 1449 ± 553 mmol/kg H2O; P<0.0001), and lower UKV (0.4 ± 0.2 vs. 0.9 ± 0.3 µmol/gBW/2hrs; P=0.002) compared to controls (n=8), whereas UNaV and UpH were similar. Furthermore, WIN reduced the apical/cytoplasmic ratio of AQP2 expression in PCs of the inner medulla by 43% (n=209 cells in 4 mice) compared to controls (n=183 cells in 4 mice) (P<0.0001).

Conclusion

ICs express CB1R. Although UpH and Na+ excretion were unaffected in either acid‐loaded or non‐acid loaded mice by WIN, the cannabinoid receptor agonist induced diabetes insipidus, a disorder characterized by the excretion of large volumes of dilute urine, and reduced K+ excretion in non‐acid loaded mice. Efforts are underway to determine whether this effect is central or nephrogenic. These results promise to reveal how cannabis affects kidney function and to identify potential targets for novel therapeutics to treat water and electrolyte disorders.
A Strategy to Enhance Confidence in Respiratory Chemoreflex Characterization by Modified Rebreathing
Authors: Guluzade Nasimi A. , Keltz Randi R. , Huggard Joshua D. , Keir Daniel A.
Published: May 2022

Modelling breath‐by‐breath ventilation (V̇E) in response to carbon dioxide rebreathing in hyperoxic and hypoxic conditions (modified rebreathing) is used to characterize the central and peripheral respiratory chemoreflexes in humans. However, breath‐to‐breath variability produces uncertainty in the estimation of model parameters. We explored whether signal‐averaging of repeated rebreathing trials would improve chemoreflex sensitivity parameter estimation and confidence. Seven healthy males (age: 27±5 years) performed 6 repetitions of modified rebreathing tests in isoxic‐hypoxic (end‐tidal PO2 (PETO2)=50 mmHg) and ‐hyperoxic conditions (PETO2=150 mmHg) over the course of 4 days. End‐tidal PCO2 (PETCO2), PETO2 and V̇E were measured breath‐by‐breath by dual gas analyser and pneumotach. The slope of the V̇E vs PETCO2 relationship above the PETCO2 threshold in the hyperoxic test gave the central chemoreflex sensitivity (L∙min‐1∙mmHg‐1) and the difference in hypoxic versus hyperoxic test slopes provided peripheral chemoreflex sensitivity. Chemoreflex slopes modelled from a single trial versus ensemble‐average of 6 trials were compared by paired t‐test and effect size (Cohen’s d). Breath‐by‐breath data from the 6 trials were aligned to the same starting PETCO2, linearly interpolated to 0.1 mmHg of PETCO2, ensemble‐averaged, and averaged into 0.2 mmHg bins. Central chemoreflex sensitivity did not differ (p=0.94) between single (4.10±2.98 L∙min‐1∙mmHg‐1, 95%CI: 3.82–4.39) versus ensemble‐averaged data (4.13±2.14 L∙min‐1∙mmHg‐1, 95%CI: 3.99–4.27) but the 95%CI was reduced (p=0.031) by 0.29 L∙min‐1∙mmHg‐1 (effect size=1.5) with the ensemble‐averaged approach. Similarly, peripheral chemoreflex sensitivity did not differ (p=0.83) between single (1.69±1.14 L∙min‐1∙mmHg‐1, 95%CI: 1.54–1.84) versus ensemble‐averaged data (1.58±1.32 L∙min‐1∙mmHg‐1, 95%CI: 1.5 –1.66); 95%CI of the ensemble‐averaged data was 0.15 L∙min‐1∙mmHg‐1 lower than a single trial but this was not different (p=0.38; effect size=0.5). Signal averaging of multiple modified rebreathing trials reduces the confidence interval of respiratory chemoreflex sensitivity. This strategy could enhance the ability to detect differences in respiratory chemoreflex control in comparative or interventional studies where modified rebreathing is used.
Distinct Spatial Conformations and Trafficking Patterns of β‐arrestin Isoforms in AT1R Signaling
Authors: Pham Uyen , Chundi Anand , Rajagopal Sudarshan
Published: May 2022

Excessive activation of the AT1R by the hormone Angiotensin II (AngII) is implicated in the pathogenesis of hypertension by promoting vascular smooth muscle cell migration, hypertrophy, and cardiac remodeling. A limitation of current drug development to target this receptor is the complex pattern of its downstream signaling pathways, the majority of which are mediated by G proteins and β‐arrestins. Although the two β‐arrestin isoforms, β‐arrestin 1 and 2, share ~78% amino acid sequence homology, they have been shown to differentially regulate signaling pathways such as receptor internalization and the mitogen‐activated protein kinase pathways, leading to distinct physiologic and pathophysiologic outcomes. In our study, we investigated the functional divergence of the β‐arrestin isoforms at the AT1R and its link to physiologic function. We hypothesized that β‐arrestin isoforms adopt distinct recruitment patterns and spatial conformations that lead to differential regulation of downstream signaling and physiologic outcomes and “biased responses” by different agonists. Using the split NanoLuciferase complementation assay (NanoBiT), we determined that β‐arrestin 1 and 2 are recruited differentially to the receptor, the plasma membrane and the endosomes following AT1R stimulation with AngII and the β‐arrestin‐biased agonist TRV023. Compared to TRV023, AngII was found to be more potent and efficacious in inducing the recruitment of both β‐arrestin isoforms to the receptor. We also developed a new conformational biosensor, called the complex Fluorescent Arsenical Hairpin (FlAsH) assay, to assess the spatial conformational signatures of the β‐arrestin isoforms when they are bound to the AT1R or localized at the plasma membrane and endosomes. β‐arrestins 1 and 2 were found to adopt distinct conformations at all three cellular locations following agonist treatment. Our finding provides new insights into the mechanisms underlying the functional differences between the β‐arrestin isoforms at the AT1R. Further studies will be necessary to elucidate the link between the trafficking and conformational differences of the β‐arrestin isoforms and their distinct physiological roles in AT1R signaling.
Exercise Responsiveness in Obese Adults
Authors: Gries Kevin , Hart Corey , Kunz Hawley , Ryan Zachary , Zhang Xiaoyan , Parvizi Mojtaba , Liu Yuanhang , Dasari Surendra , Lanza Ian
Published: May 2022

Obesity is associated with several skeletal muscle impairments which can be improved through an aerobic exercise prescription. The possibility that exercise responsiveness is diminished in people with obesity has been suggested but not well‐studied.

PURPOSE

The purpose of this study was to investigate how obesity influences acute exercise responsiveness within skeletal muscle.

METHODS

Non‐obese (NO; n=19; 10F/9M; BMI=25.1 ± 2.8 kg/m2) and Obese (O; n=21; 14F/7M; BMI=37.3 ± 4.6 kg/m2) adults performed 30 minutes of single‐leg cycling at 70% of VO2peak. Serial muscle biopsies (vastus lateralis) were collected before exercise and 3 and 6 hours post‐exercise to measure protein synthesis and gene expression.

RESULTS

The exercise‐induced fold change in mixed muscle protein synthesis trended (p=0.058) higher in NO (1.28 ± 0.54‐fold) compared to O (0.95 ± 0.42‐fold) and was inversely related to BMI (r=‐.374, p=0.027). RNA sequencing revealed 331 and 280 genes that were up or downregulated after exercise in NO and O, respectively. Gene set enrichment analysis showed O had blunted post‐exercise pathways related to MAPK signaling, chemokine‐mediated signaling, FC receptor mediated stimulation signaling, cell proliferation, apoptosis, and RNA polymerase II promotion. Quantitative polymerase chain reaction of select metabolic (PGC1a, PGC1b, TFAM, PDK4, IRS, MAPK, and PRKAA1) and muscle growth (SLC, TBC, MSTN, MyoD, TRIM32, FOX03, and FBXO3) genes were similar between groups 3 and 6 hours after exercise.

Conclusion

These data highlight several unique pathways in individuals with obesity that result in a blunted exercise response.
Spontaneous Cardiovagal Baroreflex Sensitivity in Females with Multiple Sclerosis
Authors: Trotter Claire E. , Jarrard Caitlin P. , Smith Alex D. , Knoll Helena D. , Young Benjamin E. , Huang Mu , Davis Scott L.
Published: May 2022

Multiple sclerosis (MS) is a progressive demyelinating disease of the central nervous system that is known to disrupt autonomic function. Females with MS are at an increased risk of cardiovascular disease partly due to altered autonomic regulation of arterial blood pressure. Longitudinal data suggest that low spontaneous cardiovagal baroreflex sensitivity is a predictor of cardiovascular disease. Therefore, the purpose of this investigation is to test the hypothesis that spontaneous cardiovagal baroreflex sensitivity (cBRS) is less in females with MS compared to healthy controls. Electrocardiogram (ECG) and beat‐to‐beat blood pressure (Finometer) were continuously recorded during 10 minutes of quiet supine rest in 3 females with relapsing‐remitting MS (Expanded Disability Scale Score < 6.0) and 3 sex‐, age‐, height‐, weight‐ and race‐ matched healthy controls. Spontaneous cBRS was determined using the sequence method (CardioSeries v2.7). Spontaneous cBRS gain is calculated as the average slope of the relationship between R‐R interval and systolic blood pressure from individual sequences of 3 or more consecutive cardiac cycles where both variables are increasing (up‐up sequences) or decreasing (down‐down sequences). Females with MS had similar resting mean arterial pressures (P=0.55), systolic blood pressures (P=0.53) and heart rates (P=0.18) compared to healthy controls. Overall spontaneous cBRS gain was greater in females with MS compared to healthy controls (27.1 ± 6.3 ms/mmHg vs. 18.8 ± 1.1 ms/mmHg respectively; P=0.04). Spontaneous cBRS gain from up‐up sequences was also greater in females with MS compared to healthy controls (27.6 ± 5.1 ms/mmHg vs. 17.1 ± 2.1 ms/mmHg respectively; P=0.01). Spontaneous cBRS gain from down‐down sequences trended to be greater in females with MS compared to healthy controls but was not statistically significant (27.2 ± 7.8 ms/mmHg vs. 20.1 ± 2.4 ms/mmHg respectively; P=0.10). Counter to our hypothesis, these preliminary findings in a small sample suggest that females with MS have increased spontaneous cBRS compared to healthy controls. If this relationship continues with additional data collection, it may indicate that females with MS have enhanced cardiac responses to regulate arterial blood pressure.
Chronic Circadian Disruption Contributes to Excess Aldosterone Production and Loss of Diurnal Electrolyte Excretion
Authors: Benjamin Jazmine I. , Pati Paramita , Tao Binli , Meek Rachel , Pollock Jennifer S. , Pollock David M.
Published: May 2022

Disruptions in circadian rhythms contribute to a number of cardiometabolic health risks, including kidney disease. While chronic circadian disruption (CCD) has been recognized as a health risk in a number of other organ systems, we do not understand the effects of CCD on kidney function and sodium handling factors. Individuals with CCDs such as rotating shift workers, have an increased incidence of kidney and cardiovascular disease including impaired sodium excretion patterns. We hypothesized that this impaired sodium excretion is due in part to increased activation of the renin‐aldosterone‐angiotensin system (RAAS). We recently observed that CCD interrupts blood pressure, heart rate, and activity rhythms. Male 8‐week‐old C57BL/6J mice were placed under either a normal 12:12 light/dark cycle (control) protocol or a 10:10 light/dark cycle (CCD, T20) protocol, each with ad libitum food and water for 12 weeks. Previous experiments with this model have demonstrated that after 10 weeks the T20 mice did not exhibit a significant difference in mean arterial pressure (MAP) between the light and dark cycles (114±13mmHg during dark cycle, 113±12mmHg during light cycle, p=0.86) as opposed to control animals, who did display a typical circadian pattern in MAP averaging 119±3 mmHg during the dark cycle and 101±2 mmHg during the light cycle (p>0.001). The same pattern persisted in the heart rate (HR) of these animals, with the T20 animals lacking a diurnal variation in HR (537±14 BPM during dark cycle, 526±5 BPM during light cycle, p=0.48), and the control animals maintaining a dark/light period difference (584±11 BPM during dark cycle, 511±4 BPM during light cycle, p=0.001). Prior studies indicate that the circadian timing system controls sodium reabsorption throughout the nephron by effecting aldosterone effects on collecting duct sodium transport systems via the molecular clock. Therefore, aldosterone excretion was assayed in urine from control(n=6) and T20 mice (n=4). Interestingly, T20 animals excreted an order of magnitude more aldosterone than control animals (p<0.015 2‐way ANOVA) and lacked a diurnal variation in aldosterone excretion (115.1±51.5 µg/hr during the dark cycle, 64.1±13.1 µg/hr during the light cycle, p=0.17 dark v. light) and during both dark and light phases (0.080±0.014 µg/hr and 0.012±0.003 µg/hr, respectively, p=0.02 dark v. light). T20 mice also lacked a diurnal variation in sodium (UNaV) and potassium (UKV) excretion as compared to control mice. These results suggest that CCD disrupts diurnal sodium handling, at least in part, by disruptions in the RAAS. We further propose that the loss of blood pressure rhythms may be due to hyperaldosteronism in CCD mice.
The Relationship Between Central and Peripheral Chemoreflex Sensitivities and V̇E‐V̇CO2 Slope Below and Above the Respiratory Compensation Point of Incremental Exercise
Authors: Keltz Randi R. , Guluzade Nasimi A. , Huggard Joshua D. , Keir Daniel A.
Published: May 2022

During incremental exercise, the ventilation (V̇E) versus carbon dioxide production (V̇CO2) relationship exhibits a double‐linear response with an inflection of slope at the transition from hyperpnea to hyperventilation (i.e., respiratory compensation point (RCP)). Whether peripheral or central chemoreflexes contribute to the hyperventilatory response to incremental exercise remains undetermined. This study tested the hypothesis that peripheral and not central chemoreflex sensitivity would relate to the magnitude of the V̇E‐V̇CO2 slope above RCP and that neither would relate to the sub‐RCP V̇E‐V̇CO2 slope. Seven healthy, caffeine‐free males (age: 27±5 years) performed a ramp‐incremental test to exhaustion on a cycle ergometer, during which ventilation and gas exchange were measured by metabolic cart. The V̇E‐V̇CO2 slope above and below RCP were determined by linear regression from the estimated lactate threshold to RCP, and from RCP to end‐exercise, respectively. On four separate days, 12 modified rebreathing tests were performed: six in isoxic‐hyperoxia (PO2=150 mmHg) and six in isoxic‐hypoxia (PO2=50 mmHg). Using ensemble‐average data, central chemoreflex sensitivity was measured as the mean hyperoxic V̇E versus end‐tidal partial pressure of carbon dioxide (PETCO2) slope (in L∙min‐1∙mmHgPETCO2‐1) and the peripheral chemoreflex sensitivity was determined from the difference of hypoxic and hyperoxic slopes. The mean V̇E‐V̇CO2 slopes above and below RCP were 35.0+3.7 (range: 28.3‐39.9) and 61.8+9.2 (range: 51.9‐78.3), respectively. Mean peripheral and central chemoreflex sensitivities were 1.6±1.3 L∙min‐1∙mmHgPETCO2‐1 (range: 0.1‐3.7) and 4.1±2.1 L∙min‐1∙mmHgPETCO2‐1 (range: 2.3‐8.4), respectively. Peripheral chemoreflex sensitivity did not relate to V̇E‐V̇CO2 slope below (r=0.44, p=0.32) or above (r=‐0.02, p=0.96) RCP. No relationships were found between central chemoreflex sensitivity and V̇E‐V̇CO2 slope below (r=0.06, p=0.90) or above (r=0.04, p=0.93) RCP. These preliminary observations suggest that neither central nor peripheral chemoreflex sensitivity factor into the magnitude of the hyperpneic or hyperventilatory response to incremental exercise.
Social Stress Upregulates Histamine H3 Receptor mRNA in Murine Urinary Bladder Smooth Muscle
Authors: Jones B Malique , Mingin Gerald C. , Tykocki Nathan
Published: May 2022

In mice, social stress causes mast cell degranulation and alters voiding behavior. Histamine, a mast cell‐derived biogenic amine, is hallmarked as a modulator that can drive neurogenic bladder overactivity. Additionally, histamine causes a rapidly desensitizing contraction of the urinary bladder smooth muscle (UBSM) in normal mice that is H1 receptor‐dependent. However, histamine receptor expression in UBSM after social stress is unclear. We sought to determine if the expression of histamine receptors change in urinary bladder smooth muscle cells after social stress. We hypothesized that histamine H1 receptor expression would be upregulated in UBSM cells collected from stressed mice as compared to non‐stressed. Prior to cell isolation and collection, 4‐week‐old C57BL/6 male mice were randomly housed with CD1 adult male aggressor mice for 5 minutes, after which both mice were placed in barrier housing for 1 hour. Social stress was repeated with different aggressor mice for 14 days. Afterward, bladders were enzymatically dissociated, and urinary bladder smooth muscle cells were isolated for single cell qRT‐PCR. Non‐stressed UBSM cells expressed only Hrh1and Hrh2 mRNA. However, UBSM cells from stressed mice also expressed Hrh3 mRNA. These data suggest that social stress alters histamine receptor expression in UBSM, specifically by upregulating histamine H3 receptor expression. Since H3 receptors are involved in inflammatory peptide release in other organ systems, the functional role of H3 receptors in stress‐induced bladder dysfunction warrants further investigation.