bluuebunny/crossref_metadata_2025 huggingface dataset:
Maternal Exercise May Improve Metabolic Outcomes in Offspring in Mice
Authors: Prentice Danielle M. , Bingaman Sarah , Ural Serdar , Browning Kirsteen , Arnold Amy
Published: May 2022

Objective

It is well recognized in both human epidemiological and animal studies that the maternal in utero environment has significant effects on development of offspring. This includes sedentary lifestyle, which has been linked to developmental programming of metabolic dysfunction. The aim of this study is to investigate metabolic outcomes in offspring of sedentary mice versus mice that exercised in pregnancy.

Study Design

C57BL/6J female mice were placed on a control diet and allocated to sedentary versus exercise cages at 17 weeks of age. Exercise cages included a voluntary running wheel, with daily exercise measured via activity trackers. Mice were allowed to exercise for 2 weeks prior to breeding and throughout weaning. Offspring underwent body composition measurement and insulin and glucose tolerance tests at 6 weeks of age. At 14‐16 weeks of age, body composition was reassessed and mice were euthanized for tissue collection. Outcomes were analyzed between sedentary (SED) and exercise (EX) offspring using an unpaired t‐test, with p<0.05 considered significant.

Results

We studied metabolic function in 10 SED and 9 EX offspring. Insulin sensitivity, measured as the area under the curve for the decrease in glucose over time in response to insulin, tended to be improved in EX versus SED offspring (‐188 vs. 2991; p=0.062), with no difference in fasting glucose levels or glucose tolerance. While body mass was similar at end of study (23.4 EX vs. 23.1 SED; p=0.83), the percentage of subcutaneous white adipose tissue tended to be lower (0.94 EX vs. 1.67 SED; p=0.054) and brown adipose tissue was higher (0.57 EX vs. vs 0.30 SED; p<0.001) in EX offspring.

Conclusions

These data suggest that maternal exercise may improve insulin sensitivity in offspring even under normal diet conditions. Further, despite similar body mass, offspring from mice that exercised during pregnancy had a shift in adipose depot composition with lower white adipose and higher brown adipose tissue. This change in body composition may have important implications for energy metabolism and is an important area of future study.
Dietary Bioflavanol Supplementation Does Not Improve Cold‐Induced Vasodilation in Healthy Adults
Authors: Greenfield Andrew , Yurkevicius Beau , Castellani John , Alba Billie
Published: May 2022

Background

Cold‐induced vasodilation (CIVD) is a cyclical rise in blood flow to glabrous skin that occurs during cold exposure of the extremities. Nitric oxide (NO) is a proposed mediator of the CIVD response through active vasodilation and/or withdrawal of sympathetic vascular smooth muscle tone. Dietary flavanols augment bioavailable NO by upregulating NO synthase, the main source of NO in the vasculature, and by enhancing removal of reactive oxygen species that are known to breakdown NO. Although acute dietary flavanol intake has been shown to augment skin blood flow, at least in part through increased NO, no studies have examined the impact of flavanol intake on skin perfusion during cold exposure. Improving skin blood flow to the extremities may enhance manual dexterity and reduce the risk of peripheral cold injury, benefits pertinent to warfighters or other populations that spend time in cold environments.

Purpose

To examine the effect of high‐dose flavanol supplementation on CIVD. We hypothesized that acute and 8‐day flavanol supplementation would augment CIVD during single‐digit cold water immersion.

Methods

Eleven healthy adults (24±6 yrs; 10M/1F) ingested cocoa‐based flavanols (FLV; 900 mg/d) or caffeine and theobromine‐matched placebo (PLA) for 8 days in a double‐blind, randomized, crossover design. On Days 1 and 8, CIVD was assessed 2 hours after treatment ingestion. Subjects immersed their middle finger in warm water (42°C) for 15 minutes before cold water immersion (4°C) for 30 min, during which nail bed and finger pad skin temperature were measured continuously.

Results

Flavanol ingestion had no effect (all P > 0.05) on CIVD event frequency (Day 1, FLV: 3±2 vs. PLA: 3±2; Day 8, FLV: 3±2 vs. PLA: 3±1) or amplitude (Day 1, FLV: 4.3±1.7 vs. PLA: 4.9±2.6 °C; Day 8, FLV: 3.9±1.9 vs. PLA: 3.9±2.0 °C) in the finger pad following acute or 8‐day supplementation. Furthermore, average (Day 1, FLV: 9.2±1.5 vs. PLA: 9.2±1.8 °C; Day 8, FLV: 8.3±1.7 vs. PLA: 8.9±1.9 °C), maximum (Day 1, FLV: 11.9±3.0 vs. PLA: 11.2±2.3 °C; Day 8, FLV: 10.1±1.9 vs. PLA: 10.6±2.3 °C), and minimum (Day 1, FLV: 6.2±1.3 vs. PLA: 6.0±1.5 °C; Day 8, FLV: 5.8±1.8 vs. PLA: 6.2±1.7 °C) finger pad temperatures during cold water immersion were not different (all P > 0.05) between treatments on Day 1 or Day 8 of supplementation. Similarly, no differences (all P > 0.05) in CIVD parameters were observed in the nail bed following acute or 8‐day supplementation.

Conclusion

Dietary ingestion of cocoa‐based flavanols does not improve the CIVD response to single‐digit cold water immersion.
Exercise Preconditioning Attenuates Doxorubicin‐induced Diaphragm Contractile Dysfunction Independent of Phrenic Nerve‐stimulated Force Production
Authors: Nguyen Branden , Doerr Vivian , Montalvo Ryan , Ozdemir Mustafa , Smuder Ashley
Published: May 2022

Doxorubicin (DOX) is a highly effective anthracycline antibiotic used to treat a wide variety of cancers. Unfortunately, clinical use of DOX is often associated with respiratory muscle weakness characterized by fatigue, dyspnea and exercise intolerance. Investigation into the cause of DOX‐induced respiratory insufficiency has focused primarily on altered diaphragm muscle function; however, these symptoms of respiratory distress may also result from neural impairment. Recent preclinical evidence supports the postulate that neuromuscular function may be compromised following DOX administration, as soleus expression of key proteins required for neuromuscular transmission is negatively impacted. Interestingly, exercise preconditioning is a sufficient stimulus to prevent these adverse changes while also improving muscle quality and function. Although the functional impact of these findings is currently unclear, this data supports the premise that DOX‐induced neuromuscular toxicity contributes to the development of respiratory muscle dysfunction. To test this hypothesis, adult female Sprague‐Dawley rats were randomized into four experimental groups: 1) sedentary‐saline, 2) sedentary‐DOX, 3) exercise‐saline, or 4) exercise‐DOX. Exercise preconditioning consisted of treadmill running for 1 hour/day at 70% VO2Max for 10 days. 24 hours after the last bout of exercise, animals were injected with DOX (20 mg/kg I.P.) or saline (equal volume I.P.). Forty‐eight hours following saline or DOX treatment, the phrenic nerve was carefully isolated and remained innervated to the medial costal diaphragm to determine both phrenic nerve‐dependent diaphragm force and diaphragm force production independent of phrenic nerve activation. Our results demonstrate that maximal diaphragm muscle specific force production was significantly reduced in the sedentary‐DOX group in response to both phrenic nerve and direct diaphragm stimulation compared to both saline‐treated control groups. Importantly, exercise preconditioning in DOX treated animals significantly improved phrenic nerve‐independent diaphragm contractility, while phrenic nerve‐dependent contractile force remained depressed compared to sedentary DOX animals. These results reveal that DOX damages both the muscle contractile apparatus as well as neural input to the diaphragm, and that exercise training is sufficient to improve sarcomere contractility without identical improvements to neural function.
Structural Investigation of Allosteric Regulation in Class III Ribonucleotide Reductases
Authors: Andree Gisele A. , Levitz Talya S. , Funk Michael A. , Dawson Christopher D. , Drennan Catherine L.
Published: May 2022

Ribonucleotide reductases (RNRs) are essential enzymes that use radical‐based chemistry to catalyze the reduction of ribonucleotides to deoxyribonucleotides. Each RNR class uses a different cofactor to generate a catalytically‐essential thiyl radical species in the enzyme active site. Anaerobic (class III) RNRs employ an oxygen‐sensitive glycyl radical cofactor installed within the glycyl radical domain (GRD) that is adjacent to the active site, whereas aerobic (class Ia) RNRs employ long‐range radical transfer between the catalytic subunit and a di‐iron/tyrosyl radical cofactor housed in the b2 subunit. To maintain intracellular nucleotide pool balance, RNRs are allosterically regulated. For the prototypical class Ia RNRs from Escherichia coli, this allosteric activity regulation involves dATP binding to the N‐terminus of the catalytic subunit in a region known as the ‘cone domain’, resulting in an association between the cone domain and the b2 subunit that prevents radical transfer between subunits. Despite not requiring a b2 subunit, some class III RNRs (e.g. Streptococcus thermophilus)have a cone domain and are allosterically inhibited by dATP. In this study, we investigate allosteric activity regulation for such a class III RNR, the class III RNR from Streptococcus thermophilus(StNrdD). We use a newly developed LC‐MS/MS based activity assay to show that dATP downregulates StNrdD activity and ATP upregulates activity as is true for class Ia RNRs. Using crystallography, we obtain structural data showing that ATP and dATP both bind to the cone domain, and that both the cone domain and GRD are flexible. In particular, in one monomer of StNrdD dimer, the cone domain contacts the GRD, and both domains are ordered, and in the other monomer, the cone domain is positioned away from the GRD and the GRD is disordered. These observations led us to hypothesize that the cone domains of class III RNRs, like those of class Ia RNR, regulate activity through promotion or inhibition of the radical transfer step, but instead of controlling the position of b2, the position of the GRD is allosterically regulated. In this presentation, we show the data described above and show recent cryogenic electron microscopy of ATP‐bound and dATP‐bound states of StNrdD, which reveals differential positioning of the cone domains in the presence of these allosteric effectors. We also present our current thinking about the mechanism of allosteric regulation of activity in a class III RNR.
Elucidating the Kinetic Mechanism of Human METTL16
Authors: Breger Kurtis , Brown Jessica A.
Published: May 2022

Over 140 RNA modifications have been discovered, yet only recently have they been studied in depth due to recent technological advancements. N6‐methyladenosine (m6A) is an abundant RNA modification in messenger RNA (mRNA) and long non‐coding RNA that affects various cellular functions such as mRNA stability, phase‐separation of RNAs, and others. Methyltransferase‐like protein 16 (METTL16) is one of four catalytically active m6A RNA methyltransferases in humans. Two well‐known methylation targets of METTL16 are U6 spliceosomal RNA and hairpins in the 3′ untranslated region of MAT2A mRNA. However, METTL16 binds to many other RNAs, including the 3′ triple helix of MALAT1. Using in vitro assays, we have started to investigate the kinetic mechanism and other fundamental properties of METTL16. Thus far, we have determined that METTL16 is a monomer in complex with either U6 snRNA or the MALAT1 triple helix. The METTL16•RNA complex has a dissociation constant (KD) of 18 nM with the U6 snRNA and 31 nM with the MALAT1 triple helix. The apparent dissociation constant for S‐adenosylmethionine (SAM), the methyl donor, with the METTL16•U6 snRNA binary complex is 112 µM. Under in vitro conditions, the cancer‐associated MALAT1 triple helix is not a substrate of METTL16 at position A8290 and other adenosine residues. Preincubation assays suggest that there is an ordered mechanism by which U6 snRNA binds to METTL16 before SAM. Steady‐state assays established a kcat of 0.074 min‐1 and single‐turnover assays established a kchem of 0.56 min‐1. This difference in the rates suggests conformational rearrangements and/or product release may be rate limiting. Ongoing work includes the characterization of METTL16 mutants. Mutations in the METTL16 K‐loop led to a 7‐fold increase of SAM binding to METTL16•U6 snRNA. Future studies will focus on more METTL16 mutants, including critical residues in other structures and those identified in cancer patients, to ascertain how these residues affect the kinetic mechanism of METTL16. This study will enable future research on METTL16 as a therapeutic target.
Involvement of Lysyl Oxidase in the Pathogenesis of Arterial Stiffness in Chronic Kidney Disease
Authors: Sharma Ravindra K. , Krishnan Suraj , Jakkidi Nishka , Gomes Joshua , Li Shiyu K. , Mohandas Rajesh
Published: May 2022

Rationale

Increased vascular stiffness is a key mediator of cardiovascular disease (CVD) in patients with chronic kidney disease (CKD). Increased vascular stiffness in CKD is thought to be the consequence of long‐standing hypertension and vascular calcification. However, vascular stiffness is increased early in patients with CKD, even before metabolic complications develop. Lysyl Oxidase (LOX) is a key mediator of collagen crosslinking and matrix remodeling. Up‐regulation of LOX has been associated with increased tissue stiffness in cancer and wound healing. We hypothesize that increased LOX might be a key mechanism for increased vascular stiffness in CKD. β‐aminopropionitrile (BAPN), a LOX inhibitor, was used to test the hypothesis.

Methods

5/6 nephrectomy (Nx) or Sham surgery was performed in 8‐weeks old male C57BL/6 mice. 2‐weeks after Nx, mice were randomized to BAPN (300 mg/kg/day in water) or vehicle for 4‐weeks. Vascular stiffness was assessed by pulse wave velocity (PWV). LOX levels were measured by ELISA and qPCR. Collagen crosslinking was measured by mass spectrometry. Comparisons between groups were made using one‐way ANOVA, p‐Value <0.05 was accepted as significant.

Results

Nx resulted in decreased renal function at 8 weeks but no change in blood pressure or calcification. (~2‐3‐fold increase in creatinine, urea, and cystatin C) compared to control mice. Nx surgery resulted in increase in PWV (Con:179±32, Nx: 306 ± 47 cm/s, p<0.001). This was associated with an increased LOX in aorta (Con:118±8, Nx: 311±13 pg/mg dry weight, p<0.001). Mice with Nx surgery had an increased total collagen contents (183%, p<0.001) as well as cross‐linked collagen (DPD; 83%, p<0.05, PYD: 270% p<0.01). BAPN treatment significantly attenuates PWV (211± 49 cm/s, p<0.01), LOX (256±21 pg/mg dry weight, p<0.05) and total collagen contents (23%, p<0.01). BAPN treatment reduced the crosslinked collagens (DPD and PYD) but this did not reach statistical significance.

Conclusion

Our findings suggest: a) Increased vascular stiffness in CKD is independent of BP and calcification b) LOX mediated changes in extracellular matrix are at least in part responsible for the pathogenesis of arterial stiffness in CKD. Inhibition of excessive LOX may have therapeutic potential in alleviating increased vascular stiffness in CKD.
Using an HDAC Inhibitor to Alter Muscle Growth in Fetal Pigs
Authors: Cooper Elizabeth J. , Palou Rousset , Markley Grace , Stahl Chad H.
Published: May 2022

A broad spectrum histone deacetylase (HDAC) inhibitor, butyrate, has been shown to promote increased myofiber size by altering satellite cell (SC) activity. The use of dietary supplementation of butyrate to affect muscle development has been hampered by its technical and pharmacokinetic properties. Most of these can be overcome with the use of tributyrin (TB), a butyrate prodrug. Dietary supplementation of TB was able to promote muscle growth by increasing myofiber size in neonatal pigs. We hypothesized that muscle growth during fetal development may be improved by increased fiber number through the dietary supplementation of TB to gestating sows. Nine sows of 53.6 ±1.08 gestational days (gd) were randomly assigned to one of three dietary treatment groups (n=3). These dietary treatments were a basal diet, basal diet + 1% TB, and the basal diet + 2% TB. All diets were made isocaloric with the addition of oil and wheat flour. Fetal piglets were removed at 107.6 ±1.5 gd. Weights and body lengths were recorded for all fetuses. Sections of Longissimus dorsi and Psoas major were collected from the median male, median female, the smallest, and the largest piglets of each litter and frozen for future histological analysis. Two novel methods for objective description of muscle fiber organization were evaluated, including fiber eccentricity (measure of elongation), and proportion of primary myofibers. The most significant difference in cross sectional area (CSA) was found within LD muscle with TB supplementation causing a significant reduction compared to control animals (p value < 0.05). Fibers from 1% TB animals were significantly more elongated than control (p value < 0.05). The proportion of primary to secondary myofibers was significantly higher in muscle from 2% TB treated piglets compared to control (p value < 0.05). The smaller CSA together with a similar body weight across individual piglets (p >0.05) suggest that TB supplementation increased myofiber number. The increased fiber count may be further supported by more elongated myofibers suggesting alterations to muscle fiber organization and an improved development of primary fibers. Further studies to elucidate the impact of TB supplementation during gestation and early postnatal life are warranted.
Does Macrophage Stimulation by CCL5 Alter Melanoma Migratory Behavior?
Authors: Meyer Jacob T. , Baer Robert W.
Published: May 2022

Background

CCL5 (RANTES, regulated upon activation, normal T‐cell expressed and secreted) is secreted not only by T‐cells, but also by melanoma cells into the tumor microenvironment. The current project is part of a bigger effort by our laboratory to determine how the stimuli present in the tumor microenvironment may contribute to the progression of melanoma. Here, we explore CCL5 receptor expression on monocytes, macrophages, and melanoma cells, and whether endogenous or exogenous CCL5 stimulation of THP‐1 macrophages produce phenotypical changes that affect melanoma migratory behavior.

Methods

We used WM278 and LM‐Mel‐45 melanoma cells and THP‐1 monocytes before and after conversion to macrophages by 2 days exposure to 50 ng/ml PMA. To examine expression of the 3 key CCL5 receptors, we compared median fluorescence intensity (MFI) of antibody‐exposed cells to MFI of unlabeled cells using antibodies for CCR1, CCR3, and CCR5. We gated out debris and multiples, corrected for spillover, and analyzed the samples on a Bio‐Rad S3e cell sorter. We used recombinant CCL5 (100 ng/ml), the CCR1 & CCR3 receptor antagonist, UCB35625 (100 nM) and the CCR5 antagonist, maraviroc (100 ng/ml). Melanoma migration rate was measured by a 16 h wound‐closure assay. Treatments included: Control, CCL5 only, maraviroc only, UCB35625 only, maraviroc and UCB35625 together, and CCL5 plus both blockers. Wound closure of LM‐Mel‐45 melanoma alone, a 1:4 mixture of macrophages to melanoma, or LM‐Mel‐45 melanoma treated with macrophage‐conditioned medium were compared in each treatment condition.

Results

CCL5 receptor expression was not found on WM278 melanoma cells. LM‐Mel‐45 melanoma express CCR1 but not CCR3 or CCR5. CCR1 and CCR5, but not CCR3, are expressed by both THP‐1 monocytes and THP‐1 macrophages. Melanoma cells alone or when co‐cultured with previously treated macrophages migrated at 14.3 ± 0.4 μm/h, but neither CCL5 stimulation nor receptor blockade changed migration significantly. When LM‐Mel‐45 melanoma cells were treated with conditioned macrophage medium, velocity increased to 16.8 ± 0.7 μm/h.

Conclusions

This work suggests that CCL5 does not directly impact melanoma migration either by endogenous expression or by exogenous addition. Even in the presence of CCL5 or antagonist‐exposed macrophages, we found no phenotypical changes that altered the physical interactions of macrophages on melanoma migratory rate. There is a small but significant increase in velocity of melanoma cells when treated with conditioned macrophage medium.
Investigation of Disease‐Causing Point Variants of Vaccinia‐Related Kinase 1 (VRK1)
Authors: Frederick McKinzie M. , Mai Elissa N. , Hurley David P. , Schoeneman Laura L. , Ruff Emily F.
Published: May 2022

Vaccinia‐related kinase 1 (VRK1) is a serine/threonine kinase that plays a variety of roles in transcription regulation and the cell cycle. Its substrates include histones, p53, and coilin. The kinase activity of the VRK1 protein is largely controlled by its C‐terminal tail, which interacts with the enzyme active site. Several rare point mutations in VRK1 (including L195V, R89Q, and Y213H) are associated with neurodegenerative disorders, and questions remain as to how these mutations affect VRK1’s intrinsic stability and activity. For this in vitro study, mutations were generated in a His‐tagged VRK1 kinase domain construct, and proteins were expressed in E. coliand purified. The mutant proteins’ folding and stability were analyzed by circular dichroism, and ligand binding was investigated using differential scanning fluorimetry. Protein modeling in PyMOL was also used to visualize the kinase and its associated changes.
Results of Screening of Goats in Diagnosing of Ketosis
Authors: Nikitina Anastasia
Published: May 2022

Obtaining high‐quality and environmentally friendly livestock products must be provided with healthy livestock of animals. With ketosis, all types of metabolism are disturbed, the diagnosis and prevention of its various forms in ruminants are relevant. Dairy goats, like cattle, are prone to ketosis. Many representatives of dairy goat farms mistakenly believe that ketosis is not common among the dairy goat population. However, our initial studies of the prevalence of ketonemia among the population showed that at least 40% of animals in a herd had increased the concentration of ketone bodies in the blood. This fact indicates subclinical ketosis, which is promising for further research. In general, studies of metabolic disorders in dairy goats are not widespread; in this direction, the study of the metabolism of cattle occupies a leading position. The research was carried out on one of the goat farms in the North‐West region of Russia. We examined 5% of the total amount of animals at the farm (n=45). Goats were randomly selected, all from the milk group, from 1 to 8 lambing/lactation. Blood was obtained, and the concentration of β‐ketones was determined using a FreeStyle Optium Neo glucometer, researched the amount of total protein and its fractions, enzymes, lipids, and bilirubin RL‐Clima 15. Analysis of the study results and their comparison with the indicators of the referents revealed the following changes: the concentration of β‐ketones in 22 goats (48%) was in the range of 0.6 ‐ 0.9 mmol / L. Further, it was determined: the concentration of total protein was 13% higher than the limiting reference value and amounted to 86.4 ± 4.2 g / l, while no signs of dysproteinemia were observed. The urea level was also higher by 26.8% ‐ 9.29 ± 1.45 mmol / L. The activity of blood enzymes was within the normative values. The total bilirubin concentration was 10.5% higher than the reference value ‐ 5.7 ± 0.25 mmol / L, and the level of cholesterol was within the physiological range ‐ 2.11 ± 0.27 mmol / L. A violation of the calcium‐phosphorus ratio was noted. However, the concentrations of these substances were included in the reference values. Thus, it was determined that subclinical ketosis is prevalent up to about 50% among the goat population on a particular farm. Sick animals have protein metabolism disorders ‐ hyperproteinemia without dysproteinemia, and pigment metabolism disorders ‐ hyperbilirubinemia. It should be noted that no violations in the behavior and general condition of the animals were observed, which indicates precisely the subclinical form or stage of the disease. Timely prevention will reduce the percentage of sick animals.
Bilateral Nephrectomy Impairs Cerebral Oxygen Delivery After Acute Hemodilution Anemia in Rats
Authors: Chin Kyle , Steinberg Benjamin E. , Goldenberg Neil M. , Baker Andrew J. , Mazer C. D. , Hare Gregory M. T.
Published: May 2022

Hypothesis

We hypothesize that renal sensing of hypoxia is essential for activation of cardiovascular responses needed to maintain cerebral oxygen delivery during acute anemia and that bilateral nephrectomy will negatively affect brain oxygen delivery during acute hemodilutional anemia.

Methods

All experiments were approved by the Animal Care and Use Committee at St. Michael’s Hospital and conducted in accordance with ARRIVE‐2 guidelines. Sham or bilateral nephrectomy (n=5) were performed on isoflurane anesthetized Sprague‐Dawley rats prior to performing acute hemodilution (50% estimated blood volume) 1:1 with 6% hydroxyethyl starch. Heart rate (HR), mean arterial pressure (MAP) and rectal temperature were monitored continuously. Arterial blood gases and cooximetry samples were measured before and after hemodilution. Continuous measurements of brain microvascular oxygen tension (PbrO2) were measured via a phosphorescence quenching method using an intravascular oxygen probe, Oxyphor PdG4, and an OxyLED phosphorometer. Data were tested for normality and analysis performed by two‐way ANOVA with p<0.05 taken to be significant.

Results

Bilateral nephrectomy resulted in a reduction in baseline HR (p<0.004). Hemodilution resulted in a comparable reduction in hemoglobin concentration in both groups (Sham: 5.1±0.3 g/dL; Nephrectomy: 4.2±0.4 g/dL) with no differences in arterial PaO2 or PaCO2. After hemodilution, HR increased significantly in both groups (p<0.05). MAP tended to be lower in the bilateral nephrectomy group and decreased transiently in both groups following hemodilution (p<0.01). Brain microvascular pO2 (PbrO2)was not significantly different between nephrectomy and sham at baseline. Post‐hemodilution the PbrO2 was significantly lower in nephrectomised rats (11.5±5.1 mmHg) compared to sham operated rats (25.4±11.3 mmHg) (p=0.031).

Conclusions

We observed that bilateral nephrectomy resulted in changes in baseline physiology, including a lower HR. Hemodilution resulted in a comparable reduction in hemoglobin concentration, without difference in PaO2 or PaCO2. MAP decreased more profoundly immediately after hemodilution in nephrectomised animals, and recovered with time. Nephrectomy resulted in a significant drop in brain microvascular pO2 (PbrO2) immediately after hemodilution. This was not observed in sham controls. These data suggest that the kidney serves an important sensing role during acute anemia which supports cardiovascular responses and preserves the adequacy of brain tissue oxygen delivery. Hypoxia sensing by the kidney, and other integrative systemic mechanisms, may be central to these findings.
Endothelin‐1 via ETA receptor activation promotes renal iron deposition in murine models of iron overload
Authors: Kasztan Malgorzata , Hyndman Kelly A. , Binning Elizabeth , Pollock Jennifer S. , Pollock David M.
Published: May 2022

Renal iron deposition correlates with elevated plasma ET‐1 in patients and mouse model of sickle cell disease (SCD). Additionally, excessive renal iron accumulation in sickle cell mice (HbSS) is ameliorated by ETA receptor antagonism. Thus, we hypothesized that ET‐1 via ETA receptor activity leads to dysfunctional renal iron trafficking, promoting iron accumulation in the kidney. We conducted time‐ and concentration‐dependent in vitro studies on mouse proximal tubule (PT) cells and observed that ET‐1/ ETA signaling promotes cellular iron uptake (by increasing transferrin receptor 1 expression) and inhibits cellular iron removal pathways (65% reduction of ferroportin‐1 (FPN‐1) and doubled hepcidin (Hamp) expression). In vivo studies demonstrated that altered expression of iron transporters in PT of male HbSS mice was consistent with iron accumulation phenotype. ETA receptor blockade attenuated expression of the cellular iron uptake mediator, DMT‐1, and also decreased renal iron accumulation (1.43±0.06 vs. 2.62±0.12; p=0.097) by preserving FPN‐1, reducing Hamp expression, and increasing urinary iron excretion (10.9±1.0 vs 6.4±1.2 μg/24h; p=0.010). To determine the long‐term effect of ET‐1 on chronic renal iron accumulation, we generated HbSS mice lacking ET‐1 in vascular endothelial cells mice (HbSS VEET KO; using Tie‐2 Cre). There were no differences in anemia status between 20 weeks old male HbSS VEET KO and flox mice. However, plasma iron concentration (117.3±4.3 vs. 103.9±4.3 μg/dl, p=0.04) and urinary iron excretion (10.4±1.9 vs. 7.4±1.1 μg/24h; p=0.059) were elevated and renal iron deposition attenuated (24.3±1.3 vs. 33.8±2.3; p=0.008) in HbSS VEET KO mice. To further elucidate ET‐1/ETA receptor‐mediated renal iron accumulation already observed with pharmacological and genetic approaches in HbSS mice, using PEPCK Cre we generated PT specific ETA receptor knockout (PT ETA KO) mice. At baseline, 16 weeks old male and female PT ETA KO mice presented with higher plasma iron concentration (1.0±0.2 vs. 0.5±0.1 μg/ml; p=0.03) and urinary iron excretion (4.5±0.7 vs. 1.7±0.5 μg/24h; p=0.002), whereas renal iron deposition remained unchanged (2.26±0.48 vs. 2.44±0.73 Kpix/ μm; p=0.85). To induce hemolysis with acute iron overload, mice were injected with phenylhydrazine (PhZ, 40 mg/kg, IP; 2 consecutive days) or saline. PhZ significantly reduced hemoglobin in both PT ETA KO and littermate controls when compared to saline injected mice. Moreover, PT ETA KO mice given PhZ had increased plasma iron concentration (4.4±0.3 vs. 3.0±0.5 μg/ml; p=0.03) and urinary iron excretion (5.6±0.7 vs. 3.4±0.7 μg/24h; p=0.05) and decreased renal iron accumulation (0.50±0.22 vs. 0.90±0.20; p=0.011) when compared with saline injected KO control mice. Together, these data demonstrate that ET‐1, via ETA receptor activity, contributes to renal iron accumulation in murine models of iron overload.
Role of Intestinal Stearoyl‐CoA Desaturase 1 in Whole‐Body Lipid Metabolism and Metabolic Health
Authors: Burchat Natalie , Ye Hong , Akal Tasleenpal , Sampath Harini
Published: May 2022

Stearoyl‐CoA desaturase 1 (SCD1) is an ER‐resident enzyme that converts saturated fatty acids into monounsaturated fatty acids (MUFAs). These MUFAs are the preferred substrates for the synthesis of lipid species such as cholesterol esters and triglycerides. The intestine plays an important role in both lipid absorption and assimilation. Cholesterol and free fatty acids must be esterified into cholesterol esters (CE) and triglycerides (TG), respectively, for efficient secretion of chylomicrons for eventual lipid assimilation. Given the important role of SCD1 in modulating the esterification of both CE and TG, we hypothesized that intestinal SCD1 may regulate lipid secretion from the intestine. Our studies show that SCD1 protein and gene increases along the length of the intestine and this expression is induced by fasting followed by refeeding a high sucrose very low‐fat diet. To study the role of intestinal SCD1 in lipid metabolism, intestine‐specific knockout (iKO) mice were generated by crossing SCD1 floxed mice with mice expressing Cre recombinase under the control of the villin promoter. Our studies reveal that these mice have a 13% reduction in steady‐state plasma lipids, with a specific reduction in plasma triglycerides and free cholesterol. They also have reduced hepatic diacylglycerols and cholesterol esters, with a particular reduction in species containing the MUFA myristoleic acid (14:1). iKO mice have altered lipid composition in the distal intestine, where SCD1 is more highly expressed and regulated. In addition to these alterations in lipid handling, iKO mice have increases in plasma and hepatic bile acids, potentially due to an inability to efficiently esterify cholesterol. Downstream signaling via bile acids is elevated in iKO mice, resulting in activation of TGR5 signaling in brown adipose tissue and the ileum. Thus, our results indicate that deletion of intestinal SCD1 has significant impacts on whole‐body energy balance and lipid metabolism.
Type I interferon co‐stimulation enhances γδ intraepithelial lymphocyte IFNγ production
Authors: Fischer Matthew , Jia Luo , Edelblum Karen
Published: May 2022

Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor (TCR) constantly survey the intestinal epithelium, facilitating a localized effector response to limit pathogen invasion. Despite constant γδ TCR triggering in vivo, these IELs remain immunologically quiescent, suggesting that a co‐stimulatory signal is required to overcome their activation threshold. Type I interferon (IFN) is a family of pro‐inflammatory cytokines that are critical for host defense against bacterial and viral infection. Activated γδ IELs limit viral replication in intestinal epithelial cells by producing type I IFNs, such as IFNα; however, the role of IFNα in mediating γδ IEL antiviral effector function remains unknown. To determine whether IFNα directly activates these sentinel lymphocytes, murine small intestinal γδ IELs were isolated, sorted, and stimulated ex vivo with 1μg/mL αCD3, 10 ng/mL IFNα, or both for 5 h. Following treatment with αCD3 and IFNα, we observed a 27%±9.3 increase in IFNγ+ γδ IELs (p<0.0001) and a 2.5‐fold increase in IFNγ mean fluorescence intensity (MFI)(p<0.0001) compared to αCD3 stimulation. Interestingly, IFNα treatment alone was not sufficient to induce IFNγ production. To determine the threshold of TCR signaling necessary for this enhanced IFNγ production, we treated γδ IELs with suboptimal concentrations of αCD3 (0.01 or 0.1 μg/mL), to mimic constant TCR triggering in vitro.At 0.1 μg/mL αCD3, IFNα induced a 27%±7.2 increase in frequency of IFNγ+ γδ IELs (p<0.01) and a significant increase in MFI of γδ IEL IFNγ production over αCD3 treatment alone (p<0.001). With a low level of TCR priming (0.1 μg/mL αCD3), a low dose of IFNα (0.1 ng/mL) was able to stimulate a 22%±6.8 increase in IFNγ+ γδ IELs compared to αCD3 treatment alone. However, higher concentrations of IFNα failed to further enhance γδ IEL IFNγ production. Stimulation of γδ IELs with IFNα ex vivo was sufficient to induce STAT1 and STAT4 phosphorylation; therefore, we asked whether STAT1 or STAT4 is required for IFNα‐induced IFNγ production. To this end, we treated WT, STAT1 KO, or STAT4 KO γδ IELs with 1 μg/mL αCD3, 10 ng/mL IFNα, or both and found that the IFNα‐induced increase in IFNγ production was abrogated in STAT4‐, but not STAT1‐deficient γδ IELs (p<0.0001). To assess changes in STAT1 and STAT4 expression in γδ IELs during acute viral infection, WT mice were infected orally with 1.5x106 PFU murine gamma herpesvirus (MHV). We observed a 16%±3 increase in the frequency of γδ IELs 3 days post‐infection compared to controls, which corresponded with a decrease in total STAT1 (MFI: 905±95 vs 638±102, p<0.01). Taken together, our data demonstrate that γδTCR signaling is required for IFNα‐induced IFNγ production in a STAT4‐dependent manner. These findings suggest that IFNα signaling in conjunction with constant TCR triggering promotes the rapid activation and expansion of γδ IELs, which may enhance their effector response to microbial pathogens.
Mechanical stretch‐driven ROS generation stimulates endothelin production in neonatal pig renal epithelial and vascular endothelial cells
Authors: Kumar Ravi , Afolabi Jeremiah A. , Kanthakumar Praghalathan , Soni Hitesh , Adebiyi Adebowale
Published: May 2022

The circulating level of endothelin (ET), a potent vasoactive peptide, decreases with postnatal age. ET is also of pathophysiological significance in the kidney. ET is generated via endothelin converting enzyme (ECE)‐induced proteolytic processing of pro‐molecule big ET to biologically active peptides. Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), transactivates the promoter of ECE1. Oxyradicals contribute to kidney pathophysiology, but whether ROS regulates ET production in the kidneys remains unresolved. The present study shows that ECE1 is the predominant isoform in neonatal pig kidneys. We also demonstrated that H2O2 induces ECE1‐dependent ET1‐3 production in the neonatal pig renal proximal tubules (PT) and vascular endothelial cells (EC). A uniaxial stretch of neonatal pig PT and EC increased NADPH oxidase (NOX) 2 and 4 levels and stimulated H2O2production. Mechanical stretch increased cellular ECE1 production in the PC and EC cells, which catalase mimetic EUK 134 and NOX inhibitor apocynin reversed. Mechanical stretch also triggered cellular ET1‐3 generation in PT and EC. Mechanical stretch‐induced ET1‐3 production was attenuated by EUK 134, apocynin, and a selective ECE1 inhibitor CGS 35066. Short‐term unilateral urinary tract obstruction (UUO), an inducer of the mechanical stretch of kidney cells and oxyradical production, increased ECE1‐dependent ET1‐3 production in neonatal pig kidneys. Despite removing the obstruction, acute UUO generated ROS, reduced kidney perfusion, and increased renal vascular resistance (RVR). UUO also elevated early biomarkers of acute kidney injury (AKI). Inhibition of ECE1 and ET receptors protected against UUO‐induced increase in RVR and early AKI. These data suggest that mechanical stretch‐driven ROS generation stimulates ET production in neonatal pig renal epithelial and vascular endothelial cells. ET then promotes AKI by causing renal hypoperfusion. This signaling pathway may underlie UUO‐induced renal insufficiency in infants.
Extradiol cleavage of L‐DOPA as strategy for natural product biosynthesis
Authors: Jones Paige A. , Johnson Nyjah M. , Klugh Kameron L. , Peterson Larryn W. , Colabroy Keri L.
Published: May 2022

L‐DOPA dioxygenase was first discovered as part of a biosynthetic gene cluster to the natural product antibiotic, lincomycin. Within the larger biosynthetic context, L‐DOPA dioxygenase is part of a mini‐pathway to the synthon 3‐vinyl‐2,3‐pyrroline‐5‐carboxylic acid (VPCA). This synthon is elaborated and embedded within the final product structure of lincomycin, and also within the natural product structures of anthramycin, sibiromycin, tomaymycin and hormaomycin. Using the VPCA mini‐pathway as a starting point, we searched sequence space to identify 1) novel natural product pathways containing a VPCA synthon and 2) instances of L‐DOPA dioxygenase homologs that were not associated with a known VPCA containing natural product pathway, and that might provide new sources of natural product diversity and answer questions about the evolutionary origins of L‐DOPA dioxygenase. Using bioinformatic methods, including PSI‐BLAST, genome context analysis and structurally informed multiple‐sequence alignment, we identified a variety of known and novel natural product pathways that utilize an L‐DOPA dioxygenase ‐ many with the context of a VPCA synthon. Representative examples from these pathways were isolated and tested for iron binding and L‐DOPA dioxygenase activity.
A novel integrated pharmacokinetic‐pharmacodynamic model for the determination of in vivo synergism of combination therapy
Authors: Choi Young Hee , Tu Mei‐Juan , Yu Aiming
Published: May 2022

Establishment of pharmacokinetic‐pharmacodynamic (PK‐PD) relationship is critical for dose optimization during drug development and clinical therapy. While combination therapy is common, especially in oncology, conventional PK‐PD models do not recognize quantitative contributions from individual drugs. Rather, only one “interaction factor” is used in those models and arbitrarily assigned to a specific drug. Meanwhile, the interaction factor is overstretched to define pharmacologic synergism in vivo. In this study, we developed a novel PK‐PD model for combination therapy by considering apparent contributions from individual drugs or mutual interactions. Doxorubicin (Dox) and sorafenib (Sor) were used as model drugs whose PK data were obtained in mice and fit to two‐compartment models. Xenograft tumor growth showed biphasic features in mice and anticancer drug responses were described by three‐compartment transit models. This PK‐PD model revealed that Sor (contribution factor = 1.62) had much greater influence than co‐administered Dox (0.644) on overall tumor growth inhibition, which explains the mysterious clinical findings on a remarkable benefit for cancer patients when Sor is added to Dox treatment, whereas none when Dox is added to Sor therapy. Furthermore, we incorporated the combination index method into this PK‐PD model to critically determine in vivo synergism which, however, cannot be accurately defined by interaction factor method. Additionally, this new PK‐PD model readily predicts optimal dosage combinations to achieve greater extents of tumor growth inhibition that are consistent with stronger synergy. These results support the utilities of this new PK‐PD model and reiterate the application of valid method for the evaluation of in vivo synergism.
Impaired Proteostasis, not Protein Synthesis, Limits Recovery of Aged Skeletal Muscle After Disuse Atrophy
Authors: Fuqua Jordan D. , Lawrence Marcus M. , Hettinger Zachary , Borowik Agnieszka K. , Peelor Frederick F. , Confides Amy L. , Dupont‐Versteegden Esther E. , Miller Benjamin F.
Published: May 2022

Older individuals undergo more frequent inpatient hospitalizations and bedrest compared to younger persons, causing rapid losses of muscle mass and strength. Evidence suggests that only aged muscle fails to completely recover following disuse despite aged and adult muscle having similar levels of muscle loss. Current efforts to improve muscle recovery in older individuals are commonly aimed at increasing myofibrillar protein synthesis via mTOR stimulation despite recent evidence demonstrating that old muscle has chronically elevated levels of mTORC1 activity. We hypothesized that old muscle fails to fully recover muscle mass due to impaired proteostasis and not due limitations in protein synthesis.

Methods

Adult (10 month) and old (30 month) F344BN rats were hindlimb unloaded (HU) for 14‐days to induce atrophy, followed by subsequent reloading (RE) to study muscle regrowth. During RE the rats were labeled with deuterium oxide (D2O) for 7, 15, 30, 45 or 60 days (n=4‐6 per timepoint per group) to determine bulk and individual protein synthesis and RNA synthesis. We also assessed muscle mass, fiber size by immunohistochemistry, mTORC1 signaling by western blot, and aggregate formation by tissue fractionation.

Results

Adult and old gastrocnemius (GA) muscle had significant losses of muscle mass (359 mg ±55 and 724 mg ±60 loss respectively,p<0.05) and fiber size (1296 um2±202 and 1480 um2 ±142 loss respectively, p<0.01) with HU. While adult muscle fully recovered GA mass and fiber size by day 15, old GA muscle did not fully recover during the entire 60 days of RE (18% CSA and 11% mass loss still present). Despite limited mass and fiber size recovery in old muscle, GA myofibrillar protein synthesis rates (1.45 ±0.18 vs 2.28 ±0.24, FSR%, p<0.05) and mTORC1‐related signaling were higher in old muscle compared to adult. Additionally, GA RNA synthesis rates (2.79 ±0.139 vs 5.18 ±0.684, p<0.05) and RNA concentration (183 ng ±3.6 vs 249 ng ±13.9, p<0.05), which are markers of translational capacity, were also elevated in old muscle relative to adult during RE. Old muscle had higher levels of insoluble protein aggregates, a marker of impaired proteostasis, during RE compared to adult (1.2 ±0.09 vs 1.5 ±012 fold increase, p<0.05). Lastly, we assessed individual protein synthesis rates of the whole muscle proteome and discovered that old GA muscle had a larger number of proteins with increased synthesis rates compared to adult. In conclusion, these data strongly suggest that limitations in old muscle to recover after disuse are not due to limitations in protein synthesis but are instead due to impaired proteostasis with age. Therefore, understanding how proteostasis responds during these periods surrounding unloading in old muscle are critical to improve muscle recovery after disuse.
Predicting Protein Function and Orientation on a Gold Nanoparticle Surface Using a Residue‐Based Affinity Scale
Authors: Xu Joanna Xiuzhu , Alom Md. Siddik , Yadav Rahul , Fitzkee Nicholas C.
Published: May 2022

Gold nanoparticles (AuNPs) associate strongly with proteins when used as a nanoscale drug carrier in vivo. It is recognized that the structure and function of a protein can be affected upon surface adsorption. To understand the structural properties of the adsorbed protein, we developed a residue‐based affinity scale obtained from competitive binding to predict the bound orientation of a given protein and therefore its function on AuNP. A total of 20 K13X GB3 variants are generated by mutating the key binding residue K13 into each of the 19 amino acids, and a series of host‐guest binding experiments are performed to quantify the affinity scale of each residue for AuNP with NMR spectroscopy. The effects of size, charge, and hydrophobicity of each residue on AuNP‐binding are systematically evaluated. Our results show that, for nonpolar residues, the steric size is the dominant factor during competition for an AuNP surface, as the affinity scale decreases linearly with increasing side chain volumes. Importantly, time‐resolved 2D NMR indicates the adsorption kinetics and thermodynamics are drastically different for each variant when competition is present. Combined with the surface accessibility of a residue, the affinity scale is applied to predict the binding surface and function of two selected enzymes on an AuNP. With its predictability successfully validated by activity assays, the affinity scale should be widely applicable for guiding the fabrication of AuNP‐based nanomedicines using active biomolecules.