bluuebunny/crossref_metadata_2025 huggingface dataset:
Interdisciplinary Research in Physiology: Insights into the Physiological Society's (UK) Report on “The Future of Interdisciplinary Research beyond REF 2021”
Authors: Witchel Harry J.
Published: May 2022

The role of interdisciplinary research (IDR) to address research questions posed by global social, economic, ecological and political changes is widely recognised (e.g. addressing Covid‐19). Physiology is by nature an interdisciplinary science – understanding how fundamental processes work and interact with other systems in the body in good health to then understand how to respond to ill health. In November 2021 the Physiological Society (UK) published a report that has since been presented to the UK Parliament on the specific challenges faced by bioscience researchers (including but not exclusively physiologists) whose work is interdisciplinary. These problems are experienced by both senior researchers and especially early career researchers. There are prominent issues relating to funding, peer review, bibliometrics, risk in career progression, and team research. Particularly with the UK's discipline‐based Research Excellence Framework (REF), there has been a perception that interdisciplinary researchers have been disadvantaged. The author, who was a member of the committee that oversaw the drafting of the report, will provide a personal view of the drives to present the report to Parliament, and its conclusions. The proposals include more research into the nature of interdisciplinary research, more flexibility in the assessment of research, and the use of narrative to contextualise the potential impacts of such research. The report in its entirety can be found here:

https://www.physoc.org/policy/research‐landscape‐and‐funding/interdisciplinary‐research/
Lactic metabolism and swimming. Comparison between freestyle swimming and swimming with fins in french athletes
Authors: Duvallet Alain , Duvallet Emilie , Lhuissier François , Beaudry Michèle
Published: May 2022

Compare lactatemia after freestyle and swimming with fins competitions among French athletes specialized in these sports. Fifty two athletes participated in the study, 32 athletes in freestyle and 20 with fins, 28 male and 24 female athletes. Lactatemia was measured on samples (n=102) obtained by micro puncture from the earlobe at 5th minute recovery [LA].

Comparisons are made based on the race distance. Speed for the same distance is always higher when swimming with fins versus freestyle swimming. We calculated the working power (distance/speed) and the power provided by lactic metabolism ((LA)/time, M/min) to analyze the lactatemia as a function of work.

Mean lactatemia increased during both swimming exercices with maximal values after 100m race. The lactatemia after swimming with fins is always highter than after freestyle swimming. Significant differences were found only for the 800‐1500m races (p<0.001).

The power provided by lactic metabolism was maximal for short races. For 50,100, 200 and 400m it was higher when swimming with fins. For 1500 m the values were identical. The differences were not significant.

The increase of lactatemia indicates the sollicitation of energy source producing lactic acid. According to Margaria and Edward and Di Prampero et al., a lactic process, i.e., the ATP and CP splitting may be sufficient to provide all the energy necessary for 5 to 10 sec physical exercise. And during short exercices of 30 sec, the oxygen stores may play an important rôle in postponing lactate production.

The results indicate that lactic metabolim condition is identical in both exercise. This metabolim in more important during short race. With the power of work more important when swimming with fins, the lactic power is always higher, but not‐significant. So the most determinant parameter for lactic metabolim is the work power and not the type of exercice.
Toxic form of steatosis in dairy cows (clinical, hematological and histological data)
Authors: Nikitina Anastasia , Kovalev Sergey , Trushkin Vyacheslav , Vasiliev Roman , Vasilieva Svetlana , Gaponova Viktoriya
Published: May 2022

Steatosis is a common disease in dairy cattle, especially during the transit period. Usually, the subclinical form of hepatosis is evenly distributed in the population throughout the year: 30‐70% of cows can be affected. However, severe (toxic) steatosis is rare. The study was carried out on 505 dairy cows in the North‐West of Russia in July‐August. In the blood serum, the concentrations of the main substances were investigated, and the morphological examination of the blood was carried out. Liver samples for histological examination were collected posthumously. 47% of cows had subclinical hepatopathy, 1.78% (9 cows) ‐ a toxic form of steatosis. Clinical signs of severe hepatopathy were observed from 1 to 10 days after calving: anorexia, icterus, ventroflexia, excitability, convulsions, exophthalmos. In the blood: total protein 59.8±0.9 g/l, albumin ‐ 35.1±2.5%, total bilirubin ‐ 23.8±0.8 μmol/l, triglycerides ‐ 0.329±0.01 mmol/l, cholesterol ‐ 8.7±1.0 mmol/l, RBC 3.9±0.1*1012/l, PLT ‐ 190.1±39.8*109/l, WBC ‐ 8.36±0.75*109/l, HCT – 29.1±0.5%. Autopsy: icterus of the mucous membranes, petechiae in the kidneys, intestines, brain membrane, injection of blood vessels, enlargement of the liver and kidneys, the liver capsule breaks easily, the parenchyma is loose. Liver’s parenchyma imprint smears: staining with Saathof solution ‐ much neutral fat. Rumen: detachment of the mucous membrane. Liver: large and small vacuole diffuse fatty infiltration of hepatocytes, thickening of the portal tracts with expressed mononuclear infiltration. The liver has an ordered lobular structure, is moderately full‐blooded, the sinusoids of the liver and the Disse spaces are moderately dilated, staining with Schiff‐iodic acid according to McManus ‐ the absence of glycogen in hepatocytes. Kidneys: hyaline‐drop dystrophy of the epithelium of the renal tubules, fibrosing interstitial glomerulonephritis. Rumen: the mucous membrane forms convex structures of the main plate of various sizes and shapes, covered with multilayer squamous epithelium; there are areas of degeneration. The muscle plate was formed by separate bundles of smooth myocytes lying at the base of the papillae; individual muscle cells were found in the main plate of the papilla. The plate consisted of an inner annular and an outer longitudinal layer of smooth muscle cells, among which there were individual fibers of striated muscle tissue. The serous membrane of the rumen had a typical structure; it was represented by loose connective tissue covered with mesothelium. According to the results of this study, it was revealed that the toxic form of hepatosis is accompanied by multiple organ failure: an expressed violation of the nervous system, hypoprotein‐, dysprotein‐, hyperbilirubin‐ and anemia, large and small‐drop fatty infiltration of hepatocytes, glomerulonephritis, lesions of the rumen and intestinal wall.
Identification of Genes Essential for Sulfamate and Fluorine Incorporation During Nucleocidin Biosynthesis
Authors: Pasternak Ola , Zechel David
Published: May 2022

Nucleocidin, a secondary metabolite of Streptomyces calvus(1), is a derivative of adenosine that contains 4’‐fluorine and 5’‐O‐sulfamate groups. Both substituents are rarely observed in natural products. A minimal biosynthetic gene cluster has yet to be defined for nucleocidin, and at least 24 genes appear to be involved. To date, only two functions have been assigned, neither of which are directly involved in sulfamate biosynthesis or fluorination (2, 3). In this presentation, we present biosynthetic studies on two newly discovered producers of nucleocidin, Streptomyces virens B‐24331 and Streptomyces aureorectusB‐24301 (4). Both strains are shown to be significantly (up to 30‐fold) better producers of nucleocidin than S. calvus. Gene disruption experiments in S. virens have identified, for the first time, genes differentially impacting sulfmate and 4’‐fluorine biosynthesis. These and additional results will be presented.

References:

(1) Zhu, X. M., et al., Biosynthesis of the Fluorinated Natural Product Nucleocidin in Streptomyces calvus Is Dependent on the bldA‐Specified Leu‐tRNA(UUA) Molecule. ChemBioChem 2015, 16, 2498–2506.

(2) Feng, X., et al. Two 3’‐O‐b‐glucosylated nucleoside fluorometabolites related to nucleocidin in Streptomyces calvus.Chem. Sci. 2019, 10, 9501‐9505.

(3) Ngivprom, U., et al. Characterization of NucPNP and NucV involved in theearly steps of nucleocidin biosynthesis in Streptomyces calvus. RSC Adv., 2021, 11, 3510‐3515.

(4) Chen, Y., et al., Streptomyces aureorectus DSM 41692 and Streptomyces virens DSM 41465 are producers of the antibiotic nucleocidin and 4’‐fluoroadenosine is identified as a co‐product. Org. Biomol. Chem. 2021, DOI: 10.1039/d1ob01898a.
EGFR Inhibition in Cardiomyocytes Stabilizes Cardiomyocyte Cohesion in a Murine Model for Arrhythmogenic Cardiomyopathy
Authors: Shoykhet Maria , Dervishi Orsela , Hiermaier Matthias , Waschke Jens , Yeruva Sunil
Published: May 2022

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase ubiquitously expressed in different tissues, including the heart. EGFR is dysregulated in several kinds of cancer, as well as in heart diseases. Arrhythmogenic cardiomyopathy (ACM) is a familial heart disease and one of the underlying mechanisms is impaired desmosome turnover. The intercalated disc (ICD) not only provides cellular cohesion but can also provide the structural framework of a signaling hub. Thus, stabilizing desmosome integrity, thereby enhancing cardiomyocyte stability, may provide potential new treatment options. In this study, we investigated the role of EGFR inhibition on cardiomyocyte cohesion under physiological as well as under pathophysiological conditions using the cardiomyocyte‐specific plakoglobin knockout (Jup‐/‐) ACM model in which EGFR was upregulated. Dissociation assays in HL‐1 cells and murine cardiac slice cultures showed that EGFR inhibition by erlotinib similar to SRC inhibition enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR, desmoglein 2 (DSG2) and desmoplakin (DP), indicating that altered EGFR signaling might affect desmosomes. Immunostainings and atomic force microscopy (AFM) revealed enhanced DSG2 localization and binding at the cell borders upon EGFR inhibition. In addition, during desmosome assembly induced by a Ca2+‐switch, DSG2 and DP were augmented at the cell borders after EGFR or SRC inhibition. Our study shows that EGFR inhibition by erlotinib leads to increased DSG2 binding and desmosome assembly, which stabilizes cardiomyocyte cohesion in the ACM mouse model. Because EGFR inhibition by erlotinib is already used in cancer patients, these data reveal a potential new therapeutic strategy in ACM.
Adult mouse kidney stem cells orchestrate the de novo assembly of a nephron via Sirt2‐ modulated canonical Wnt/β‐catenin signaling
Authors: Han Xiaobin , Sun Zhongjie
Published: May 2022

End‐stage renal disease affects an estimated 2.4 million people annually worldwide. With cases increasing 8% every year, regenerative strategies are needed to help alleviate, and potentially replace, the increasing demand for kidney transplants. An important question remains whether stem cells reside in the adult mammalian kidney. Here we developed a methodology for isolation of kidney stem cells from adult mouse kidney by using Sca1 and Oct4 stem cell markers. Using these cells, we generated three types of kidney‐like structures. First, we observed the assembly of self‐organizing three‐dimensional kidney organoids. These kidney organoids show characteristic of kidney structures and contain podocytes and endothelial cells that form networks of capillary loop‐like structures. We also observed the de novogeneration of a self‐organizing nephron (and its associated collecting duct) from monolayer cultures. Comma‐ and S‐shaped bodies that expressed activated Notch1 were present during this formation, suggesting that Notch signaling played a key role in driving nephrogenesis. This is consistent with our current knowledge of nephron formation in the mouse embryo. Furthermore, we observed the differentiation of monolayer cells into functionally mature tubules and self‐organizing kidney‐shaped structures that selectively endocytose dextran. Finally, we demonstrated that expression of Sirt2 was upregulated, which modulated the dynamics of canonical Wnt/β‐catenin signaling during kidney organoid development. Altogether, our observations provide the first evidence for the existence of stem cells in the adult mouse kidney. These findings offer powerful tools for future applications, including drug screening, disease modelling, cell therapy, and kidney regeneration.
Rapid deployment of smartphone‐based augmented reality tools for field and online education in structural biology
Authors: Sung Rou‐Jia , Adamala Katarzyna Z. , Engelhart Aaron E.
Published: May 2022

Structural biology education commonly employs molecular visualization soft‐ ware, such as PyMol, RasMol, and VMD, to allow students to appreciate structure–function relationships in biomolecules. In on‐ground, classroom‐ based education, these programs are commonly used on University‐owned devices with software preinstalled. Remote education typically involves the use of student‐owned devices, which complicates the use of such software, owing to the fact that (a) student devices have differing configurations (e.g., Windows vs MacOS) and processing power, and (b) not all student devices are suitable for use with such software. Smartphones are near‐ubiquitous devices, with smartphone ownership exceeding personal computer ownership, according to a recent survey. Here, we show the use of a smartphone‐based augmented real‐ ity app, Augment, in a structural biology classroom exercise, which students installed independently without IT support. Post‐lab attitudinal survey results indicate positive student experiences with this app. Based on our experiences, we suggest that smartphone‐based molecular visualization software, such as that used in this exercise, is a powerful educational tool that is particularly well‐suited for use in remote education.
SRI 22136, A Novel Delta Opioid Receptor (DOR) Antagonist for the Treatment of Alzheimer’s Disease
Authors: Tanguturi Parthasaradhi R. , Ananthan Subramaniam A. , Pathak Vibha , Venukadasula Phanindra K. , Zhang Sixue , Chafiq Omar Moukha , Szafran Corinne E. A. , Streicher John M.
Published: May 2022

Alzheimer’s disease (AD) is the most common form of dementia, which affects 47 million people worldwide. There are over five million AD patients over the age of 65 in the U.S. which is predicted to increase to 16 million by 2050. There are currently no approved disease‐modifying therapies for AD and attempts to prevent or slow the progression of the formation of beta‐amyloid plaques by targeting both the beta‐secretase 1 (BACE1) and gamma‐secretase enzymes have not yet achieved clinical success.

Studies suggest that indirect modulation of the function of these enzymes via G‐protein coupled receptors (GPCRs) may provide a novel strategy to reduce A‐beta peptide production with potentially less side effects. Among GPCRs that influence amyloidogenesis, the delta opioid receptor (DOR), in particular, has been shown to play an important role in the trafficking and function of BACE1 and gamma‐secretase and in the production of A‐beta peptide. DOR activation increases BACE1 and gamma‐secretase activity in vitro and in a mouse model of AD, and antagonism of DOR specifically blocks the amyloidogenic pathway and efficaciously prevents AD progression in mice. These effects were demonstrated using a known DOR antagonist, naltrindole. However, the potential of DOR antagonists as therapeutic agents for AD has yet to be explored.

The aim of this project is to create novel DOR antagonists via medicinal chemistry and identify the most promising lead compound based on binding, selectivity, and functional profile in vitro. Further, select a small set of the most promising compounds and evaluate the compound’s ability to mitigate AD‐like pathology in vivo using APP/PS double‐transgenic mice. We report here the identification of several selective DOR antagonists with low nanomolar affinity/potency that have been shown to block BACE1 activity in vitro. Lead compound SRI‐22136, showed systemic stability and blood brain barrier penetration using the in vivo tail flick assay in CD‐1 mice. SRI‐22136 also showed efficacy in Novel Object Recognition (NOR) behavioral studies and in brain histological analysis studies we observed greater reduction in the AD pathological markers at a dose of 1mg/kg twice daily for 90 days continuous treatment in vivo using the APP/PS AD model double‐transgenic mice. Also, in our brain histological analysis the compound exhibited greater efficacy in reduction of the AD pathology markers such as Aβ signal, and secondary inflammation and activated glia detected by GFAP (astrocytes) and CD11b (microglia) in APP/PS AD model double‐transgenic mice. We further report our progress in testing this compound’s effect on biochemical marker evaluation using the cortex and hippocampus regions of the APP/PS AD model double‐transgenic mice.
Lower‐limb venous function in hypoxia and hyperoxia: effect of healthy ageing
Authors: Fan Jui‐Lin , Sayegh Ana L. C. , Kaur Manpreet , Dawes Mathew , Paton Julian F. R. , Fisher James P.
Published: May 2022

Oxygen is an important modulator of vascular function, but its influence on the veins is incompletely understood. While age‐related impairments on arterial function have been well‐established, the effects of ageing on the venous circulation have not been examined. The purpose of this study was to determine the effects of hypoxia and hyperoxia on lower limb venous function in healthy young and older humans. We tested the hypothesis that the venomotor response to hypoxia and hyperoxia is attenuated with ageing.

In fifteen young (29±8 years, mean±SD) and seven older (age: 66±6 years) healthy individuals, we assessed peripheral oxygen saturation (SpO2) and the cross‐sectional area (CSA) of the great saphenous vein (GSV; Doppler ultrasound) during a standard thigh cuff inflation‐deflation protocol (11 min) while breathing room air, hypoxia (fraction in inspired oxygen [FIO2]: 0.10) or hyperoxia (FIO2: 0.50), in a single‐blinded, randomized manner. The pressure‐CSA relationship was modelled using an established quadratic regression equation and GSV compliance derived.

Resting SpO2 was decreased by hypoxia (80.9±5.6%) and increased by hyperoxia (98.8±0.7%) compared to room air (96.3±1.1%, p<0.001) in both young and older groups. In the young group, hypoxia selectively decreased GSV CSA (index of capacity) (13.4±5.7 mm2, p<0.001) compared to room air (17.0±7.9 mm2), while GSV compliance was unchanged (p=1.000). In contrast, hyperoxia did not alter GSV CSA (17.1±8.7 mm2, p=0.883) in the young group, but GSV compliance was increased by 2‐3 fold compared to room air and hypoxia (p=0.007). In room air, GSV capacity was almost double in older participants (+19.6m2, p=0.016), but GSV compliance was not different from the younger group (p=0.423). Neither hypoxia nor hyperoxia affected GSV CSA and compliance in the older group (p>0.05 vs. room air).

These preliminary data suggest that in young healthy individuals, hypoxia reduces GSV capacity and hyperoxia increases GSV compliance. However, such manipulations in blood oxygenation do not alter venous function in older healthy individuals. Further investigations are required to determine whether local and/or chemoreflex‐mediated sympathetic mechanisms underpin such alterations in venous function.
Ferroptosis Regulation by the NGLY1/NFE2L1 Pathway
Authors: Murray Magdalena , Forcina Giovanni , Pope Lauren , Dong Wentao , Abu‐Remaileh Monther , Bertozzi Carolyn , Dixon Scott
Published: May 2022

Cells are equipped with various mechanisms to maintain cell viability upon exposure to stress. The Cap ‘n’ Collar (CNC) family includes five vertebrate stress‐responsive transcription factors. The CNC member nuclear factor erythroid 2 like 2 (NFE2L2) is considered the master regulator of the cellular oxidative stress response. Fellow CNC member nuclear erythroid 2 like 1 (NFE2L1) is better known for its role in responding to proteasome dysfunction. We hypothesized that NFE2L1 also responds to oxidative stress. We demonstrate that genetic disruption of NFE2L1 sensitizes cancer cells to death by a non‐apoptotic, oxidative form of cell death termed ferroptosis. We also show that genetic disruption of N‐glycanase 1 (NGLY1), which catalyzes NFE2L1 deglycosylation and sequence editing, sensitizes cancer cells to ferroptosis through an NFE2L1‐dependent mechanism. Expression of a sequence‐edited NFE2L1 mutant, which mimics NGLY1‐processed NFE2L1, rescues cellular resistance to ferroptosis in NFE2L1 gene‐disrupted cells. Finally, we provide evidence that the ferroptosis protection conferred by NFE2L1 is mediated by the expression of glutathione peroxidase 4 (GPX4). All together, our results highlight the role of NFE2L1 in ferroptosis resistance and implicate NFE2L1 and NGLY1 as potential targets for cancer therapy.
Comparison of del Nido and Histidine‐tryptophan‐ketoglutarate cardioplegic solutions on cardiac and endothelial function
Authors: Xue Hong‐Mei , Hou Hai‐Tao , Sun Wen‐Tao , Wang Shi‐Fu , Guo Shan , Yang Qin , He* Guo‐Wei
Published: May 2022

Objectives

This study was designed to compare cardiac and endothelial protection of three clinically used cardioplegias del Nido (DNC), Histidine‐tryptophan‐ketoglutarate (HTK), and cold blood (BC) cardioplegia followed by HTK (BC+HTK) in ischemia/reperfusion(I/R)rat model.

Methods

Sixty male Wistar rats were subjected to either 120‐min global ischemia at 4°C followed by 90‐min reperfusion at 37°C or no I/R (Control) in Langendorff apparatus and were randomly allocated into five groups: Control, I/R, DNC, HTK, and BC+HTK. Hemodynamic parameters were continuously recorded. The endothelium‐dependent relaxation to acetylcholine (ACh) was measured in the left anterior descending artery thereafter in myograph. The protein expression of cardiac Troponin T (cTnT) and creatine Kinase MB (CKMB) were measured by western blotting.

Results

During reperfusion, HTK had higher LVSP whereas DNC had lower LVEDP, better LVDP, and best +dp/dtmax and ‐dp/dtmax than other two groups but the differences disappeared at the end of the reperfusion. HTK or BC+HTK preserves the ACh‐induced endothelium‐dependent relaxation better than DNC (Emax=48.2±8.0% in DNC vs. 75.0±8.0% in HTK, p<0.05; vs. 96.9±3.5% in BC+HTK, p<0.001). The protein level of cTnT and CKMB was downregulated in the three groups.

Conclusions

DNC shows a better recovery in diastolic function, whereas HTK or BC+HTK exerts better protection in coronary endothelial function. All these three cardioplegias could ameliorate the myocardium injury at the protein level. These findings may suggest that searching for “perfect” cardioplegia is warranted.
Postinjury Inhibition of Endothelin‐1 Dependent Renal Vasoregulation Mitigates Rhabdomyolysis‐Induced Acute Kidney Injury
Authors: Afolabi Jeremiah M. , Williams Jada , Kanthakumar Praghalathan , Kumar Ravi , Adebiyi Ade
Published: May 2022

Rhabdomyolysis is a life‐threatening condition resulting from the breakdown of skeletal muscle fibers leading to the release of myoglobin into the blood. Increased circulating myoglobin can cause kidney damage, and acute kidney injury (AKI) occurs in 33‐50% of patients with myoglobinuria. Persistent constriction of the renal microvessels, which reduces renal blood flow (RBF) and glomerular filtration rate (GFR), contributes to rhabdomyolysis‐induced AKI. Since activation of G‐protein‐coupled receptors (GPCR) and ion channels regulate renal vascular resistance (RVR), rhabdomyolysis may likely modulate RVR via GPCR and vascular ion channel mechanisms. Pretreatment of rats with an endothelin receptor antagonist has been shown to alleviate rhabdomyolysis‐induced AKI. However, the downstream and upstream links between rhabdomyolysis and increased ET‐1 synthesis are unknown. The downstream effectors of ET‐1‐induced vasoconstriction are smooth muscle cell (SMC) Ca2+‐permeable ion channels. Yet, the ion channel mechanisms that trigger renal vasoconstriction in rhabdomyolysis‐induced AKI remain unknown. Also, most cases of rhabdomyolysis‐induced AKI cannot be predicted. Whether post‐injury inhibition of the vascular mechanisms of ET‐1 ameliorates, rhabdomyolysis‐induced AKI is unknown. This study investigates post‐injury renal production and vascular regulation of the ET system in rhabdomyolysis‐induced AKI. Six h of rhabdomyolysis increased peptidase endothelin converting enzyme (ECE‐1)‐dependent biosynthesis of ET‐1 in the kidneys. Rhabdomyolysis‐induced AKI was not sex‐dependent. At 24 h, rhabdomyolysis also increased renal reactive oxygen species (ROS), ECE‐1, and ET‐1 levels. Furthermore, ET‐1 activated rat renal vascular smooth muscle cell TRPC3 leading to a reduction in RBF and increased RVR. Protein expression levels of the ET receptors (ETA and ETB) and TRPC3 channels in renal microvessels were unaltered. However, postinjury inhibition of ECE1, ET Receptors, and TRPC3 mitigated rhabdomyolysis‐induced GFR impairment, renal hypoperfusion, AKI biomarker elevation, and morphological kidney damage. To further explore the role of TRPC3 ion channels in rhabdomyolysis‐induced AKI, we subjected TRPC3 knock‐out (KO) rats to rhabdomyolysis. Compared to their wild‐type counterparts, KO rats show protection against rhabdomyolysis‐induced AKI. Our findings suggest that ECE‐driven proteolytic ET‐1 production contributes to rhabdomyolysis‐induced AKI. Downstream, ET‐derived DAG activates renal vascular smooth muscle cell TRPC3 channels leading to extracellular calcium entry, vasoconstriction, increased RVR, and reduced GFR.
Effects of Fumarate on Blood Pressure and Renal Hemodynamics in Normotensive Rats: Possible Contribution of Sodium Potassium Co‐transporter (NKCC)
Authors: Edosuyi Osaze , Choi Myung , Igbe Ighodaro , Oyekan Adebayo
Published: May 2022

The tricarboxylic acid cycle product, fumarate, and its related products, have been implicated in the etiology of genetic and non‐genetic models of hypertension. Fumarate showed a mechanistic link to nitric oxide (NO) production and thus impacted blood pressure and renal hemodynamics in both models. This study evaluated the possibility of a role of fumarate in regulation of blood pressure and renal hemodynamics in normotensive rats and elucidate possible mechanisms involved. Animals were anaesthetized with Thiobutabarbital (100 mg/kg, ip). The carotid artery was cannulated for mean arterial blood pressure (MABP) measurement and the left kidney was exposed to measure cortical blood flow (CBF) and medullary blood flow (MBF). Blood and urine were collected to determine glomerular filtration rate (GFR), urine volume (Uv) and sodium excretion (UNaV). The fumarase inhibitor, pyromellitic acid (PMA) (0.1 mg/kg/h), or fumarate (0.1 mg/kg/h) were infused in these animals. To determine renal sodium excretory mechanisms involved with fumarate, fumarate (1 mg/kg, po) was administered to normotensive rats treated with hydrochlorothiazide (HCTZ) (10 mg/kg, po) or furosemide (10 mg/kg, po), urine was collected hourly for five (5) hours and assayed for sodium. Fumarate elicited a hypotensive effect, with peak effect at the 45th minute (126 ± 6.2 vs 93 ±2.6 mmHg, p<0.05). Similarly, inhibition of fumarase caused a time‐dependent decrease in MABP (p<0.05). There was a consistent drop in CBF in fumarate‐infused rats and although PMA caused an initial rise in CBF at 15 mins, there was a 10‐fold increase in CBF at 60 min when compared to fumarate‐infused rats (p<0.05). MBF also decreased in a time‐dependent fashion in rats infused with fumarate. Consistent with the drop in MABP and CBF, GFR reduced (0.89 ± 0.26 vs 0.39 ± 0.11 mL/min, p<0.05) at 30 mins before to returning to baseline levels at 60 mins in fumarate‐infused rats. Uv was unchanged in rats infused with fumarate but there was a 1‐fold reduction in UNaV at 30 mins (p<0.05). Also, PMA, tended to increase UNaV at 30 mins, compared to baseline. However, there was an exacerbation in UNaV in PMA‐infused rats (24.2 ± 3.8 µmol/L) compared to fumarate‐infused rats at 30 mins (4.56 ±1.4 µmol/L, p<0.05). Fumarate increased UNaV compared to control (p<0.05), however, there was no significant difference in UNaV between furosemide‐treated and furosemide‐treated fumarate rats. These data indicate that fumarate and its products elicited vasodilatory effects that reduced blood pressure and exerted vascular and tubular effects on renal hemodynamics. Also, the tubular effects, possibly involves inhibition of sodium potassium co‐transporter (NKCC).
SIRT1‐mediated Lysine Crotonylation is Involved in the Regulation of CaMKII Activity in Myocardium
Authors: Wang Xiang‐Chong , Chen Huan‐Xin , Hou Hai‐Tao , Xue Hong‐Mei , Sun Wen‐Tao , Yang Qin , He Guo‐Wei
Published: May 2022

Objectives

Post‐translational modifications (PTMs) can modify proteins and regulate their functions. Lysine crotonylation is a novel protein PTM that can be regulated by silent mating type information regulation 2 homolog 1 (SIRT1). Ca2+/calmodulin‐dependent protein kinase II (CaMKII) plays a significant role in mediating cellular responses to external signaling molecules. Little is known regarding the effect of SIRT1 on CaMKII crotonylation and the impact of crotonylation on the activity of this enzyme.

Methods

Cre‐Loxp technology was used to generate conditional cardiac‐specific SIRT1 knockout mice (SIRT1 KO). Protein crotonylation modification was studied by PTMomics. CaMKII activity was determined by ELISA. Western blot and co‐immunoprecipitation (CO‐IP) were performed to examine the expression and crotonylation of CaMKII

Results

PTMomics showed changes in protein crotonylation profiles in the myocardium of cardiac‐specific SIRT1 knockout mice. Crotonylation of CaMKII was significantly increased at lysine 301 and the enhanced CaMKII crotonylation was further confirmed by CO‐IP (2.37±0.25 in SIRT1 KO vs. 0.96±0.10 in wild‐type and 1.04±0.10 in SIRT1fl/fl mice, p<0.01). Cardiac‐specific knockout of SIRT1 did not affect the protein expression of CaMKII whereas significantly increased CaMKII activity (2.75±0.17 m units/μL in SIRT1 KO vs. 2.10±0.13 m units/μL in wild‐type and 1.98±0.17 m units/μL in SIRT1fl/fl mice, p<0.05). CO‐IP showed no physical interaction between SIRT1 and CaMKII.

Conclusions

SIRT1 deficiency increases CaMKII activity in myocardium at least partially through enhancing lysine crotonylation of CaMKII, which advances our understanding of the role played by PTM in the regulation of CaMKII.
Neutrophil‐Derived Microvesicles Enhance Pulmonary Vascular Inflammation via a Toll‐like Receptor 4 Signaling‐Dependent Mechanism
Authors: Tsiridou Diianeira Maria , Sachouli Eirini , Takata Masao , O'Dea Kieran P.
Published: May 2022

Background

Circulating neutrophil‐derived microvesicles (NMVs) are markedly elevated during sepsis and therefore could have a role in the development of indirect acute lung injury (ALI). We recently found that NMV‐enriched CD11b+ MVs, immunoaffinity isolated from lipopolysaccharide (LPS)‐stimulated healthy volunteer blood, have potent pro‐inflammatory activity in a human peripheral blood mononuclear cell (PBMC) and lung microvascular endothelial cell (HLMEC) coculture model of pulmonary vascular inflammation (1). By contrast, immunoaffinity isolated platelet‐MVs (CD61+) produced negligible responses in these assays, suggesting specificity of the NMV‐enriched CD11b+ MV, activity. Here, we investigated the signaling mechanisms responsible for NMV‐mediated activation of monocytes and HLMECs in this model.

Methods

Heparinized blood from healthy donors was treated with LPS (100 ng/ml, 3 h) and NMVs (CD66b+) were isolated by positive immunoaffinity selection. NMVs were then incubated in PBMC‐HLMEC cocultures for 4 h. NMV‐induced responses were determined by flow cytometric quantification of cell surface activation markers (ICAM‐1 for HLMECs, ICAM‐1 and tissue factor for monocytes) and pro‐inflammatory cytokine release by ELISA (TNFα, IL‐8, IL‐6, MCP‐1).

Results

NMV‐induced HLMEC activation required the presence of monocytes and was completely attenuated by anti‐TNFα neutralizing antibody (ICAM‐1: isotype control 302±103 vs anti‐TNFα 66±29; p<0.01). Pharmacological inhibition of mitogen‐activated protein kinases p38, MEK1‐MEK2 and phosphoinositide‐3 kinase ablated monocyte and HLMEC activation. Monocyte activation was prevented in the presence of the Toll‐like receptor 4 (TLR4) inhibitor TAK‐242 (e.g., TNFα release: vehicle 1108±257 vs TAK‐242 16±12; p<0.001) and partially inhibited by an anti‐TLR4 neutralizing antibody. However, the LPS inhibitor polymyxin‐B had no effect on these responses, indicating that LPS contamination was unlikely. Furthermore, NMV activity was found to be sensitive to proteinase K and heat‐treatment (55 °C, 15 min) (e.g., TNFα release: untreated 474±267 vs proteinase K 12±15; p<0.0001 vs heat‐treatment 200±84; p<0.01).

Conclusions

We have demonstrated that NMVs produced within LPS‐stimulated blood are potent mediators of lung microvascular endothelial cells via monocyte‐derived TNFα signaling. NMV‐induced monocyte activation involves TLR4 signaling and is protease sensitive, suggesting that MVs serve as vehicles of damage‐associated molecular pattern (DAMP) activity, mediated by surface proteins. Together these findings support a major role for NMVs as novel long‐range mediators for systemic propagation of inflammation, contributing to the pathogenesis of sepsis‐induced indirect acute lung injury.

(1) Tsiridou et al. European Respiratory Journal. 2020;56:4468
A‐Kinase anchoring protein 2 (AKAP2) and Pendrin; cross‐talk between cell signaling and renal physiology
Authors: Belaid Houwayda , Wang Dongmei , Mendes Carraco Ana , Li Na , Van Bemmelen Miguel , Diviani Dario , Staub Olivier
Published: May 2022

Kidney tubules play a pivotal role in the maintenance of body fluid homeostasis and pH regulation. Accordingly, kidney disorders are known to be strongly associated with hypertension and acid‐base imbalance. In the kidney, the intercalated cells of the nephron are the main site of acid‐base balance: type A are acid‐secreting whereas type B are base‐secreting. The chloride bicarbonate exchanger Pendrin is present in type B intercalated cells and participates actively in regulating blood pressure and NaCl balance. It is still not entirely deciphered how Pendrin activity is regulated; nevertheless, it was previously demonstrated that Pendrin is regulated by c‐AMP/Protein Kinase A (PKA) signaling pathway. Consequently, in this study, our hypothesis is that the A‐Kinase anchoring protein 2 (AKAP2), strongly expressed in the kidney, regulates in time and space the intracellular signal transduction.

In order to investigate AKAP2 involvement in Pendrin regulation and trafficking to the apical plasma membrane, we generated an inducible and nephron‐specific Akap2Pax8/LC1 mice knockout model. By confocal microscopy, fluorescent immunostaining on kidney tissue sections showed that AKAP2 is present in the tubules and most importantly colocalizes with Pendrin at the apical plasma membrane of the intercalated cells. To show that Pendrin and AKAP2 are interacting, we used the Proximity Ligation Assay (PLA) and, positively, we could reveal the association between the two proteins. The latter finding was also confirmed in vitro taking advantage of the Opossum Kidney Cell line (OKP) stably expressing Pendrin and co‐transfected with AKAP2, we could co‐immunoprecipitate Pendrin and AKAP2 after crosslinking on live cells. Fluorescent immunostaining in Akap2Pax8/LC1 show that Pendrin tends to be shifted from the apical membrane (seen in control mice) to intracellular compartments.

In conclusion, our data suggest that AKAP2 and Pendrin are interacting, and AKAP2 may play a key role in Pendrin trafficking to the apical plasma membrane, hence in regulating its activity.
CXCR7 Promotes Adipogenesis and Insulin Resistance in Human Visceral Adipocytes
Authors: Shi Yi , Qian Shenqi , Alqahtani Saud , Peng Qianman , Shen Jianzhong
Published: May 2022

Obesity is a multifactorial chronic condition that increases the risk of cardiovascular diseases, type II diabetes, and certain types of cancer. It has been known that some chemokines and their receptors facilitate the development of obesity, leading to other related comorbidities. However, the exact role of atypical chemokine receptor CXCR7 in obesity and obesity‐induced type II diabetes remains unknown. We aimed to study whether CXCR7 promotes adipogenesis and insulin resistance in human visceral adipocytes in vitro. Using RT‐PCR and flow cytometry studies, we found that both CXCR7 mRNA and its protein expressions were significantly upregulated during the differentiation of human visceral adipocytes. Stimulation of CXCR7 by SDF‐1 or LIH383 (a CXCR7‐selective peptide ligand) increased lipid droplets formation during adipocytes differentiation. In addition, we found that SDF‐1 or LIH383 treatment inhibited AKT phosphorylation in a dose‐dependent manner with no effect on ERK1/2 and JNK signaling pathways, which indicates a selective suppression of the AKT signaling axis by CXCR7. Furthermore, activation of CXCR7 by SDF‐1 inhibited insulin signaling to the AKT pathway and insulin‐induced glucose uptake. In contrast, ablation of CXCR7 by CRISPR/Cas9 system restored AKT signaling and enhanced insulin sensitivity in human visceral adipocytes. We concluded that CXCR7 plays a critical role in human visceral adipogenesis and insulin resistance by suppressing the AKT signaling pathway, highlighting CXCR7 as a potential new drug target against obesity and type II diabetes.
Enzymatic production of acetaldehyde using a Pseudomonas fluorescens mannitol‐2‐dehydrogenase mutant to regenerate NAD+
Authors: Cosse Kylee J. , Sharma Victor K. , Allgeier Alan M. , McFarlane Jeffery S.
Published: May 2022

Over the past decade, market saturation has drastically reduced the value of ethanol, which threatens the viability of industrial bioethanol facilities throughout the Midwest. In response, we are exploring enzymatic methods to convert ethanol and other biomass substrates into higher value chemical derivatives, such as acetaldehyde, to enhance the economic sustainability of bioethanol production. Acetaldehyde has a market price 2.5 times greater than ethanol and the volatility of acetaldehyde offers the potential for ready industrial separation. We are currently evaluating a coupled enzyme reaction in which horse liver alcohol dehydrogenase produces acetaldehyde from ethanol while an N191D mutant of Pseudomonas fluorescens mannitol‐2‐dehydrogenase (PfM2DH) is used to produce mannitol from fructose and to oxidize NADH regenerating NAD+. We have chosen the N191D mutant based on work by Klimacek and Nidetsky demonstrating that this mutation converts wild type PfM2DH, which is reversible, into an enzyme selective for fructose reduction (1). In previous work, we have shown that PfM2DH is active under a wide range of pH and temperature conditions making it a promising enzyme for industrial production. We are developing a GC‐MS solid phase microextraction (SPME) assay to evaluate accumulation of acetaldehyde in the head space of our reaction vessel. This assay will allow us to optimize conditions for the industrial stripping of acetaldehyde from a reactor. We are also optimizing a HILIC‐LC‐MS assay to quantify mannitol production in solution. Combining these assays allows the determination of total acetaldehyde production and the acetaldehyde fraction entering the gas phase. In addition to evaluating the viability of our horse liver ADH and PfM2DH_N191D coupled reaction, these assays will allow the characterization of future enzyme pairs in our efforts to valorize bioethanol using an industrial enzymatic process.
Chaperoning mechanism of innate immune receptor NOD2 by HSP70
Authors: Le Ha , Ukaegbu Ophelia , Grimes Catherine L.
Published: May 2022

Host pattern‐recognition receptors (PRRs) are the frontline members of the innate immune system. They are responsible for recognizing molecular structures derived from bacteria or damaged cells and producing appropriate innate immune responses against potential threats from pathogenic agents. However, their defective forms can lead to the development of inflammatory diseases. Crohn’s disease (CD), a prevalent and incurable inflammatory bowel disease, has been shown to closely link to three polymorphisms of the intracellular innate receptor nucleotide‐binding oligomerization domain containing 2 (NOD2). Biophysical and biochemical data show that CD‐associated NOD2 mutants have a much shorter half‐life and reduced signaling function compared to the wild‐type, contributing to the downstream chronic inflammation condition found in CD patients. Given NOD2’s involvement in disease development, it has become an attractive target for drug development against CD. Besides the existing effort in developing anti‐inflammatory drugs and immune suppressors, a novel approach focuses on designing peptidomimetics targeting NOD2 based on its positive modulator protein ‐ stress‐induced heat shock protein 70 kDa (HSP70). Overexpression of both full‐length and truncated HSP70 in cells stabilizes NOD2 mutants and rescues their function, indicating that HSP70 can be used as a platform for developing NOD2 peptide agonists. We first determined the direct interactions at a low micromolar affinity between HSP70 and NOD2 by surface plasmon resonance (SPR) and biolayer interferometry (BLI) technologies. The optimized binding assays will further allow screening of potential HSP70‐derived peptides that retain the same affinity for NOD2. To identify the interacting regions on the amino acid level, we will utilize cross‐linking mass spectrometry (XLMS) together with different docking algorithms. The results will give us a glance into the regulatory mechanisms of HSP70 on NOD2 and provide beneficial structural information for structure‐based peptide drug design. In the future, we plan to study the effects of HSP70‐NOD2 interactions on NOD2‐specific immune responses via RNA sequencing and RT‐PCR.
Activation of aryl hydrocarbon receptor stimulates GLUT‐mediated glucose transport in human renal proximal tubular cells
Authors: Kaewsaro Kannaree , Soodvilai Sunhapas
Published: May 2022

Aryl hydrocarbon receptor (AhR), a ligand‐activated transcription factor, plays a major role in detoxification of xenobiotics. AhR deficiency has been shown to attenuate renal injury induced by diabetes. It is interesting whether the beneficial effect of AhR deficiency on renal injury in diabetes involve in alteration of intracellular glucose in renal tubular cells. The aim of this study was to investigate the effect and mechanism of AhR on glucose uptake into human renal proximal tubular cells. Exposure HK‐2 cells, human renal proximal tubular cell line, with omeprazole, an AhR agonist, increased glucose transporter (GLUT)‐mediated [3H]‐2‐Deoxy‐D glucose uptake into the cells in concentration‐dependent manner. The stimulatory effect of omeprazole was reduced by co‐incubation with CH223191, an AhR antagonist. These results indicate that the stimulatory effect of omeprazole on GLUT transport function regards activation of AhR. The kinetic study showed that omeprazole significantly decreased half‐maximal transport (Kt) but not maximal rate of transport (Jmax) of GLUT, suggesting that AhR might increase affinity of GLUT‐mediated glucose uptake into the cells. In addition, activation of AhR significantly induced cellular level of ROS. Inhibition of ROS using N‐acetyl‐L‐cysteine (NAC) inhibited the stimulatory effect of AhR on GLUT‐mediated glucose uptake. These results support that activation of AhR increases GLUT‐mediated glucose accumulation via increase in ROS production in human renal proximal tubular cells. Inhibition of AhR might be a target for treatment of high glucose‐induced renal injury from diabetes.
Mycoplasma infection modifies attachment, proliferation, and the expression of immune checkpoint antigen CD276 (B7‐H3) on the Cal29 bladder cancer cell line
Authors: Aicher Wilhelm K.
Published: May 2022

Introduction and aim

Bladder cancer is among the most prevalent malignancies in Western societies. Despite intensive research, progress towards better diagnosis or therapy was sparse in the last decades. However, many studies documented elevated expression of immune checkpoint antigens including PD‐1, PD‐L1, CTLA‐4, or CD276 (alias B7‐H3) on bladder tumor cells. Immune checkpoint antigens are capable to suppress immune responses against the tumor. But novel therapies targeting immune checkpoint antigens are promising. Some of these agents are already part of clinical studies. At the same time mycoplasma infections were reported in about 18% of sexually experienced adults. We therefore investigated if mycoplasma contaminated urothelial carcinoma cells Cal29 differed in attachment, appearance, proliferation, and expression of immune checkpoint antigen CD276 from mycoplasma‐free Cal29.

Materials and Methods

Mycoplasma infected Cal29 cells (mcCal29) and clean Cal29 cells (Cal29, from ATCC) were expanded and infection vs. sterility was monitored by PCR throughout the experiment. Cells were seeded and attachment, appearance and size of the cells were observed by microscopy. Proliferation was enumerated in consecutive passages using a hematocytometer and trypan blue dye exclusion. Expression of CD276 was studies on the mRNA level by quantitative PCR of cDNA (RT‐qPCR). Analyses of GAPDH and PPIAγ served as controls. CD276 protein levels were explored by immunoblot, and ß‐actin served a control. CD276 expression on cell surfaces was explored by flow cytometry and is documented as mean fluorescence intensity (MFI). Unstained cells and beads served as controls.

Results

Mycoplasma‐contaminated Cal29 proliferated significantly faster than clean Cal29 (n=5, 132%, p<7,8E‐09). Upon seeding in flasks, Cal29 adhered faster then mcCal29, attached more firm to the flasks as microscopy showed up to double diameters in comparison to mcCal29. Expression of CD276 transcripts and total proteins did not differ, but on cell surfaces mcCal29 (MFI 4334) expressed less than half of CD276 in comparison to Cal29 (MFI 9729).

Conclusions

Urinary infection by mycoplasma may indirectly contribute to tumor spready by accelerating proliferation of bladder cancer cells and by reducing cell‐cell and/or cell‐matrix interactions as documented by attachment and cell growth studies. However, mcCal29 presented less CD276 on tumor cell surfaces. Thus, mycoplasma infection may facilitate immune responses to bladder cancer cells by reducing the expression of CD276. But these preliminary results must be conformed or challenged by additional studies with other urothelial cells.
May Chelated Iron Be Pro‐Inflammatory?
Authors: Tarczykowska Agata , Engström Niklas , Dobermann Darja , Powell Jonathan , Scheers Nathalie
Published: May 2022

We have shown that two iron chelates, used in iron nutrition, promote induction of the oncogenic growth factor Amphiregulin in human gut epithelial cells. Since then, we have investigated several iron compounds on the safe lists of EFSA and USFDA using a human intestinal cell assay in combination with proteomic profiling. Here we will report proteomic cell data for other iron chelates, salts and nanoparticulate iron which suggest that iron chelates may increase the cellular sensitivity to pro‐inflammatory mediators and growth promotors by increasing their receptor levels. We conclude that iron chelates may be pro‐inflammatory and pro‐oncogenic to intestinal cells.