bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of nutrient intake and food consumption patterns in normal‐weight, overweight and obese adults
Authors: Stote Kim S. , Carrico Pauline , Cole Renee
Published: June 2021

The NHANES 2007–2008 show an overweight prevalence of 68%, with 34% of the population classified as obese. The objective of the study was to compare nutrient and food consumption patterns in normal‐weight, overweight and obese adults. A convenience sample of adults was surveyed utilizing an online learning management system. The NCI's automated self‐administered 24‐h dietary recall was used to assess dietary intake. Foods reported in the 24‐h recalls were converted into food groups using MyPyramid Equivalents. Results showed that participants (n=101) were 76% female and 24% male with a mean (±SD) age of 39 ± 10 years. The mean (±SD) BMI was 22 ± 2 (n=35), 27 ± 2(n=33), and 36 ± 7 kg/m2(n=33) in the normal‐weight, overweight and obese participants, respectively. Participants completed six 24‐h recalls, over 15 weeks, for a total of 606 used for this analysis. No significant differences were observed in calorie, carbohydrate, and fat consumption between the three groups. However, consumption of protein, alcohol, caffeine, and sugars were significantly higher in the overweight and obese groups. MyPyramid Equivalents indicated increased consumption of meats, grains, solid fat and alcoholic drinks in the overweight and obese groups with the normal‐weight group consuming increased amounts of vegetables, soy, nuts, legumes and oils. Food consumption patterns may contribute to obesity in this adult population.
Cariogenic bacteria and diet according to nutritional status in pre‐school children
Authors: Argentieri Angela , Mateo María , Iglesias María , Manto María , Friedman Silvia , Lifshitz Fima , Molgatini Susana , Rodriguez Patricia
Published: June 2021

Dental caries is a multifactorial disease, and diet, closely associated with nutritional status (NS), plays an essential role. However, the relation between these factors has not been thoroughly studied to date. To clarify the relationship between NS and cariogenic bacteria, eighty four children aged 59.2 ± 12.8 months, attending a day care center, were examined. They were classified by Body Mass Index Z‐scores (WHO, 2007) in adequate (A), overweight (OW) or obese (O). Carbohydrate consumption frequency between meals/day (CH), cariogenic risk (CR), and colony‐forming unit/ml saliva (CFU) of streptococci and lactobacilli were assessed.

NS showed 46% of children A, 30% OW and 18% O. Although CH did not differ significantly among groups (p>0.05), O presented less children with high CR (A:62, OW:77 and O:50%, p<0.05). The number of CFU of Streptococcus mutans group did not differ significantly, but CFU of Lactobacillus acidophilus was reduced in O (O:2241±1586 vs A:3700±1710 and OW:2715±1793, p<0.05).

Thus, these results suggest an association among NS, diet and microbiology composition of dental biofilm that leads to a negative relationship between obesity and caries risk.
Ways of educating, informing and networking with health professionals about nutritional sciences: The Nestlé Nutrition Institute
Authors: Klassen Petra , Haschke Ferdinand
Published: June 2021

The aim of the Nestlé Nutrition Institute (NNI) is to deliver latest scientific information in the area of nutrition and health and to provide training for capacity building and continuous medical education. The NNI is a not‐for‐profit organization which is run separately from the business.

The information provided is based on educational needs which were identified by consumer research. Results led to restructuring the content of the NNI website by nutrition topics. Consequently traffic to the site doubled over a 6 months period. All information and training is prepared by independent experts in their fields. Global distribution of information is achieved via the website, distribution of materials by a global NNI network reaching out to tens of thousands of health professionals. E.g. a recently published practical guide book for pediatric nutrition (Pediatric Nutrition in Practice) has been distributed over 60’000 times.

The NNI plays a role in education of health professionals by organizing nutrition trainings in the field of clinical nutrition and mother and child nutrition, a global scholarship program and accredited continuous medical education. Since the NNI supports these activities in conjunctions with renowned institutions and societies (e.g ESPGHAN or NASPGHAN) it can be an important partner to lever and foster ongoing efforts in nutritional education in the academic and public health sector globally.
Underserved minority children are not meeting the moderate‐to‐vigorous physical activity (MVPA) recommendation
Authors: Wong William W. , Ortiz Christina L. , Lathan Debra , Moore Louis A. , Konzelmann Karen L. , Adolph Anne L. , Stuff Janice E. , Mikhail Carmen , Smith E. OˈBrian , Butte Nancy F.
Published: June 2021

Childhood obesity is more prevalent among underserved minority children. It is recommended that children should spend a minimal of 60 min/d in MVPA in order to maintain a healthy body weight. Based on a minimal of 5 days of Actical measurements, we quantified the amount of MVPA in 483 underserved minority children (10.3±1.0 y; 51.1% male; 31.1% Black/68.9% Hispanic; 66% overweight or obese) who took part in our community childhood obesity intervention program (Healthy Kids‐Houston). The majority of children (89.4%) were in the reduced cost/free meal program and 71.1% had no health insurance or were insured under programs for low‐income families. The children spent more time on MVPA (p<0.01) on weekdays (46.8±1.2 min/d, mean±SE) than on weekend days (41.0±1.6 min/d). Time spent on MVPA decreased with age (−5 min/d per y, p< 0.01) with boys spending more time (12.6 min/d, p<0.01) in MVPA than girls. Black children also spent more time on MVPA (6 min/d, p=0.03) than Hispanic children. MVPA also decreased with BMI z‐score (p<0.01). Most alarmingly, the majority of children (76.4%) spent less than 60 min/d on MVPA. Therefore, to combat childhood obesity, public and private resources should be invested to promote MVPA and to enhance the environment for underserved, minority children.

National Research Initiative of the USDA National Institute of Food and Agriculture grant #2008‐55215‐18875 and USDA/ARS grant #6250‐51000‐053. Clinical trial: NCT00994084.
Aryl hydrocarbon receptor‐dependent IL‐10 production in response to BCG exposure: a novel immune modulatory pathway
Authors: Gray Essanna Sequel , Gonzalez Rosa Salazar , Aliberti Julio
Published: June 2021

Mycobacterium tuberculosis (MTB) is a slow growing pathogen spread through inhalation of air droplets from cough or sneeze of an infected person. MTB typically causes a chronic infection that can be reactivated at anytime. Tuberculosis is highly prevalent in areas with poor nutrition and unsanitary living conditions. Mycobacterium bovis Bacillus Calmette‐Guérin (BCG) is an attenuated strain of Mycobacterium bovis that is widely used as a vaccine against tuberculosis in many parts of the world. However, currently used vaccines differ in characteristics and immunogenicity due to differences in the process of culturing bacterial strains. Furthermore, the efficacy of BCG as a vaccine in adults and adolescents is significantly less than in children as well as more effective against tuberculosis meningitis and miliary tuberculosis than against pulmonary tuberculosis. This low efficacy is hypothesized to be a result of BCG inducing a high level of anti‐inflammatory cytokines. Signaling through the Aryl hydrocarbon receptor, AhR, promotes the expression of Suppressor of Cytokine Signaling 2 (SOCS2), an important mediator of cytokine signaling, which attenuates the inflammatory response. Here, we hypothesized that the activation of AhR during BCG exposure modulates protective type 1 response induction, thereby weakening the generation of protective memory cells. To test the role of AhR in the immune response induced by BCG, splenocytes were isolated and cultured from spleens of both wild type and AhR deficient mice and levels of IL‐12 (p40), TNFα, IL‐6, and IL‐10 were measured using ELISAs after in vitro infection with BCG. The results suggest that AhR plays a crucial role in the induction of IL‐10 by BCG therefore, inhibiting the Type 1 immune response. Understanding the role of IL‐10 modulation by AhR signaling can lead to a more efficient vaccine that will admit an effective TH1 type response while preventing tissue damage to the host.
REEPs, an ER adapter protein family, have differential effects on GPCR expression
Authors: Angelotti Timothy , Ho Vincent , Hurt Carl
Published: June 2021

Introduction

REEPs are members of a growing class of proteins termed ER morphogens or adapter proteins. REEP1 mutations have been implicated in Hereditary Spastic Paraplegia (HSP). To further understand the role of REEPs in ER membrane trafficking and HSP, we have examined their role using two model proteins, alpha2A and alpha2C adrenergic receptors (ARs).

Methods

Transiently transfected HEK293 cells expressing individual Flag‐REEP proteins and HA‐alpha2A&C ARs were examined by FACS analysis to determine REEP effects on receptor expression, correlated with ligand binding and deglycosylation analysis.

Results

Analysis of individual cells expressing both REEPs and ARs revealed a complex effect. Co‐expression of alpha2A ARs with REEP1, REEP2, and REEP6 caused a small increase in plasma membrane AR expression (1.0×, 1.0×, and 0.5× respectively), however there was a larger increase in total AR expression within the cell (1.8×, 1.8×, and 1.2× respectively). A similar analysis of alpha2C AR expression showed a larger REEP effect on surface expression (1.3×, 1.4×, and 1.9× respectively) coupled with a smaller increase in total AR expression (1.2×, 1.2×, and 1.5× respectively). Ligand binding of total membranes showed a similar pattern. Lastly, deglycosylation analysis of ARs revealed that co‐expression of REEPs led to an increase in a minimally glycosylated form of alpha2C ARs.

Conclusions

REEPs were originally identified by their ability to enhance GPCR surface expression; however, their effects cannot be generalized. REEPs appear to enhance the production or stability of a minimally glycosylated alpha2C AR. Lastly, co‐IP analysis has revealed that REEPs interact preferentially with alpha2C, but not alpha2A ARs.
Elucidation of the phosphorylation profiles of the long and short isoforms of the omega‐3 fatty acid receptor‐1 (GPR120)
Authors: Burns Rebecca N. , Moniri Nader H.
Published: June 2021

The omega‐3 fatty acid receptor‐1 (O3FAR1), formally termed GPR120, is a G protein‐coupled receptor that has been thrust to the forefront of diabetes research due to its ability to stimulate release of the insulin secretagogue glucagon‐like peptide‐1 (GLP‐1) upon agonism by the family of omega‐3 fatty acids. We have identified long and short splice variants of human O3FAR1 and demonstrated that the long variant contains additional amino acids within the third intracellular loop, a GPCR region typically associated with signaling and desensitization. Our previous work also showed four additional phosphorylation sites within this region of the long isoform, suggesting varied desensitization between the two variants. While we did not detect notable differences in agonist‐induced phosphorylation of either isoform, basal phosphorylation was significantly higher for the shorter variant, evoking the possibility that O3FAR1‐long and short are differentially regulated by receptor phosphorylation. Here, we present data investigating the phosphorylation profiles of both O3FAR1 isoforms. Our results are the first to reveal the molecular mechanisms of agonist‐induced and agonist‐independent phosphorylation of O3FAR1.

This project was funded by a grant from the Diabetes Action Research and Education Foundation. Mrs. Burns is supported by a fellowship from the American Foundation for Pharmaceutical Education.
Development of a reporter assay system to screen chemicals for claudin‐4 modulator activity
Authors: Kondoh Masuo , Watari Akihiro , Yagi Kiyohito
Published: June 2021

Claudins (CLs), a tetra‐transmembrane protein family that contains 27 members, are key functional components of tight junction (TJ) seals. Modulation of CL‐4‐based TJ‐seals is a potent strategy for non‐invasive drug delivery. The barrier function of CL‐4 is disrupted in intestinal inflammation. These findings strongly suggest that CL‐4 modulators are promising candidates for CL‐4– targeted drug development; however, CL‐4 modulators have never been fully developed. In the present study, we developed a simple screening system for CL‐4 modulators by using a reporter gene. We cloned the promoter region of the CL‐4 gene, and prepared cells that stably expressed the luciferase reporter gene driven by the CL‐4 promoter. Treatment of the cells with a CL‐4 inducer or repressor resulted in an increase or decrease, respectively, in the luciferase activity in the cells. These findings suggest that the reporter‐expressing cells are a potent screening system for modulators of CL‐4 expression. We used the cells to screen 86 chemicals for CL‐4 modulator activity, and identified several CL‐4 modulator candidates. Potassium carbonate is a CL‐4 repressor; and curcumin, thiabendazole and carotene are CL‐4 inducers. We demonstrated that these four chemicals also modulate the TJ‐barrier. Thus, this simple reporter system will be a useful tool for the development of CL‐4 modulators.
Purification of human cytochromes P450 and their interactions in vesicular lipid reconstituted systems
Authors: Park Ji Won , Reed James R. , Brignac-Huber Lauren M. , Cawley George F. , Eyer Marilyn , Backes Wayne L.
Published: June 2021

P450s are membrane‐bound proteins found in the endoplasmic reticulum, requiring interaction with NADPH‐cytochrome P450 reductase (CPR). Previously, using rabbit enzymes, we demonstrated that one P450 can influence the behavior of another P450 by forming P450•P450 complexes. The goal of this study was to determine (1) if similar P450•P450 complexes are found with human P450s, and (2) how these interactions are affected by membrane lipids. Human CYP1A2 and CYP3A4 were incorporated into different reconstituted systems at subsaturating CPR, and the activity of CYP1A2 and CYP3A4 was examined. Substrate metabolism by both CYP1A2 and CYP3A4 were sensitive to alterations in lipid composition, and may affect interactions among P450s. These interactions were examined by comparing simple systems, containing CPR and a single P450, and mixed systems contain multiple P450s in the same vesicles. Interestingly, when CYP1A2 and CYP3A4 were present in the same vesicles, an inhibition of CYP3A4 function was observed, consistent with studies with rabbit P450s. The result of altered function of CYP3A4 with CYP1A2 is consistent with P450•P450 complex formation. (Supported by NIH R01 ES004344 & P42 ES013648)
Identification and Characterization of Novel Arylamine NAcetyltransferase Small Molecule Inhibitors
Authors: Leggett Carmine S. , Doll Mark A. , States J. Christopher , Trent John O. , Hein David W.
Published: June 2021

Many environmental arylamines play a vital role in the etiology of cancer and require metabolic activation by acetylation. Arylamine N‐acetyltransferase 1 (NAT1), a xenobiotic metabolizing enzyme, catalyzes the N‐acetylation or O‐acetylation of arylamine carcinogens. O‐acetylation, which is preceded by cytochrome P450 N‐hydroxylation, results in nitrenium ions that form DNA adducts. If unrepaired, these adducts can lead to mutagenesis and initiate cancer. Molecular epidemiology studies suggest that NAT1 expression and genetic polymorphisms play an important role in individual risk for many cancers, including breast cancer. Studies also suggest that NAT1 may play an important role in cell growth and survival, as well as in cell invasion, which is a hallmark of metastatic cancer. The purpose of this study is to use NAT1 as a molecular target for the development of small molecule inhibitors for cancer prevention. We utilized in silico screening of 17 million compounds to identify those that interact with NAT active sites. A total of 150 lead compounds were identified, scored and ranked based on their active site association. Sixty of these compounds were tested for their ability to inhibit recombinant human NAT1. The IC50 values of the most efficacious inhibitors (2, 10, 11 and 16 ) of human NAT1 in vitro were >2 mM, and 0.75, 191, and 202 μM, respectively. The most potent inhibitor (10 ) was further tested for inhibition of N‐acetylation in situ in Chinese hamster ovary cells. The IC50 values of inhibitor 10 were 118 μM and 81.6 μM towards the N‐acetylation of p‐aminobenzoic acid and the arylamine carcinogen 4‐aminobiphenyl. We report the in silico identification and verification of compound 10 as a potential new small molecule for cancer prevention. [NIH/NIEHS (T32‐ ES011564) and UofL Clinical and Translational Sciences Program]
Activity of LUF6000 and LUF6096 as positive allosteric modulators (PAMs) for the A3 adenosine receptor (AR) is species‐dependent
Authors: Du Lili , Gao Zhan-Guo , van Veldhoven Jacobus P. D. , IJzerman Adriaan P. , Jacobson Kenneth A. , Auchampach John A.
Published: June 2021

PAMs including the imidazoquinolinamine derivative LUF6000 and the quinoline derivative LUF6096 have recently been discovered for the human A3AR. These agents increase the maximal efficacy of orthosteric agonists and slightly increase or have not effect on potency. Given that orthosteric pharmacology of the A3AR differs markedly among species, the goal of this study was to characterize the activity of LUF6000 and LUF6096 for the A3AR from non‐human species commonly used in preclinical drug research. Functional modulation was assessed with a [35S]GTPγS binding assay using membranes prepared from HEK 293 cells stably expressing recombinant A3ARs. We found that both LUF6000 and LUF6096 produced substantial enhancing activity with the canine and rabbit, but not with mouse or rat A3ARs. In responsive species, both modulators increased the maximal efficacy of the partial A3AR agonist Cl‐IB‐MECA as well as the native ligand adenosine more than two‐fold. A decrease in potency was detected with the canine (but not the rabbit) A3AR explained by an effect of the modulators to decrease orthosteric binding affinity determined in equilibrium displacement binding assays with the orthosteric agonist radioligand [125I]I‐AB‐MECA. Our results reveal that allosteric pharmacology of the A3AR is species‐dependent. Supported by the NIH (R01HL077707 and NIDDK Intramural Research Program).
Preparation of a broad‐specific claudin binder by using Clostridium perfringens enterotoxin
Authors: Suzuki Hidehiko , Takahashi Azusa , Matsushita Kyohei , Li Xiangru , Tsujino Hirofumi , Watari Akihiro , Kondoh Masuo , Aoyama Hiroshi , Uno Tadayuki , Yagi Kiyohito
Published: June 2021

Claudins (CLs), tetra‐transmembrane proteins, are potent targets for drug development. However, development of CL binders has been delayed because of their low antigenicities. A C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE) is the most characterized CL binder. We previously developed a screening system for CL binders by using a C‐CPE mutant library and CL‐displaying baculovirus (CL‐BV), and used this system to identify a novel CL‐1 binder (m19). In the present study, we characterized the mode of binding of m19. Functional domain mapping of m19 revealed that substitution of three amino acids in C‐CPE was critical for the CL‐1 binding function of m19. We showed that m19 bound not only to CL‐1 but also to CL‐2 and ‐5. These findings suggest that m19 is a broad‐specific CL binder, the first reported to date. The X‐ray structural analysis of m19 showed that the structure of the putative CL‐binding region of m19 is similar to that of C‐CPE. In contrast, the electrostatic maps differed between C‐CPE and m19. The putative CL‐binding region of m19 is more positively charged than that of C‐CPE, and the putative C‐CPE binding regions of CL‐1, ‐2 and ‐5 are more negatively charged than that of CL‐4, suggesting that electrostatic forces are involved in the interaction between m19 and CLs. These findings will contribute to the further development of CL binders for CL‐targeted drug development.
SRJ23, a potent inducer of cell cycle arrest and apoptosis in prostate cancer cells
Authors: Wong Hui Chyn , Sagineedu Sreenivasa Rao , Loke Seng Cheong , Lajis Nordin Haji , Stanslas Johnson
Published: June 2021

SRJ23 exhibited selectivity against prostate cancer cells in the USA NCI in vitro anticancer screen (Jada et al., 2008. Br J Pharmacol 155: 641–654). MTT assay was used in assessing in vitro growth inhibition of compounds against PC‐3, DU‐145 and LNCaP cells. Flow cytometry was used to analyse cell cycle distribution whereas fluorescence microscopy was performed to determine morphological cell death. DNA fragmentation and annexin V‐FITC/PI flow cytometry were performed to confirm apoptosis induced by SRJ23. Quantitation of cell cycle and apoptotic proteins were determined by immunoblotting. SRJ23 exerted selectivity towards PC‐3 cells. Mechanistically, SRJ23‐treated PC‐3 cells down‐regulated CDK1 without affecting levels of CDK4 and cyclin D1 to induce G2/M arrest which led to apoptosis. Apoptosis was further confirmed by DNA fragmentation and annexin V‐FITC which displayed inhibition in the presence of caspase 8 inhibitor (Z‐IETD‐FMK). Apoptosis was associated with increase in caspase 8 expression and activation. This thought to have induced cleavage of Bid into t‐Bid. Additionally, expression and activation of caspase 9 and Bax proteins were apparent, with concomitant down‐regulation of Bcl‐2 protein. Taken together, these support the potential of SRJ23 as a new anti‐prostate cancer agent. This research project is supported by Universiti Putra Malaysia via RUGS grant scheme.
Distinct roles of mitochondria‐ and ER‐localized Bcl‐XL in apoptosis resistance and Ca2+ homeostasis
Authors: Eno Colins Ogwandi , Eckenrode Emily F , Olberding Kristen E , Zhao Guoping , White Karl , Li Chi
Published: June 2021

Bcl‐2 proteins are major regulators of cellular responses to intrinsic and extrinsic apoptotic stimuli. Among them, increased expression of the anti‐apoptotic protein Bcl‐XL is involved in tumorogenesis of human prostate, colorectal, and intestinal cancer cells. Although Bcl‐XL has been shown to modulate intracellular Ca2+ homeostasis and organelle‐specific apoptotic signaling pathways, its specific activities at mitochondria and the endoplasmic reticulum (ER) have not been fully defined. To explore how endogenous Bcl‐XL functions to regulate apoptosis, we generated mouse embryonic fibroblast (MEF) cell lines deficient in Bcl‐XL expression (Bcl‐XL‐KO). Deficiency in Bcl‐XL expression did not induce compensatory changes in the expression of other Bcl‐2 proteins. Bcl‐X‐KO cells showed increased sensitivity to various apoptotic stimuli compared with wild‐type cells. When expressed at the endogenous Bcl‐XL level, mitochondria‐targeted but not ER‐targeted Bcl‐XL restored apoptosis protection of Bcl‐XL‐KO cells to that observed in wild‐type cells. Despite lacking effect on apoptosis, expression of ER‐targeted Bcl‐XL was required to restore Ca2+ homeostasis in Bcl‐ XL‐KO cells. These data indicate that Bcl‐XL localized to mitochondria and the ER can serve to regulate apoptosis and ER Ca2+ homeostasis independently. Overall, our studies improve our understanding of how Bcl‐XL regulates apoptosis, which is crucial to develop new anti‐cancer therapeutic strategies to induce apoptosis in Bcl‐XL‐expressing tumor cells.
Systemic adenosine‐induced impairment of endothelial glycocalyx barrier properties is associated with a reduction in functional capillary density in hindlimb skeletal muscle of rats
Authors: van Haare Judith , Vink Hans , Eskens Bart , Cleutjens Jack , Cobelens Hanneke , van Teeffelen Jurgen
Published: June 2021

Objective

To determine the effect of systemic adenosine infusion on dextran accessibility in capillaries of rat skeletal muscle.

Methods

A bolus of fluorescently‐labeled small (40 kDa) dextrans (Dex40) and large (2000 kDa) dextrans (Dex2000) was i.v. infused in anesthetized rats. This was done in control rats (N=4) and in rats that received continuous adenosine infusion (2 mg/ml; 1.5 ml/h) (N=3) for 15 minutes. 3 minutes after dextran infusion, calf muscles were removed and sectioned. Images were captured using fluorescence microscopy. For both tracers, the number as well as the diameter of tracer‐filled capillaries was determined.

Results

In control rats the diameter of Dex2000 filling in capillaries was smaller compared to Dex40, 3.27 ± 0.05 μm versus 3.48 ± 0.08 μm (MEAN ± SEM). During adenosine infusion, diameters for both tracers did not differ anymore, 3.24 ± 0.24 (Dex40) versus 3.34 ± 0.27 μm (Dex2000). The number of dextran perfused capillaries in control animals was smaller for Dex2000 than for Dex40, 357 ± 31 versus 658 ± 51 and decreased more for Dex2000 (270 ± 22, P<0.05) than for Dex40 (572 ± 60, NS) during adenosine infusion.

Conclusion

Glycocalyx recruitment during systemic adenosine infusion in rats is associated with a decrease in vascular anatomic volume in resting hindlimb muscle.

Sponsored by Dutch Heart Foundation (NHS2009B56)
Capillary pericytes in cochlear lateral wall express strong Kir current and electrical coupling suggest its role in K+‐mediated local blood flow regulation
Authors: Yang Yu-Qin , Nuttall Alfred L , Jiang Zhi-Gen
Published: June 2021

Microvessels in spiral ligament (SL) and strial vascularis (SV) are densely populated with pericytes (PC) but their functioning mechanisms are not well understood. Using capillary segments isolated from guinea pig cochlear lateral wall and whole‐cell recording techniques, we found the PCs showed a robust inward K+‐current, which would play a key role in regulation of local blood flow.

Results

1) With physiological solutions, the PCs, but not the endothelial cells (EC), showed a Ba2+‐sensitive inward rectification in I/V domain <‐60 mV. [K+]o 20 mM enhanced the rectification and induced a ~15 mV hyperpolarization. 2) The PCs’ input conductance and capacitance indicated an 18β‐glycyrrhetinic acid‐sensitive electrical coupling. 3) TEA and 4‐AP showed little effect on IV relations. 4) La3+ and niflumic acid (0.1 mM) slightly reduced I/V slope with a net current reversal potential ~−15 and 0 mV, respectively. 5) Acetylcholine, norepinephrine, nitrendipine and glipizide caused no significant change in I/Vs or membrane potentials in the PCs. We conclude that local physio‐pathological [K+]o fluctuation can act on Kir and regulates PC's membrane potential and thus its motor tone. By this mechanism and the spreading effect of gap junction coupling, the PCs may sense the level of K+ recycling driven by hair cell acoustic transduction thus to adjust the vasotone and lateral wall blood flow. In addition, cholinergic and adrenergic neuro factors may play little role in the microcirculation control at PC level. Supported by NIH grants R01 DC004716 (ZGJ) and DC00105 (ALN).
Endothelial bioeffects from acoustic droplet vaporization for gas embolotherapy
Authors: Bull Joseph L , Seda Robinson , Samuel Stan , Fowlkes J. Brian
Published: June 2021

The dynamics of vascular microbubbles within the microcirculation is examined using a combination of experiments and theoretical analyses. This work is motivated by a developmental gas embolotherapy technique for cancer treatment by infarction of tumors with selectively formed gas bubbles. Additionally, the transport component of this work is relevant to air embolism. The bioeffects resulting from acoustic droplet vaporization (ADV) of a superheated dodecafluoropentane microdroplet is investigated using endothelial cells cultured in Opticells and in vivo in the rat cremaster muscle. The subsequent transport and lodging of microbubbles are examined in the cremaster. Acoustic pressures sufficient for ADV, but below the threshold for inertial cavitation of the resulting bubbles results in smaller damage regions in the Opticell experiments. The onset of inertial cavitation results in a larger region of endothelial bioeffects. The occurrence of bioeffects in the in vivo studies is found to depend on the size vessel in which the droplets are vaporized, with smaller vessels (relative to the initial droplet radius) leading to larger bioeffects. The subsequent lodging of the bubbles agrees with predictions from our mathematical models.

This work is supported by NIH grant R01EB006476.
Erythrocytes from Older Healthy Humans Fail to Release ATP during Hemoglobin Deoxygenation
Authors: Kirby Brett S , Garcia Leora J , Crecelius Anne R , Richards Jennifer C , Luckasen Gary J , Larson Dennis G , Dinenno Frank A
Published: June 2021

Deoxygenation of human erythrocytes (red blood cells; RBCs) from young healthy adults stimulates the release of vasoactive ATP in proportion to the number of unoccupied hemoglobin (Hb) binding sites. Aging humans have diminished plasma [ATP] and impaired skeletal muscle vasodilation to stimuli in which RBCs become deoxygenated (systemic hypoxia and exercise). Therefore, we tested the hypothesis that RBCs from older vs young adults release less ATP during Hb deoxygenation. RBCs from young (n=8, age=27±1 yrs, BMI=23±1 kg/m2, HbA1c=5.1±0.1%) and older (n=8, age=66±3, BMI=26±1, HbA1c=5.1±0.1) subjects were exposed to 16% O2 (NORM) and two levels of low O2 (HYP1 = 3% O2 and HYP2 = 1.5% O2) to facilitate Hb deoxygenation. No difference across age was observed for the variables of pH, pO2, pCO2, and the fraction of oxyHb (FO2Hb) within each condition. ATP (nmol/4•108 RBCs) release from RBCs of young adults increased by 71% during HYP1 (FO2Hb = ~71%) and by 205% during HYP2 (FO2Hb = ~43%) from NORM (FO2Hb = ~95%). In contrast, RBCs from older adults did not significantly release ATP during either HYP condition (Δ 35% and 43%; P<0.05 vs young). Intracellular ATP was not different with subject age. We conclude that RBCs from humans of advanced age fail to release ATP during Hb deoxygenation, and thus may explain the impaired plasma [ATP] and vasodilation during hypoxemia with age.

Support: HL102720
Oxidized lipid derived aldehyde, 4‐hydroxy‐trans‐2‐nonenal causes vascular inflammation by inducing endoplasmic reticulum stress
Authors: Sithu Srinivas D , Vladykovskaya Elena , Haberzettl Petra , Wickramasinghe Nalinie S , Agarwal Abhinav , McCracken James , Dougherty Susan M , Gordon Sharon A , Merchant Michael L , Hill Bradford G , Schuschke Dale A , DˈSouza Stanley E , Bhatnagar Aruni , Srivastava Sanjay
Published: June 2021

Oxidized lipoproteins‐derived aldehydes, such as 4‐hydroxy‐trans‐2‐nonenal (HNE) increase endothelial activation, however, mechanisms that mediate the effects of lipid oxidation products on the endothelium remain unclear. We observed HNE modified ER proteins in atherosclerotic lesions of apoE‐null mice. In vitro, treatment of endothelial cells (EC) and macrophages with HNE led to the modification of several proteins that colocalize with the endoplasmic reticulum (ER). HNE also caused the splicing of XBP‐1, an increase in the phosphorylation of PERK and eIF2á, and induction of ATF3 and ATF4. Phosphorylation of eIF2á by HNE was prevented by transfection with PERK siRNA. Treatment with HNE was also associated with a significant increase in the expression of ER chaperones, GRP78 and HERP. Pre‐treatment with a chemical chaperone, phenylbutyric acid (PBA) or adenoviral transfection with ATF6 attenuated HNE‐induced monocyte adhesion and IL‐8 induction. IL‐8 induction was also prevented by IRE1 siRNA. Moreover, PBA and taurine‐conjugated ursodeoxycholic acid (TUDCA) attenuated HNE‐induced leukocyte rolling and their firm adhesion to the endothelium in rat cremaster muscle. These data suggest that endothelial activation by HNE is mediated in part by ER stress. ER stress, induced by oxidized lipids may be a significant cause of vascular inflammation during atherogenesis.
Polyamine supplementation reduces oxidative stress and reverses vascular endothelial dysfunction with aging
Authors: Gioscia-Ryan Rachel A. , Zigler Melanie C. , Seals Douglas R. , Sindler Amy L. , LaRocca Thomas J.
Published: June 2021

Aging is associated with endothelial dysfunction, mediated in part by increased oxidative stress. Polyamines (PA) are natural biomolecules with antioxidant effects that also activate autophagy, a cellular damage repair process important for oxidative stress defense. To determine if PA therapy improves endothelial function with aging, we studied endothelium‐dependent dilation (EDD) to acetylcholine in carotid arteries of young (Y: 6 mo, n=7), old (O: 28–29 mo, n=7) and old PA‐treated (OPA: 29 mo, n=7; 4 weeks 3 mM spermidine drinking water) male C57/BL6 mice. Max EDD was reduced with age (O: 79 ± 5% vs. Y: 95 ± 2%, p<0.05), but PA treatment restored EDD to levels similar to Y (OPA: 99 ± 1%, p<0.05 vs. O). EDD was similar among groups in the presence of the superoxide dismutase mimetic TEMPOL, suggesting that the observed differences were superoxide‐mediated. Accordingly, aging was accompanied by increased aortic superoxide production (EPR, O: 4886 ± 630 vs. Y: 2701 ± 516 AU, p<0.05) and greater expression (western blot) of oxidative stress marker nitrotyrosine (NT, + 1.96‐fold vs. Y, p<0.05) and pro‐oxidant enzyme NADPH oxidase (+1.55‐fold vs. Y, p<0.05). PA treatment normalized superoxide production (OPA: 3296 ± 582 AU, p<0.05 vs. O) and reduced NT and NADPH oxidase expression (both p<0.05, OPA vs. O). Further, PA treatment increased expression of the antioxidant enzyme EC‐SOD (OPA: +3.52‐fold vs. Y, p<0.05) and autophagy markers LC3‐II and LAMP‐2a (both p<0.05, OPA vs. O). PA may be a novel treatment for age‐related endothelial dysfunction by reducing superoxide‐associated oxidative stress and stimulating antioxidant defenses, including autophagy. NIH AG013038, AG039210
A physiological role of p38 on aldosterone‐dependent regulation of ENaC endocytosis in renal epithelial A6 cells
Authors: Niisato Naomi , Marunaka Yoshinori
Published: June 2021

Aldosterone (ALD) is a crucial hormone to regulate ENaC‐mediated Na+ reabsorption in the kidney for controlling ECF volume and blood pressure. So far, we found ALD activated p38 and pretreatment with SB202190 (a specific p38 inhibitor: SB) abolished ALD‐stimulated benzamil‐sensitive Isc (INa ) by reducing benzamil‐sensitive conductance (GNa). In this study, we investigated a physiological role of p38 in the surface expression of ENaC through exocytic and endocytic pathways in ALD‐treated renal epithelial A6 cells by using MG132 (a proteasome inhibitor) and brefeldin A (an inhibitor of protein translocation). Pretreatment with MG132 mostly recovered the SB‐induced reduction of INa, whreas brefeldin A showed further an inhibitory effect on the SB‐decreased INa. Moreover, SB decreased both phosphorylated SGK1 and Nedd4‐2. These suggest that the p38‐ mediated signal pathway suppresses the endocytic/proteasome‐dependent ENaC degradation contributing to the stimulation of Na+ reabsorption by ALD. Supported by JSPS (19590212, 20390060), SSRF (0837, 1035) and FFPR.
Functional analysis of CLCN5 mutations in patients with Dent's disease
Authors: Grand Teddy , Teulon Jacques , Lourdel Stéphane
Published: June 2021

Dent's disease is an X‐linked renal disease characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, which can be due to loss‐of‐function mutations in the CLCN5 gene encoding the ClC‐5 2Cl−/H+ exchanger. Here, we investigated three published CLCN5 mutations: E267A, S270G and S270R (Hoopes et al., Kidney Int., 2004; Igarashi et al., Kidney Int., 1998).

We studied wild‐type (WT) and ClC‐5 mutants in X. laevis oocytes using two‐electrode voltage‐clamp for measuring currents and chemiluminescence for evaluating surface expression. Protein expression and subcellular localization was investigated in HEK293 cells using western blots and immunocytochemistry.

No significant current was recorded with the S270R mutant. The E267A and S270G mutants produced 20–50% lower currents as compared to WT. We found no difference between these mutants and WT ClC‐5 in terms of subcellular targeting and protein expression levels. They trafficked normally to the cell surface and to the early endosomes and displayed complex N‐glycosylation like WT ClC‐5.

Our study shows that these CLCN5 mutations considerably alter electrical activity of ClC‐5.
Endogenous and pharmacological inhibition of calpain decreases migration of hypoxic pulmonary arterial smooth muscle cells (PASMCs)
Authors: Maylor Julie , Zaiman Ari , Shimoda Larissa
Published: June 2021

Prolonged exposure to hypoxia causes pulmonary hypertension due to contraction, proliferation and migration of PASMCs, which result from hypoxia‐induced elevations in basal intracellular calcium concentration ([Ca2+]i). Calpain, a Ca2+‐dependent cysteine protease, has two major isoforms, μ‐ and m‐calpain, that require μM and mM [Ca2+]i, respectively, for activation. Calpain is known to participate in fibroblast and endothelial cell migration and proliferation, and PASMCs exhibit increased calpain levels during hypoxia (Ma et al, JCI 2011). In this study, we hypothesized that reducing calpain activity through in vivo overexpression of calpastatin (CAST), an endogenous calpain inhibitor, or in vitro inhibition with the pharmacological agent, calpeptin, would impair migration of PASMCs. For in vivo experiments, wild type (WT) and CAST overexpressing mice were exposed to normoxia or chronic hypoxia (10% O2; 21 d). For in vitro experiments, PASMCs were isolated from WT or CAST mice before exposure to normoxia or hypoxia (4% O2; 24 h). PASMC migration was measured using a modified Boyden chamber transwell assay. For both in vitro and in vivo experiments, WT, but not CAST, PASMCs exhibited increased migration in response to hypoxia. In WT PASMCs, calpeptin prevented hypoxia‐induced migration. These results suggest that calpain activity is necessary to facilitate the migration of hypoxic PASMCs.
Mice with targeted deletion of NBCe2 are hypertensive
Authors: Damkier Helle H , Rojek Alexandra , Christensen Henriette L , Iversen Nina K , Füchtbauer Ernst-Martin , Wang Tobias , Praetorius Jeppe
Published: June 2021

Single nucleotide polymorphisms in the electrogenic Na+ HCO3− cotransporter NBCe2 gene are associated with human hypertension. NBCe2 mRNA has been shown in many tissues including brain and kidney, all though the function of the transporter in these tissues remains unknown. The aim of this study was to determine whether deletion of NBCe2 causes hypertension in mice and to investigate whether the hypertension is salt sensitive. A full NBCe2 knockout mouse (NBCe2 ko) was generated and validated by quantitative PCR analysis of kidney and brain tissue. Tail‐cuff blood pressure measurements over a 10 day period revealed that the NBCe2 ko mice were hypertensive compared to wildtype (wt) mice (MAP (mmHg): 117±2 in NBCe2 ko mice, 83±−2 in wt ). Telemetry measurements confirmed this difference in blood pressure. Preliminary data suggest that 4% dietary Na+lead to an increase in blood pressure in both genotypes, while 0.02% Na+ diet decreased blood pressure in both genotypes. In conclusion, deletion of NBCe2 in mice causes increased systemic blood pressure. The hypertension is augmented by increasing dietary sodium, but is not abolished by low sodium diet. The mechanism causing the phenotype requires further investigation. Supported by the Lundbeck Foundation.
Hydration state of bats may explain frequency of arousals from torpor
Authors: Ben-Hamo Miriam , Muñoz-Garcia Agustí , Korine Carmi , Pinshow Berry
Published: June 2021

Bats hibernate to cope with unfavorable conditions, such as low ambient temperature (Ta) and low food availability. However, hibernation is frequently interrupted by arousals that are believed to be responsible for more than 85% of the animal's winter energy expenditure. To date, arousal in bats has been associated with either variation in body temperature (Tb), in Ta, or in both. Considering either Tb or Ta to be the cue to arousal has led to a single testable prediction, i.e. that torpor bout length (TBL) is negatively correlated with Ta and Tb. It is now known that Ta and Tb are both correlated with TBL, but correlations alone cannot establish a causal link between arousal and Tb or Ta. Since hydration state too has been implicated in arousal from hibernation, we hypothesized that water loss while hibernating creates the need to restore body water and electrolyte balance, and bats arouse to drink. We measured the TBL of bats (Pipistrellus kuhlii) kept in different conditions of humidity, but at the same Ta. We found an inverse relationship between TBL and total evaporative water loss that is independent of metabolic rate, thus supporting the hypothesis that hydration state is a cue to arousal in bats.
A Novel pH Conditioned Cl‐ Conductance in Nodose Ganglia Neurons
Authors: Wang Runping , Whiteis Carol A. , Benson Christopher J. , Chapleau Mark W. , Abboud François M.
Published: June 2021

pH sensitivity has been studied extensively in spinal afferents of dorsal root ganglia neurons but rarely in vagal afferents of nodose ganglia (NG) neurons. Here, we report a large and prolonged inward current evoked following 2 or 3 brief (10s) exposures to low pH (6.0) in 16 of 22 (70%) isolated nodose neurons using whole‐cell patch‐clamp technique. This pH‐conditioned current (pH‐I) reached its peak of 904.3±159.9pA (n=15) at ~5 minutes and returned to baseline within 10–15 minutes. Replacing Ca2+ and Na+ in the extracellular solution with NMDG or intracellular K+ with Cs+ did not significantly decrease the current. However pH‐I was eliminated by using a low Cl− (4mM) pipette solution. The I‐V plot showed a reversal potential of ~0mV with equivalent intra‐and extracellular [Cl]− (133mM) in the absence of permeable cations. Replacing [Cl]o− with aspartate shifted the reversal potential to a more positive voltage. pH‐I was reduced by tamoxifen, an inhibitor of “swell‐activated Cl− current” (SAC), but not by niflumic acid (blocker of the Ca2+ activated Cl− channel). Adding hypoosmotic solution, which activates SAC, did not further increase peak pH‐I. Conversely evoking the pH‐I did not increase the SAC current. Opening of this pH conditioned Cl− conductance may contribute to sustained depolarization of vagal afferents and potentiate a beneficial reflex sympathoinhibition during myocardial and gastrointestinal ischemia/acidosis (NIH‐HL14388).
Neuronal uptake and subcellular localization of functional nanoformulated copper/zinc superoxide dismutase (SOD nano)
Authors: Rosenbaugh Erin G , Manickam Devika S. , Batrakova Elena V , Kabanov Alexander V , Zimmerman Matthew C
Published: June 2021

Dysregulation of angiotensin II (AngII) signaling and elevated superoxide (O2 •−) in the brain are associated with cardiovascular diseases, such as hypertension. To improve brain delivery of the O2 •− scavenging enzyme SOD1, we developed SOD nano, a polyion complex composed of a polyethylene glycol corona and polyethyleneimine core that electrostatically binds SOD1 protein. We hypothesized that SOD nano delivers functional SOD to neurons via active endocytosis. Using fluorescently‐labeled SOD1 and CATH.a neurons, we observed a significant increase in the neuronal uptake of SOD nano (70 ± 1 arbitrary fluorescent units, AFU) versus free SOD (22 ± 1 AFU, P<0.05) with 6 h incubation. SOD nano uptake was inhibited by chloropromazine, a clathrin endocytosis inhibitor (14 ± 0.6 AFU, P<0.05 vs. SOD nano), and nystatin, a caveolae endocytosis inhibitor (28 ± 1 AFU, P<0.05 vs. SOD nano). SOD nano co‐localized in lysosomes (38.5%), endosomes (17.1%), mitochondria (9.8%), golgi (7.8%) and ER (5.2%). Electron paramagnetic resonance (EPR) studies show that the AngII‐induced O2 •− in neurons (88 ± 10 EPR arbitrary units, EAU) was attenuated to pre‐AngII levels with 6 h SOD nano (66 ± 11 EAU) but not free SOD protein incubation (73 ± 13 EAU). These data indicate that SOD nano is internalized by neurons through clathrin and caveolae endocytosis and localizes to various organelles where it scavenges AngII‐generated O2 •−.

NIH F31 NS073398
Crucial role of phospholipase C gamma 2 in hypoxic pulmonary vasoconstriction
Authors: Yadav Vishal Rambharos , Zheng Yun-Min , Wang Yong-Xiao
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) may ensure the physiological regional alveolar ventilation‐pulmonary perfusion matching, but can also serve as a key pathological mechanism leading to pulmonary hypertension and associated right ventricular failure. An increase in [Ca2+]i in pulmonary artery smooth muscle cells (PASMCs) is vital for HPV. However, the underlying molecular mechanisms remain incompletely understood. We have found that acute hypoxia significantly increases the activity of total phospholipase C (PLC) in isolated, endothelium‐denuded mouse resistance pulmonary arteries. The activity of PLCγ2 is largely augmented as well following hypoxic exposure. The PLC inhibitor U73122 (1 μM) greatly diminishes the hypoxia‐induced increase in [Ca2+]i in PASMCs, whereas U73433 (1 μM), an inactive analog of U73122, has no effect. 2‐aminoethoxydiphenyl borate (2‐APB) and xestospongin C to block inositol trisphosphate receptors (IP3Rs) also inhibit the hypoxic Ca2+ response. Excitingly, shRNA‐mediated PLCγ2 gene silencing diminishes the hypoxia‐induced increase in [Ca2+]i. Using myxothiazol to inhibit mitochondrial ROS production blocks the hypoxic activation of PLCγ2. In conclusion, hypoxia can specifically activate PLCγ2 and then IP3Rs to increase [Ca2+]i in PASMCs by enhancing mitochondrial ROS production, which may play an important role in HPV and hypoxic pulmonary hypertension. This study was supported by AHA EIA 0340160N and NIH R01 HL64043, HL064043‐S1, and HL075190.
Identification of a regulatory region within the EMX2 locus that is active in the neural tube
Authors: Schober Jared P. , Pira Charmaine U. , Oberg Kerby C.
Published: June 2021

Development requires regulation of essential genes to ensure functional morphology. Conservation of the developmental pathways across species suggests retention of regulatory mechanisms. Thus, conserved non‐coding regions (CNR) may play a role in the genetic regulatory process. EMX2 is a homeodomain transcription factor critical to the development of the central nervous system (CNS). We have previously identified EMX2 as a target of LMX1B, a transcription factor required for brain development and. We report on a CNR ~ 335 kb downstream of EMX2 (CNRZ1), that is highly conserved across species and that also contains putative Lmx1b binding sites (FLAT sequences). We hypothesize that CNRZ1 may play a role in regulating EMX2, possibly mediated by LMX1B binding. To test enhancer function, we isolated CNRZ1 by PCR and ligated it into a plasmid containing a thymidine kinase minimal promoter linked to a GFP reporter. The plasmid was introduced into chick embryos by electroporation (EP). Whole embryo EP was performed on Hamburger and Hamilton (HH) stage 4 chick embryos for CNS evaluation. Fluorescence was visible by microscopy in the neural tube beginning at 12 hrs post‐EP (stage 6), with the strongest signal observed at 48 hrs (stage 10–11). Co‐localization of CNRZ1 enhancer activity with EMX2 and LMX1B expression in the neural tube implicates CNRZ1 as an enhancer for EMX2 that is potentially regulated by LMX1B.

Grant Funding Source : National Institutes of Child Health and Human Development