bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of processing, cooking, and storage on β‐carotene retention and bioaccessibility in biofortified cassava (Manihot esculenta) (646.4)
Authors: La Frano Michael , Zhu Chenghao , Burri Betty 
Published: June 2021

Biofortification of cassava with β‐carotene is currently being tested in African populations where cassava is a staple food and vitamin A deficiency is a public health problem. Measuring the impact of traditional African processing and cooking on β‐carotene concentration and bioaccessibility of cassava‐based foods can help direct plant breeding efforts and estimate the potential impact of biofortified cassava on vitamin A status. Biofortified cassava was processed and cooked to prepare the following traditional dishes: gari, eba, fermented and non‐fermented fufu, and boiled cassava. Eba, non‐fermented fufu, and gari retained the most β‐carotene (43.6, 42.2, and 41.0%, respectively) while boiled cassava and fermented fufu retained the least (24.9, and 21.7%, respectively). Gari and boiled cassava bioaccessibility was 17‐ and 12‐fold higher than raw cassava, respectively (P < 0.001), while increases in bioaccessible β‐carotene concentrations in eba, and fufu products were not significant. Unfortunately, after just three days of storage at room temperature fermented flour and gari preparations lost >62% β‐carotene, while nonfermented flour was more stable. These results suggest that biofortified cassava prepared as gari or boiled might provide useful amounts of β‐carotene to alleviate vitamin A deficiency, if storage conditions are optimized.Grant Funding Source: Supported by HarvestPlus
Soybean cake as a source of stable bioactive compounds (647.43)
Authors: Silva Fabrício , Silva Cecília , Torres Alexandre , Teodoro Anderson , Monteiro Mariana , Perrone Daniel 
Published: June 2021

Soybean cake is a byproduct of soybean oil processing rich in bioactive compounds. In this study we evaluated the effect of soybean cake extracts on cell viability, cycle and apoptosis in human colon adenocarcinoma cell line (HT‐29) and their stability under different storage conditions. Soybean cake from soybean oil industry (SCI) and produced under laboratory conditions (SCL) were extracted with a ternary mixture (water:ethanol:ethyl acetate), lyophilized and incubated with HT‐29 cells at concentrations (10 to 400 mg/g) from 6 to 96 h. The dry extracts were stored in freezer or room temperature (light or dark) and the following analysis were conducted at regular intervals during 120 days: spectrophotometric assays for phenolic compounds, flavonoids and saponins; antioxidant capacity (AC) by FRAP and TEAC assays; isoflavones and soyasaponins by LC‐DAD‐MS. SCL and SCI extracts showed high levels of phenolic compounds (12.36 and 10.45 mg gallic acid/g), flavonoids (7.24 and 9.98 mg genistein/g), saponins (224.86 and 253.99 mg/g) and AC (FRAP: 71.35 and 91.57 µmol Fe+2/g; TEAC: 105.02 and 103.90 µmol Trolox/g). LC‐DAD‐MS analysis revealed that SCI extract presented higher levels of β‐glycosylated isoflavones (3.67 mg/g) than SCL (0.72 mg/g). Aglycone isoflavones were present in higher levels in SCL extract (3.16 mg/g) than in SCI (0.82 mg/g). Both extracts reduced in a time‐dose dependent fashion cell viability when compared to control at concentrations starting from 100 mg/g. These reductions ranged from 4% to 84% and from 24% to 85% when cells were incubated with SCL and SCI, respectively. After incubation with SCL a decrease in the percentage of cells at G0/G1 phase and an increase in G2/M phase was observed, as well as an increase in the apoptosis rate. After 120 days of storage, bioactive compounds’ levels did not change, with the exception of FRAP values. Extracts showed anticancer effects in vitro and soybean cake is a potential source of stable bioactive compounds for in vivo studies.Grant Funding Source: FAPERJ, CNPq, CAPES, UFRJ
Concussive injury elicits JNK‐mediated human astrocyte retraction (651.10)
Authors: Augustine Claudia , Cepinskas Gediminas , Fraser Douglas 
Published: June 2021

Background: The pathology of traumatic brain injury (TBI) is poorly understood, but likely involves blood‐brain barrier (BBB) dysfunction. The BBB is lined with microvascular endothelial cells, ensheathed by astrocyte endfeet. We investigated human astrocytes and endothelial cell responses to concussive injury in vitro.
Methods: Concussive injury in vitro was elicited by using a Cell Injury Controller II, a device which induces regulated and repeatable pulse of gas pressure to deform a flexible membrane. Human astrocytes (HA) or human immortalized cerebral microvascular endothelial cells (hCMEC/D3) grown on flexible culture membranes were subjected to a single (20, 35, or 55% deformation) or repeated (20% deformation; 6‐consecutive injuries spanned every 2 minutes) concussive injuries. Cells were assayed for morphological changes (retraction), detachment/cell death, and MAPK activation (phosphorylation of JNK1/2, ERK1/2, and p38).Results: Significant HA retraction was elicited 30 min following either a single (55% deformation) or repeated concussive injury. HA retraction was not associated with cell death or detachment. HA recovered morphologically after 2 hrs post‐injury. Concussive injury increased phospho‐JNK1/2, but not phospho‐ERK1/2 or phospho‐p38. Pre‐treatment of HA with JNK inhibitors (SP600125 or SU3327) reduced injury‐induced HA retraction. In contrast, concussive injury failed to induce either morphological changes or MAPK activation in hCMEC/D3 Conclusion: Concussive injury induces JNK‐mediated astrocyte retraction in vitro, while sparing morphological changes in endothelial cells. In vivo, astrocyte retraction from endothelial cells may result in BBB dysfunction. JNK inhibition may be a potential therapeutic target for TBI.Grant Funding Source: Supported by the Children's Health Foundation (London ON) and HSFO Grant NA6914
Characterization of electroporation for unbiased delivery of small molecules in high throughput screening applications (654.13)
Authors: Pihl Johan , Karlsson Anna , Karlsson Mattias , Lardell Susanne , Karila Paul 
Published: June 2021

In cell‐based high throughput screening applications, the assumption is typically made that all molecules have equal and complete access to the target of interest. In the case of intracellular targets, this is most likely not the case, as the molecules in a diverse screening library will exhibit varying degrees of cell permeability, or even lack thereof. This is likely to result in in false negatives.Electroporation is the use of brief electric pulses to overcome the barrier of the cell membrane. By the application of external electric fields, transient and reversible breakdown of the membrane can be induced. This transient state can be used to load cells with a variety of different molecules. Initially developed for gene transfer, electroporation can be used for delivery of a variety of other molecules ranging from ions to drugs, dyes, tracers and antibodies. Electroporation has proven useful both in vitro, in vivo and in patients, where drug delivery to tumors has been performed.In this work, we have explored the use of a high throughput electroporation platform, Cellaxess Elektra, for unbiased delivery of small molecules into cells in a cell‐based assay. The molecules selected were fluorescent and were representative of those that can be found in a small molecule library. The cell permeability with and without electroporation was characterized by means of fluorescence microscopy, and in the selected collection of ~400 compounds, 20 exhibited low or no cell permeability which could be enhanced by means of electroporation. We believe that this could prove to be a valuable tool in identifying families of new leads previously undiscovered.
BEZ235, a selective PI3k/mTOR inhibitor, enhances the therapeutic efficacy of doxorubicin in pancreatic cancer (655.7)
Authors: Durrant David , Das Anindita , Kukreja Rakesh 
Published: June 2021

Background: Pancreatic cancer is the fourth leading cause of cancer deaths in the U.S. and has the lowest relative survival rate (~6%) of all major cancers. Current treatment options have had limited success, therefore, patients are in desperate need for novel therapies which utilize both new drugs that target specific survival pathways as well as traditional therapeutics. Here we test whether BEZ235 (BEZ), a dual PI3k/mTOR inhibitor, in combination with doxorubicin (DOX) will enhance the killing of pancreatic cancer cells.Methods and results: MiaPaca2 cells were treated with BEZ and DOX either alone or in combination for 24 to 48 hours. Cell viability, measured using the MTS assay, showed a significant reduction of cells in the combination group compared to either drug alone (Fig. A). Western blot analysis showed an increase in cleaved‐PARP, a marker for apoptosis, and p‐chk2 corresponding to decreased signaling through the PI3k/mTOR pathway (Fig. B). Interestingly, BEZ caused increased intracellular DOX accumulation which also paralleled the increase in cleaved‐PARP (Fig. C). A xenograph tumor model using Athymic nude mice injected with MiaPaca2 cells showed enhanced in vivo efficacy when combining BEZ and DOX (Fig. D).Conclusion: Inhibition of survival signaling downstream of PI3k/mTOR with BEZ re‐sensitizes cells to the cytotoxic effects of DOX even in cancers not thought of to be dependent on this pathway for survival (i.e. KRAS mutants). That along with increased DOX accumulation results in enhanced cell killing. We propose that this unique combination could be a viable clinical option for patients with pancreatic cancer.$graphic_B6F300F1‐5061‐452E‐A64E‐B7890C8AC3AC$Grant Funding Source: Supported by AHA 13PRE16910038, NIH HL051045
Role of efficacy at the mu opioid receptor on the interactions between mu opioid receptor agonists and cannabinoid receptor agonists: antinociception and drug discrimination (658.4)
Authors: Maguire David , France Charles 
Published: June 2021

Cannabinoids such as Δ9‐tetrahydrocannabinol (THC) enhance some (analgesia) but not other (e.g., positive reinforcing) effects of mu opioid receptor agonists. Whether efficacy at the mu opioid receptor impacts those interactions is unknown. The effects of combining THC with agonists varying in efficacy at the mu opioid receptor were studied with antinociception and drug discrimination assays in rhesus monkeys. Etorphine, morphine, and nalbuphine increased tail withdrawal latency from warm water. THC increased the potency of high (etorphine) and moderate (morphine) efficacy agonists up to 16 fold but did not alter the potency of the low efficacy agonist nalbuphine. In monkeys discriminating fentanyl from saline, THC shifted the discrimination dose‐effect curves of etorphine, fentanyl, and nalbuphine rightward and downward. THC was 3‐fold more potent in attenuating the discriminative stimulus effects of the low efficacy agonist nalbuphine than those of the higher efficacy agonists etorphine and fentanyl. These data suggest the benefit of combining cannabinoids with opioids to treat pain is greatest with drugs having high efficacy at the mu opioid receptor and the subjective effects of high efficacy drugs are more difficult to disrupt than those of lower efficacy drugs. Given that other (abuse‐related) effects of opioids are not enhanced, these data support combining opioids with cannabinoids to treat pain.Grant Funding Source: Supported by NIH grants R01DA05018, K05DA017918 (CPF), T32DA031115 (DRM), and F32DA035605 (DRM).
Subcellular enrichment of calreticulin at myoendothelial junctions (664.2)
Authors: Biwer Lauren , Straub Adam , Isakson Brant 
Published: June 2021

In the walls of resistance arteries, cellular projections from primarily endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). The MEJ is a unique signaling microdomain responsible for multiple components of vasoreactivity. In MEJs, endoplasmic reticulum (ER) is often present; the significance of this has not been directly addressed. Using iTRAQ proteomic analysis, we discovered that calreticulin, a 50kDa calcium binding protein typically found in the ER, is highly enriched in the soluble fractions (i.e., ER) isolated from MEJs in a vascular cell co‐culture (VCCC). In the insoluble fraction (i.e., non‐ER, caveolin‐enriched lipid rafts), calreticulin was present in EC and SMC, but not MEJs. Using immuno‐TEM, mouse thoracodorsal arteries showed higher expression of calreticulin in EC (3.0 ± 0.76 beads/um2) and MEJ (2.3 ± 0.74 beads/um2) compared to SMC (0.6 ± 0.29 beads/um2). In the MEJ, beads could be identified within the ER. In order to determine a functional relevance of MEJ‐enriched calreticulin in soluble fractions, we successfully transfected EC in the VCCC with calreticulin siRNA. Next, SMC were stimulated with phenylephrine (PE) and cyclic guanosine monophosphate (cGMP) was measured. This method is known to produce a significant increase in SMC cGMP via inositol 1,4,5‐triphosphate‐based release of Ca2+ from ER at the MEJ, with subsequent activation of MEJ localized endothelial nitric oxide synthase (eNOS). The knockdown of calreticulin in EC/MEJ significantly decreased cGMP in SMC after PE stimulation. The mechanism behind this may be an interaction of calreticulin with SERCA that leads to less ER uptake of calcium, diminishing the localized calcium mobilization from the ER at the MEJ upon PE stimulation. Thus, we hypothesize that where the calreticulin is localized in cellular compartments, and not overall calreticulin expression, could be critical to MEJ function.Grant Funding Source: Supported by NHLBI 2 T32 HL007284‐36
AKAP150‐dependent cooperative TRPV4 channel gating is central to endothelium‐dependent vasodilation and is disrupted in hypertension (678.10)
Authors: Sonkusare Swapnil , Dalsgaard Thomas , Bonev Adrian , Hill‐Eubanks David , Kotlikoff Michael , Santana Luis , Scott John , Nelson Mark 
Published: June 2021

Endothelial cell (EC) dysfunction is a hallmark of hypertension. We recently discovered that local Ca2+ influx through clusters of functionally coupled TRPV4 channels (detected optically as “TRPV4 sparklets”) drives physiological vasodilation. Here, we show that stimulation of EC muscarinic receptors activated TRPV4 sparklets exclusively at discrete sites at myoendothelial projections (MEPs)—specialized regions of ECs that contact adjacent smooth muscle cells. This activation was dependent on protein kinase C (PKC) and was absent in mice lacking the PKC‐anchoring protein AKAP150 (A‐kinase anchoring protein 150), which was localized predominantly to MEPs. Cooperative gating of TRPV4 channels within a cluster amplified Ca2+ influx at MEPs by more than 2‐fold. This cooperativity was largely absent at non‐MEP sites and was virtually eliminated by chelation of intracellular Ca2+ or AKAP150 knockout, suggesting AKAP150‐dependent potentiation of TRPV4 activity by Ca2+ influx via adjacent channels. Notably, MEP‐localization of AKAP150 was disrupted in angiotensin II‐induced hypertension, leading to a complete loss of muscarinic activation of TRPV4 channels, much weaker coupling among TRPV4 channels at MEPs, and approximately an 80% reduction in carbachol‐induced vasodilation. Our results support the concept that endothelial‐dependent vasodilation of resistance arteries is enabled by MEP‐localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and coupled channel gating necessary for efficient communication of the endothelium to the smooth muscle cells in arteries—a molecular configuration that is disrupted in hypertension.Grant Funding Source: Supported by HL044455, 1P01HL095488, R37DK053832, R01HL098243 to MTN
Female mice are more sensitive to obesity‐induced vascular dysfunction than males (680.14)
Authors: Le Huy , Huby Anne‐Cecile , Anderson Ruchi , Belin de Chantemele Eric 
Published: June 2021

Over the last two decades, a three fold increased in the prevalence of cardiovascular disease (CVD) has been reported in premenopausal women and attributed to the recent epidemic of obesity. While the mechanisms involved in obesity‐related CVD are beginning to be understood in males they remain unknown and unstudied in females. We tested the hypothesis that obesity negates the protection of women from CVD by submitting 6‐week old male and female Balb/C mice to a control (CD) or a high fat diet (HFD) for 12 weeks. On CD, females were smaller than males, but exhibited higher adiposity, fasted glycemia and leptin levels than males. On HFD, females had a higher food intake (HFDF: 3.1±0.5 vs. HFDM: 2.7±0.2g/day) but displayed lower increases in body weight (HFDF: +15% vs. HFDM: +35%), adiposity (HFDF: +12% vs. HFDM: +190%), glycemia and leptin levels than males, suggesting that female mice have a higher metabolic rate than males. Aortic ring reactivity was studied via wire myography at the end of the diet. Consistent with the human literature, at baseline, females displayed a better endothelial function than males (CDF: 82±3 vs. CDM: 49±3%, p<0.05). However 12 weeks of HFD impaired endothelial function (12% reduction in relaxation) and reduced the contractility of the aortic rings to phenylephrine (‐35%), serotonin (‐14%) and depolarization (‐13%), in female mice only, suggesting that females are more sensitive to obesity‐induced vascular dysfunction than males. While reduction in vascular contractility might initially protect females from hypertension, reduced endothelial function might predispose females to atherosclerosis and further cardiovascular events.Grant Funding Source: 11SDG5060006
Activation of P2X‐purinergic receptors in the lateral parabrachial nucleus increases renal sympathetic nerve discharge and mean arterial pressure in anesthetized rats (686.13)
Authors: Menezes Miguel , Favero Michele , Yamamoto Kenta , Menani Jose , de Paula Patricia , Mifflin Steve 
Published: June 2021

The lateral parabrachial nucleus (LPBN) is an important area involved in cardiorespiratory control. ATP is an important neurotransmitter and purinergic receptors are present into LPBN. The involvement of purinergic mechanisms into LPBN in the control of mean arterial pressure (MAP) and renal sympathetic nerve discharge (RSND) is unknown. We investigated the effect of alpha, beta‐me ATP (P2X agonist) alone or following pyridoxalphosphate‐6‐azophenyl‐2',4'‐disulfonic acid (PPADS, P2 antagonist) injected into LPBN on MAP and RSND in rats. Anesthetized, vagotomized and mechanically ventilated male rats (n=3) were used. Unilateral injections of alpha, beta‐me ATP (2.0 nmol/50 nl) into LPBN increased MAP (Δ 12±2 mmHg, p<0.05) and RSND (Δ 32±8%), without changing heart rate (HR). Unilateral injection of PPADS (0.125 nmol/50 nl) into LPBN did not change MAP, HR and RSND, however, pre‐treatment with PPADS into LPBN (10 min before) abolished the pressor response (Δ 2±0.8 mmHg, p<0.05) and reduced the RSND increase (Δ 12±7%, p<0.05) produced by alpha, beta‐me ATP injected into LPBN. In 2 rats responses to alpha, beta‐me ATP recovered to control within 30 min while in the remaining rat the response had recovered 31% by 70 min. These results suggest that alpha, beta‐me ATP acting on P2X purinergic receptors into LPBN produces pressor and sympathoexcitatory responses in anesthetized rats.Grant Funding Source: CAPES (18906‐12‐5); FAPESP; CNPq
Role of O‐GlcNAcylation in regulating mitophagy in cardiomyocytes (693.14)
Authors: Wright JaLessa , Chatham John 
Published: June 2021

Acute increases in the post‐translational protein modification O‐linked‐N‐acetylglucosamine (O‐GlcNAc) have been shown to protect cardiomyocytes against oxidative stress and this has been associated with O‐GlcNAcylation of mitochondrial proteins. Mitophagy, a type of cargo‐mediated autophagy, protects against oxidative stress by removing damaged mitochondria. Therefore the goal of this study was to determine whether O‐GlcNAcylation plays a role in regulating mitophagy in AC16 immortalized human ventricular cardiomyocyte cells. Cells were treated with 2,3‐dimethoxy‐1,4‐napthoquinone (DMNQ, 20 µM), a redox cycling agent which increases mitochondrial reactive oxygen species (ROS) and FCCP (0.5 µg/mL), a mitochondrial uncoupler commonly used to induce mitophagy. Following 3 hour treatment with DMNQ or FCCP, O‐GlcNAc levels were decreased and this was associated with increased levels of total PINK1 and increased mitochondrial Parkin. Increasing basal O‐GlcNAc levels for 2 hours with thiamet‐G (3 µM) a highly selective inhibitor for O‐GlcNAcase, attenuated the loss of O‐GlcNAc induced by both DMNQ and FCCP treatment and appeared to increase basal PINK1 levels. These results support the concept that protein O‐GlcNAcylation plays a role in regulating mitophagy and that this could contribute to the protection against oxidative stress. Future studies will include quantifying the effects of DMNQ and thiamet‐G on mitochondrial function using XF24 Extracellular Flux Analyzer as well as determining whether PINK1 or Parkin are targets for O‐GlcNAcylation.Grant Funding Source: Supported by NIH grants HL101192 and HL110366
T cell mitochondrial ROS in development of hypertension (693.15)
Authors: Nazarewicz Rafal , Dikalova Anna , Bikineyeva Alfiya , Egnatchik Rob , Young Jamey , Harrison David , Dikalov Sergey 
Published: June 2021

T cell mitochondrial ROS in development of hypertension
Rafal R. Nazarewicz*, Anna Dikalova*, Alfiya Bikineyeva*, Rob Egnatchik#, Jamey Young#, David G. Harrison*, Sergey Dikalov* Division of Clinical Pharmacology*, Chemical and Biomolecular Engineering#, Vanderbilt University Medical Center, Nashville, TN Immunity plays a key role in the development of hypertension. Locally acting T cells secret pro‐inflammatory cytokines that have profound damaging effects on end‐organs and facilitate hypertension. Mitochondrial ROS may contribute to regulation of T cells activity and cytokine production by switching from aerobic mitochondria‐base metabolism to anaerobic glycolysis. The objective of this study was to establish the role of T cell mitochondrial ROS in development of hypertension. Increased mitochondrial superoxide and H2O2 in T cells from hypertensive mice was inhibited by overexpression of catalase in mitochondria or mitochondria targeted SOD mimetic mitoTEMPO. Angiotensin II‐induced hypertension led to impaired T cells respiration which was reversed by scavenging of mitochondrial H2O2. Despite impaired respiration, T cells from hypertensive mice synthesized more ATP compared to normotensive mice. Glycolysis was identified as the main source of ATP as indicated by increased lactate production. HPLC analysis showed higher glutamine uptake suggesting increased glutaminolysis. These metabolic changes facilitated T cell activity. Scavenging of mitochondrial superoxide prevented angiotensin II‐induced pro‐inflammatory cytokine production and ultimately hypertension in mice. Our data show that mitochondrial ROS‐dependent metabolic adaptation is necessary for T cell activation and their pro‐hypertensive responses. We suggest T cell mitochondrial ROS as a potential target for pharmacological intervention in hypertension.Grant Funding Source: Supported by National Institutes of Health grant R01HL094469.