bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vascular Remodeling with Reduced Renal Mass
Authors: Adam Ryan J. , Marks Amanda , Kriegel Alison J. 
Published: April 2020

Maintenance of vascular anatomy is critical for the kidneys to perform their chief function of blood filtration. It is then no surprise that compromises in kidney vascular anatomy and function are associated with chronic kidney disease (CKD), which afflicts >15% of the US population. Rat renal remnant models of CKD, including 1/3 and 5/6 nephrectomies (1/3NX, 5/6NX, respectively), recapitulate many elements of human CKD. We have observed from prior studies that remnant kidneys of 5/6NX rats spontaneously undergo remarkable hypertrophic remodeling, and by 10 weeks post NX are, on average, larger than Sham NX, intact kidneys. In contrast, the 1/3NX remnant kidney (2/3 of mass excised with contralateral kidney intact) atrophies over time. These disparate observations of whole‐organ remodeling must occur concomitantly with commensurate differences in vascular remodeling. Careful examination of differences in vascular remodeling in these two CKD models may uncover important molecular pathways and mechanisms underlying alterations in renal function. The first step toward realization of this goal, and the primary aim of this study, was to anatomically characterize the vasculature of the remnant kidneys from 1/3NX and 5/6NX rats.Methods1/3NX, 5/6NX, and Sham surgeries were performed on 10 week old male Sprague Dawley rats. Gross vascular anatomy was assessed 10 weeks post NX using two approaches. The first was in‐vivo assessment of major renal vessels with using Doppler ultrasound imaging of the short axis. The second was morphometric analysis of microfil kidney vascular cast photos.ResultsDoppler imaging revealed a reduction in renal blood flow through large vessels in the 1/3NX remnant kidney. The 5/6NX rats demonstrated pronounced vascular remodeling, with both increased diameter of larger vessels and turbulent flow. Quantitative analysis of microfil photos indicates that 1/3NX remnant kidneys have, on average, reduced vessel‐area fraction (FAV, area of photo occupied by vessels/area of tissue) compared to Sham (0.96 ± 0.2 vs. 0.53 ± 0.15; mean ± SEM), and that 5/6NX kidneys have reduced FAV compared to 1/3NX remnant kidneys (0.24 ± 0.06). Sham animal microfil casts extended to the capsule, while the mean distance from the most superficial casted vessels was greater in 1/3NX remnant kidney (0.20 ± 0.13 mm) and much greater in 5/6NX kidneys (1.57 ± 0.32 mm).ConclusionThese observations demonstrate pronounced anatomical differences between 1/3, 5/6, and Sham NX rat kidney vasculature, including apparent differences in peripheral vessel density. These anatomical differences were accompanied by functional differences in blood flow. Thus, these rat models of CKD appear to recapitulate elements of the vessel rarefaction/injury of human CKD, which is associated with hypoxia, fibrosis, tubular damage, and functional loss. Improved understanding of these anatomical changes and underlying molecular pathways may reveal novel treatment approaches.Support or Funding Information1R01HL128332‐01A1Figure 1
Investigating the CYP450‐Catalyzed Metabolism of Cannabidiol and Its Implications for Drug Safety
Authors: Murray Jessica L. , Nardone-White Dasean , Jackson Klarissa D. 
Published: April 2020

Cannabidiol (CBD) is a naturally occurring phytocannabinoid derived from Cannabis sativa. It was recently FDA‐approved as Epidiolex® for the treatment of seizures associated with Lennox‐Gastaut or Dravet syndrome in patients aged two and older. CBD’s rise in popularity among consumer products and supplements has led to an increase in safety events and concerns for toxicity. Epidiolex® is known to cause dose‐dependent hepatotoxicity, and monitoring of serum transaminases and bilirubin is recommended for six months following treatment initiation. Cannabidiol is reported to undergo hepatic metabolism by cytochromes P450 (CYP) 2C19 and CYP3A4, and UDP‐glucuronosyltransferases (UGT) 1A7, UGT1A9, and UGT2B7, respectively. However, the relative enzyme contributions have not been described. The objectives of this project were to determine the specific roles of P450 and UGT enzymes in CBD metabolism with respect to formation of the active metabolite 7‐hydroxy‐CBD (7‐OH‐CBD) and glucuronides, and to investigate the effect of CYP2C19 activity on formation of 7‐OH‐CBD in human liver microsomes. Cannabidiol (1 μM) was incubated with pooled human liver microsomes (0.2 mg/mL protein) in the presence and absence of enzyme cofactors NADPH and UDPGA over time (0–60 min). Cannabidiol and its 7‐hydroxy metabolite were detected and quantified using LC‐MS/MS. The results from substrate depletion studies indicated that P450 enzymes were faster than UGT enzymes in metabolizing CBD (depletion rate constant = 0.12 min−1 with NADPH versus 0.05 min−1 with UDPGA). Formation of 7‐OH‐CBD increased rapidly in incubations supplemented with NADPH, followed by a gradual reduction in metabolite generation. Based on substrate depletion data in the presence of UDPGA, UGTs appeared to play a slightly lesser role in CBD metabolism. To determine the influence of CYP2C19 activity on CBD metabolism, a sample of individual CYP2C19‐genotyped human liver microsomes with poor, rapid, and ultrarapid metabolizing phenotypes were incubated with CBD (1 μM) for 0–30 min. Interestingly, CYP2C19 genotype appeared to influence CBD metabolite generation, with CYP2C19 poor metabolizers producing significantly less 7‐OH‐CBD compared to rapid and ultrarapid metabolizers. Future work will investigate the metabolism, transport, and toxicity of cannabidiol in human hepatocytes.Support or Funding InformationThis work is supported by the UNC‐Chapel Hill Royster Society of Fellows and the UNC Eshelman School of Pharmacy Faculty Start‐Up Funds.Oxidative Metabolism of CannabidiolFigure 1
Short bowel upregulates oxalate and citrate transporters and increases CaOx crystal deposition in the rat hyperoxaluric kidney
Authors: Ma Ming-Chieh , Tseng Yi-Shiou 
Published: April 2020

Short bowel (SB) increases the risk of kidney stones. However, the underlying mechanism related to intrarenal changes is unclear. Here we examine SB may affect oxalate and citrate handlings on calcium oxalate (CaOx) crystal formation in the hyperoxaluric kidney. SB was induced by small intestine resection in male Wistar rats. Sham‐operation was performed the same surgery without resection. After 7 days of recovery, rats were then fed with 0.75% ethylene glycol (EG and SB+EG groups) for 28 days as a hyperoxaluric induction. The corresponding controls were fed with distilled water (control and SB groups). Blood and 24 h of urine were collected for analysis. Renal CaOx deposition was examined by Pizzolato’s staining. Oxalate transporters Slc26a2 and Slc26a6, and citrate transporter sodium‐dependent dicarboxylate cotransporter NaDC‐1 at their mRNA levels were examined (n=6 in each group). After 7 days of SB, hypocalciuria and hypocitraturia were found when compared with those in sham‐operated control rats. These were associated with hypocalcemia and low renal NaDC‐1 expression after 28 days. SB aggravated EG‐mediated CaOx crystal nephropathy by fostering hyperoxaluria, degree of supersaturation, and CaOx crystal deposition. Renal expression of Slc26a6 but not Slc26a2 was increased in SB+EG group when compared to those in EG or SB group. SB lowered NaDC‐1 expression, but it was reversed in SB+EG group. Intestinal expression of these transporters was similar among groups. We conclude that SB enhances CaOx crystal formation in the hyperoxaluric kidney. More oxalate and less citrate secretion due to Slc26a6 upregulation and insufficient NaDC‐1 expression, respectively, possibly contribute to the aggravated effect of SB on crystal formation.Support or Funding InformationThis research was supported by grants of the Far Eastern Memorial Hospital (FEMH‐2018‐C‐046, Tseng YS and Ma MC) and the Ministry of Science and Technology (MOST108‐2314‐B‐030‐004‐MY3, Ma MC), Taiwan.Hyperoxaluria and hypocitraturia in the short bowl (SB)‐treated hyperoxlauric rats.More prominent hyperoxaluria (A) and oxalate transporter Slc26a6 upregulation (B) was seen in the SB+EG group. Hypocitraturia were found in the SB group (C), this associated with citrate transporter NaDC‐1 downregulation (D). SB increased CaOx formation in the hyperoxaluric kidney (E). N=6 in each group. *P<0.05, EG vs. C group; #P<0.05, SB+EG vs. EG group; @P<0.05, SB+EG vs. SB groupFigure 1
Deduced Roles of Cardiac 14‐3‐3 Protein Interactome in Heart Metabolism, Protein Synthesis and Proteostasis
Authors: Qu Jia-Hua , Tarasov Kirill V. , Chakir Khalid , Tarasova Yelena S. , Lakatta Edward G. 
Published: April 2020

The 14‐3‐3 protein family is known to interact with many proteins in non‐cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis, and the 14‐3‐3 network is a therapeutic target of critical metabolic and proteostatic signalings in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14‐3‐3 protein interactome nor potential functional roles of 14‐3‐3 protein‐protein interactions (PPIs) has been explored in heart. Here, we evaluated both RNA expression and protein abundance of 14‐3‐3 isoforms in mouse left ventricle, followed by co‐immunoprecipitation of 14‐3‐3 proteins and mass spectrometry in left ventricle. We identified 594 proteins comprising the cardiac 14‐3‐3 interactome. Immunofluorescence assays indicated that 14‐3‐3 proteins localize in mitochondria and ribosome. Multiple bioinformatic analyses indicated that more than half of proteins bound to 14‐3‐3 are related to mitochondria, and the deduced functions of the mitochondrial 14‐3‐3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I and V, and to regulate cardiac fatty acid metabolism via interactions with enzymes within mitochondrial matrix. 14‐3‐3 protein binding to ribosomal proteins may triage energy production to coordinate protein synthesis and protein quality control. In summary, we establish the comprehensive landscape of cardiac 14‐3‐3 PPIs, dissecting its structure, deducing its function to have a major role to regulate cardiac metabolic homeostasis and proteostasis. Because 14‐3‐3 binds to proteins that are crucial to cardiac functions and heart health, the cardiac 14‐3‐3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy.Support or Funding InformationIntramural Research Program, National Institute on Aging, National Institutes of HealthConduction of 14‐3‐3 interactomeFigure 1Deduced roles of 14‐3‐3 protein‐protein interactions in metabolism and maintenance of cardiac homeostasisFigure 2
Investigating the Effects of Epoxy Fatty Acids against LPS and Pesticide Induced Toxicity
Authors: Atone Jogen , Hwang Sung Hee , Hammock Bruce D. 
Published: April 2020

Neuroinflammation is a hallmark of many neurodegenerative diseases including Parkinson’s Disease (PD) and Alzheimer’s Disease (AD), and mitigating neuroinflammation could avert the progression of these disorders. While it is commonly known that inflammation is propagated by COX metabolites of arachidonic acid (ARA) called prostaglandins, it is less known that metabolites of ARA produced by CYP450 called epoxyeicosatrienoic acids (EETs) have been shown to have an anti‐inflammatory and anti‐apoptotic effect in many disease models. Previous studies have demonstrated that increasing the biological availability of EETs by inhibiting soluble epoxide hydrolases (sEH) reduces MPTP or paraquat induced neuroinflammation and prevents the loss of dopaminergic neurons caused by these toxicants. However, the mechanisms underlying such effect is still unknown. In this study, we have treated primary rat astrocyte mixtures with LPS to determine how EETs can affect regulation of growth factors such as its BDNF, GDNF, and VEGF‐A/B. In addition, primary microglia culture was treated with LPS to determine whether microglial activation can be mitigated through the addition of EETs. Finally, N27 dopaminergic neurons were treated with a pesticide rotenone, and the effect of EETs on the survival of dopaminergic neurons were assessed. We have found that EETs and/or sEH inhibition can affect all cells in ways that are be neuroprotective against neuroinflammatory disorders such as Parkinson’s Disease, with reduced inlammatory marker, increased growth factor signaling, and increased neural viability. Future studies may investigate how inhibition of sEH can be used to combat PD in human patients.Support or Funding InformationThis work was supported by the National Institute of Environmental Health Sciences (NIEHS) Grant R01 ES002710, NIEHS Superfund Research Program P42 ES004699, and NIEHS T32 ES007059 (J.A).EETs and sEH inhibitors cause reduction in rotenone induced loss of N27 cell viability.Figure 1EETs and sEH inhibitor along with LPS stimulate growth factor signaling and reduce LPS induced IL‐1b induction.Figure 2
Optimizing the Fracture Repair Mouse Model by Using Different Sizes of Femoral Defects
Authors: Chang Chih-Hsiang , Hu Chih-Chien , Chang Yuhan , Sia Kee-Chin , Ueng Steve W. N. , Chen Mei-Feng 
Published: April 2020

Although mouse models are crucial in orthopedic studies, no study has explored the bone healing process in mice through continuous imaging. Thus, we optimized a mouse model for femoral defect by continually monitoring the bone healing process of different‐sized femoral defects. We observe bone healing images and quantify the trabecular bone value up to 1 month after surgery. In the 0.3‐mm group, significant bone bridge formation and spongy bone trabeculae were found on the day 14 after the defect operation. The bone healed on day 21 with the formation of external callus and internal callus structures and transformed to a remodeled compact bone on day 28. In the 1‐mm group, bone bridge formation occurred on day 21 after surgery. Moreover, bone healing was observed on day 28 with the formation of external callus and internal callus structures. Mice in the 1.5‐mm group were prone to fracture during feeding after bone defect surgery. The fluorescent staining results of osterix and cathepsin K also revealed that the bone formation process in the 0.3‐mm group was faster than that in the 1‐mm group. This study optimized a bone healing mouse model that can be applied for developing new strategies for accelerating bone healing.Support or Funding InformationDuring the study period, the authors received funding support from the Ministry of Science and Technology, Taiwan (grant numbers MOST 107‐2314‐B‐182A‐036‐MY3 and MOST 108‐2320‐B‐182A‐020 ‐MY3), and the Chang Gung Memorial Hospital (grant numbers CRRPG3H1311, CRRPG3H1312, CRRPG3H1313, CMRPG3F2011, CMRPG3F2012, CMRPG3F2013). The funders had no role in study design, data collection, data interpretation and analysis, decision to publish, or preparation of the manuscript.Continuous observation of the bone healing process in the 0.3‐mm bone defect group of the mouse femoral bone.Images of 3D micro‐computed tomography (CT) angiography as well as hematoxylin and eosin (H&E)‐stained and Safranin O (SFO)‐stained histopathological slides. Immunofluorescence staining of osterix and cathepsin K indicates osteoblast and osteoclast signals, respectively.Figure 1
Human Angiotensin‐(1‐12) [Ang‐(1‐12)] is a Hypertension and Cardiac Disease Biomarker
Authors: Ferrario Carlos Maria , Groban Leanne , Iyer Seethalakshmi R. , Ahmad Sarfaraz , Wright Kendra N. , Burnett John C. 
Published: April 2020

BackgroundAng‐(1‐12) is a primary non‐renin dependent substrate for direct tissue angiotensin II (Ang II) formation by chymase. This discovery challenges conventional thinking and explains the large residual risk of cardiovascular events in patients medicated with blockers of the renin angiotensin system (RAS). Here we report the first demonstration of circulating Ang‐(1‐12) as a biomarker of essential hypertension (EH) and cardio‐renal remodeling.MethodsThe cohort of patients from Olmsted County MN included 52 healthy adult volunteers (21 females), age 54 ± 8 years, and 23 essential hypertensive (EH) patients (14 females) age 61 ± 8 years with systolic blood pressure (SBP) ≥140 mm Hg prior to recruitment. Clinical examination, standardized BP measurements, and venous blood were obtained for measurements of plasma concentrations of angiotensinogen (AGT), Ang‐(1‐12), Ang II, aldosterone, renin activity, N‐terminal pro–B‐type natriuretic peptide (NT‐proBNP) and atrial and brain natriuretic peptides (ANP and BNP, respectively). All values are Means ± SD. Data were analyzed by 2‐way ANOVA for sex and hypertension.ResultsPlasma Ang‐(1‐12) levels in EHs [SBP: 134 ± 14 mm Hg; Ang‐(1‐12): 1,463 ± 446] were higher than normotensives [SBP: 117 ± 13 mm Hg; Ang‐(1‐12): 1,353 ± 379 fmol/mL, hypertension effect: P<0.05], irrespective of sex. Hypertension associated with increases in pulse pressure, PRA, and plasma AGT and NT‐proBNP concentrations (see Figure
). The elevation in circulating NT‐proBNP levels with hypertension was accounted for by a 4.6‐fold increase in hypertensive males (165 ± 134 pg/mL) while the 1.5‐fold rise in plasm NT‐proBNP did reach significance (134 ± 127 pg/mL, p> 0.05).ConclusionsWe show for the first time that Ang‐(1‐12) circulates in humans and its concentrations are elevated by chronic hypertension. We further demonstrate a sexual dimorphic pattern in hypertensives with respect to NT‐proBNP, the latter a biomarker for cardiac remodeling. Whether this represents a natriuretic system‐related compensatory mechanism, or worsening pathology, due to an elevated noncanonical RAS specific to male hypertensives remains to be studied. Further investigation to fully define the role of Ang‐(1‐12) as a cardiac disease biomarker in a larger, gender and ethnic diverse population and its relationship to hypertension and myocardial structure is warranted.Support or Funding InformationSupported by grant HL‐051952 from the NHLBI of the NIHLines are means ± SDFigure 1
Exercise promotes satellite cell contribution to myofibers in a load‐dependent manner
Authors: D’Hulst Gommaar , Masschelein Evi , Palmer Andrew , Bar-Nur Ori , De Bock Katrien 
Published: April 2020

Satellite cells (SCs) are required for muscle repair following injury and are involved in muscle remodeling during exercise. Increased muscular activity stimulates SC accumulation and function, but to what extend alternations in mechanical load control SC fusion remains to be elucidated. To investigate SC contribution to myofibers during exercise training, we labeled SCs by excising tdTomato (mT) in Pax7CreERT2/+; Rosa26mTmG/+ mice, resulting in an mEGFP (mG) labeling of all PAX7+ cells and derived myofibers (Figure
1A). Using this genetic lineage tracing model, we here show that SCs extensively contribute to myofibers during exercise and that this occurs exclusively in hindlimb plantar flexors. Upon 8‐weeks of voluntary free running (VRun), 60% of all m. soleus myofibers had at least one SC fused (mG+). Moreover, adding load to the wheel (VResRun) increased the number of mG+ myofibers to 80%, indicating that SC fusion during exercise is load dependent (Figure
1B). Since only the VResRun showed substantial muscle hypertrophy in both m. soleus and m. plantaris, this data suggests that next to hypertrophy, also endurance‐training adaptions are mirrored by extensive SC fusion. Finally, exercise training induced a shift towards an oxidative fiber type, but SC fusion was similar in oxidative and glycolytic fibers. Collectively, we show that voluntary running strongly induces SC fusion in both free and resistance running conditions, but the latter only induced muscle hypertrophy. Thus, our findings suggest that SC fusion cannot only serve hypertrophy, since we also observed significant contribution under ‘endurance’ settings.Support or Funding InformationInternal funding ETH ZurichExercise induces satellite cell fusion in a load‐dependent manner. (A) mouse model and experimental protocol. (B) mG expression in mouse hindlimb muscle under sedentary (control) and running (VRun and VResRun) conditions. *** p<0.001, ** p<0.01.Figure 1
Codon Optimization of Gene Editing CRISPR‐SaCas9 Plasmid Increases Protein Expression in Human Liver Cells to Boost in vivo Therapeutic Knockout Application Efficiency
Authors: Madejski Irene R. , Sundaresan Avirath , Groshong Tyler L. , Holmes Grace D. , Gupta Ishir G. , De Luke 
Published: April 2020

The CRISPR‐Cas9 gene editing technology shows great promise in revolutionizing the treatment of genetic disease; however, the current focus on treatment by ex vivo delivery is limited to blood diseases. In vivo treatment can substantially extend the clinical reach of the technology, but is limited by a challenging delivery process: the large size of the Cas9 gene (~4500bp) inhibits delivery by the small viral vectors and lipid nanoparticles used. We propose that a codon‐optimized saCas9 plasmid will lead to significantly higher protein expression than the presently used genes; this can compensate for the low delivery efficiency and has great potential to improve the viability of in vivo CRISPR‐Cas9 therapies. Here we present Western Blot fluorescence data showing that optimization leads to increased Cas9 production and subsequently increased CRISPR‐Cas9 editing efficiency.A commonly used parent saCas9 sequence was selected from Addgene’s public plasmid database. A novel, codon‐optimized gene sequence was engineered from the parent using GenScript’s OptimumGene™ algorithm. Both the optimized gene and parent gene were transfected into human HepG2 liver cells, and Western Blot/ELISA protein quantification of the optimized plasmid compared with three sample plasmids demonstrated that the codon‐optimized gene expressed the saCas9 protein at ~3.5× the rate of the commercial sequence. The same set of optimized and commercial plasmids were then transfected into GFP‐stable HepG2 cells in parallel with GFP‐specific sgRNA; the cells transfected with optimized saCas9 demonstrated a significantly higher GFP knockout rate than cells transfected with commercially available control plasmids, as measured through GFP detection stay and Western Blot on GFP proteins. The augmented Cas9 protein expression supported by the novel saCas9 sequence can better enable in vivo CRISPR‐Cas9 treatments.Support or Funding InformationThis research was supported by The Nueva School.ELISA protein quantification assay indicated that codon‐optimized SaCas9 displayed a 3.17‐fold and a 3.5‐fold increase in expression over its parent gene in Sample Group 1 and Sample Group 2, respectively.Figure 1