bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optical Metabolic Imaging (OMI) of Toxoplasma gondii and Cryptosporidium parvum in a 3D system
Authors: Siamof Cerise Marie , Gallego-Lopez Gina , Knoll Laura 
Published: April 2020

Both T. gondii and C. parvum are intracellular, apicomplexa parasites auxotrophic for specific metabolites. Cat intestine is the definitive host of T. gondii, and cats can produce infectious oocysts, which infect humans primarily through fecal‐oral transmission. This infection can cause fatal encephalitis in immunocompromised humans. C. parvum is another intestinal parasite with a wider range of hosts who can excrete infectious oocysts in feces. These oocyts can cause fatal diarrheal illness in cows and humans, and C. parvum infection has been linked to colon cancer in humans. How T. gondii and C. parvum change host cell metabolism has not been well‐characterized to date. Currently, there are limited treatment options available for C. parvum, and the organism has been difficult thus far to culture and propagate in vitro.For this reason, mouse intestinal organoid models have been developed to simulate the conditions a parasite would encounter in vivo. It is hypothesized that the model for studying T. gondii in vitro can be easily transferred to C. parvum, given their similarities. Using mCherry fluorescent protein labelled T. gondii and cat intestinal organoids, we have modeled intestinal infection with T. gondii. The relative ease of molecular manipulations of T. gondii makes it the ideal organism with which to build a model for optical metabolic imaging (OMI) of C. parvum. OMI of mCherry‐labelled T. gondii in 2D human fibroblasts has been completed with inhibitors such as Mycocaloide B and Cytochalasin D, and the results showed changes in NADH and FAD metabolites in vitro, showing that infection with intracellular parasites impacts metabolism of host cells.T. gondii will be imaged in vitro with additional metabolic inhibitors, including atovaquone and sodium fluoroacetate. Then, OMI will be done of T. gondii in cat intestinal organoids, a 3D system, because the materials are already available and serve as a way to test the model prior to using it on C. parvum. It is anticipated that the previously obtained results will be seen again in the 3D system. This project will label C. parvum with mCherry fluorescent protein, propagate this microorganism in a 3D system and ultimately perform OMI. Thus, the model that has been successfully developed with T. gondii will be improved, then used to understand how infection with Cryptosporidium parasite influences host metabolism, particularly in organoid models. Ultimately, this better understanding will assist in the development of therapeutics for C. parvum, an area which is currently lacking.
Plasma potassium plays a major role in controlling the concentrating ability of the kidney
Authors: Al-Qusairi Lama , Zapf Ava , Grimm P. Richard , Welling Paul A. 
Published: April 2020

Hypokalemia can induce diabetes insipidus (DI), characterized by loss of free‐water reabsorption, loss of urine concentrating ability, polyuria and polydipsia. Although the disorder has been described for decades, the pathophysiological mechanisms remain poorly understood. In addition, it still unclear if there is a sexual dimorphism.In this study, we characterized hypokalemia‐induced DI over a spectrum of plasma potassium levels in mice fed a range of low potassium diets (LKD) between 0% and 0.11% K+, over 8 days compared to control diet (1%K+). We observed that 0%K+ and 0.026%K+ diets both induced hypokalemia, polyuria and a decrease in urine osmolality starting after 5 and 6 days, respectively. However, the 0.056%K diet induced only mild DI starting at day 7. Interestingly, the 0.11%K diet did not result in DI.We then assessed the DI in males and females mice fed with LKD (0%K+) or control diet for 8 days. After 3 days of LKD, both males and females exhibited decrease in urine osmolality which was more sever in females (−21%) than in males (−12%). Profound diuresis was observed at day 5 and it was more enhanced in females (5.2 +/−0.58 mL/day) than in males (3.0 +/−0.43 mL/day), which might be a consequence of the more severe hypokalemia in females compared to males (2.6 +/−0.29 versus 3.5 +/−0.08 mmol/L) after 8 days of LKD.Moreover, it has been shown that the urinary concentration defect occurs mainly in the inner medullary collecting duct (IMCD), involving a reduction in AQP2 channel expression. Recent studies localized AQP2 protein in the autophagosome in the IMCD of DI models suggesting the autophagic degradation of AQP2 under hypokalemia‐induced DI (Khositseth S., Sci rep 2015)(Kim WY., Sci rep 2019). We confirmed and extended this observation, finding that after 8 days of LKD, the ATG12‐Atg5‐Atg16 complex, the LC3II/LC3I ratio, and P62 were all increased in the medulla but no difference was detected by western blot in the cortex. However, we observed that AQP2 levels were more severely decreased in the cortex than in the medulla of the hypokalemic versus control mice suggesting that pathways other than autophagy are at play. Together, the data indicate: i) a tight relationship between loss of the renal concentrating ability and development of hypokalemia, ii) hypokalemia in response to dietary potassium deficiency is more sever in female mice than males, and as a consequence, females are more prone to develop DI than males, iii) the autophagy is induced in the medulla of the hypokalemic mice, iv) The hypokalemia‐induced decrease in AQP2 is correlated with induction of autophagy in the medulla but not in the cortex.Support or Funding InformationNIH
β3‐adrenoreceptor as a new player in sympathetic regulation of the acid‐base homeostasis in the kidney.
Authors: Milano Serena , Gerbino Andrea , Carmosino Monica , Svelto Maria , Procino Giuseppe 
Published: April 2020

Sympathetic nervous system has an important role in the regulation of renal function.We previously showed that, in mouse, the β3‐adrenoreceptor (β3‐AR) localizes in most of the nephron segments and its selective agonism promotes a potent antidiuretic effect.Here, we report the expression of β3‐AR in distal nephron intercalated cells (ICs). Co‐localization study of β3‐AR either with H+‐ATPase or Cl−/HCO3− exchanger pendrin in mouse kidney showed that the β3‐AR was expressed in H+‐secreting type A, in HCO3− secreting type B, and in non‐A non B ICs. To unveil a possible role of β3‐AR in systemic pH regulation, we analyzed the urine pH of wild type (wt) and β3‐AR knock‐out (ko) mice. We found that the urine pH of β3‐AR ko mice was significantly higher than wt mice. In line with these results, localization and expression analysis showed that the renal H+‐ATPase significantly decreased in β3‐AR ko mice compared to wt mice supporting the idea that H+ secretion is partially blunted in these animals. Next, we investigated in kidney cells (M1‐β3‐AR) the effects of β3‐AR stimulation on the activity of H+‐ATPase monitoring the intracellular pH with the pH‐sensitive dye BCECF.Of note, exposure of M1‐β3‐AR cells to a selective β3‐AR agonist induced a 2.5 fold increase of H+‐ATPase activity compared with resting cells and this effect was prevented by a selective β3‐AR antagonist or by the H+‐ATPase inhibitor bafilomycin. Moreover, β3‐AR agonism promoted H+‐ATPase apical expression in M1‐β3‐AR cells. In addition, the PKA inhibitor H89 abolished the stimulatory effect of β3‐AR agonism, demonstrating the involvement of the cAMP/PKA pathway. We conclude that the modulation of H+‐ATPase activity in renal ICs by endogenous β3‐AR agonists may play an addition physiological role in hormonal control of systemic acid‐base homeostasis.Support or Funding InformationTelethon (project # GGP15083)
Vascular and Blood Flow Responses to Acute Upper Limb Exercise: Impact of Sex and Menstrual Cycle Phase
Authors: Garten Ryan , Weggen Jennifer , Hogwood Austin , Decker Kevin , Darling Ashley 
Published: April 2020

PurposeThis study sought to determine whether fluctuations in estrogen levels throughout the menstrual cycle (early (EF) vs late follicular (EF) phase) altered the vascular and blood flow responses to acute upper limb exercise and if these responses were different when compared to males.MethodsTen young females (YF: 21±3 yrs) were recruited and completed two identical visits (EF and LF) consisting of an acute upper limb exercise bout (progressive, rhythmic handgrip). Peripheral (brachial artery (BA) diameter, shear rate, and blood flow) and central (mean arterial pressure, heart rate) hemodynamic measurements were obtained during the last minute of each 3‐minute handgrip workload (3kg, 6kg, and 9kg) via Doppler ultrasonography and finger plethysmography, respectively. Male controls (YM: n = 21; 21±3 yrs) matched for age completed one visit to provide a biological sex comparison. Data is presented as mean ± standard deviation.ResultsIn young females, brachial artery flow mediated dilation (controlled for shear rate) [EF (3kg: 0.006±0.01; 6kg: 0.015±0.01; 9kg: 0.022±0.01); LF (3kg: 0.008±0.01; 6kg: 0.017±0.01; 9kg: 0.024±0.01)], blood flow [EF (3kg: 162±61; 6kg: 236±74; 9kg: 332±104 ml.min−1); LF (3kg: 151±23; 6kg: 237±47; 9kg: 330±104 ml.min−1)], and vascular conductance [EF (3kg: 1.8±0.7; 6kg: 2.5±0.7; 9kg: 3.1±0.7 ml.min−1.mmHg−1); LF (3kg: 1.8±0.4; 6kg: 2.6±0.6; 9kg: 3.3±1.1 ml.min−1.mmHg−1)] were significantly increased with each progressive handgrip workload but were unaltered when comparing the EF and LF conditions. When compared to young males (YM), young females reported no differences in BA FMD [YF vs YM (3kg: 0.006±0.01 vs 0.007±0.01; 6kg: 0.015±0.01 vs 0.013±0.01; 9kg: 0.022±0.01 vs 0.018±0.01)], but did report significantly lower blood flow [YF vs YM (3kg: 162±61 vs 228±64; 6kg: 236±74 vs 347±94; 9kg: 332±104 vs 474±119 ml.min−1)] and vascular conductance [YF vs YM (3kg: 1.8±0.7 vs 2.5±0.7; 6kg: 2.5±0.7 vs 3.8±0.9; 9kg: 4.9±1.1 vs ml.min−1.mmHg−1)].ConclusionThe findings of this study revealed that fluctuations in estrogen throughout the menstrual cycle do not alter vascular and blood flow regulation during exercise, but, when compared to young males, young female’s exhibited lower blood flow/vascular conductance. Additional research is necessary to determine if these sex differences in blood flow/conductance represent adequate or inadequate blood flow regulation during exercise.
Properties of Lipid Rafts in Two Epigenetically Distinct Subtypes of the Oncogenic Cell‐line SW13
Authors: Pascual Agnes S. , Espejo Luis S. , Moore Jacob L. , Leyva Kathryn J. , Hull Elizabeth E. 
Published: April 2020

IntroductionMutant GOF p53 is an epigenetic regulator which promotes oncogenesis. Many cancers with a GOF mutation accumulate cholesterol and inhibition of cholesterol synthesis with statin treatment reverses some of the pro‐oncogenic properties of GOF p53. Our central hypothesis is that lipid raft signaling is altered by cholesterol accumulation in cells expressing a GOF p53. To address the role of cholesterol in promoting oncogenesis, we use the SW13 cell line bearing a GOF p53 (H193Y). This cell line has two epigenetically distinct cell subtypes that differentially express genes coding for GPI anchors, cholesterol biosynthesis, and sphingolipids, all of which are components of lipid rafts. In addition, the SW13 subtypes have different oncogenic profiles. The SW13(Vim−) subtype has an epithelial morphology and is highly proliferative. The SW13(Vim+) subtype has a mesenchymal like phenotype and a higher metastatic potential. When treated with histone deacetylase inhibitors (HDACi), SW13(Vim−) appear to adopt a SW13(Vim+) phenotype. Therefore, we treat with HDACi to control the subtype conversion and shRNA to knockdown GOF p53 expression. This experimental approach allows the composition of raft fraction to be correlated to GOF p53 expression and oncogenic properties of each SW13 subtype, with and without p53 expression.MethodsProliferation rate, MMP expression, and migration of SW13(Vim−), SW13(Vim+), shRNA SW13(Vim−), and shRNA SW13(Vim+) were quantitated by EdU assays and in situ zymography respectively. We have isolated lipid raft fractions by the detergent resistant enrichment technique from SW13 cells of each subtype, with and without GOF p53 expression knockdown by shRNA. Proteins were identified by mass spectrometry analysis. Fluorescent cholesterol assays and p53 western blots were used to quantify cholesterol levels and p53 protein expression, respectively.ResultsData suggest that there is a shift from planar rafts in the SW13(Vim−) to caveolar rafts in the SW13(Vim+) line. Although the contribution of GOF p53 to this shift is under study, evidence suggests that raft composition is altered in the shRNA lines, in which GOF p53 has been knocked down by ~90%. In addition, p53 knockdown increases the proliferation rate in SW13(Vim−) (p = 0.01) but does not affect SW13(Vim+). In contrast, p53 knockdown decreases MMP activity in SW13(Vim+) (p = 0.05) but not SW13(Vim−).ConclusionsOur data support the hypothesis that lipid raft signaling is significantly altered between the epigenetically distinct SW13 subtypes and that these differences may correlate to oncogenic potential. In addition, statin treatment, which reverses some of the pro oncogenic properties of GOF p53, may do so, in part, by altering raft composition and signaling.
Regulation of the PAH1‐encoded phosphatidate phosphatase during lipogenesis in the oleaginous yeast Yarrowia lipolytica
Authors: Carmon Taylor , Ukey Rahul , Fakas Stylianos 
Published: April 2020

The PAH1 gene encodes for phosphatidate phosphatase (PAP) that plays a vital role in lipid biosynthesis by catalyzing the conversion of phosphatidate (PA) to diacylglycerol (DAG) which is used to synthesize triacylglycerol (TAG). Previous research has shown that PAP activity is elevated during growth in media that induce lipogenesis. In this work, the PAH1‐encoded PAP activity and protein levels were analyzed in wild type and pah1Δ cells during lipogenesis. The results showed that the PAH1‐encoded PAP activity increased in a time‐dependent manner, but the Pah1 levels declined with time. Examination of the subcellular fractions revealed that Pah1 was almost entirely responsible for the membrane‐associated PAP activity. The lipid content of the wild type cells was higher than that of the pah1Δ cells, while the pah1Δ mutation did not have a major effect on the levels of DAG and TAG.Support or Funding InformationThis work was supported, in whole or in part, by National Institutes of Health Grant R15GM124602‐01 from the United States Public Health Service. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Optical clearing and 3D‐Imaging Reveals a Sexual Dimorphism in the Structure and Remodeling Response of the Distal Convoluted Tubule
Authors: Tahaei Seyedmohammad Ebrahim , Coleman Richard , Saritas Turgay , Ellsion David H. , Welling Paul A. 
Published: April 2020

IntroductionThe abundance of the distal convoluted tubule (DCT) thiazide‐sensitive sodium chloride cotransporter (NCC) is well appreciated to be greater in females than males. Because structural remodeling of the DCT is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in DCT morphology. Until now, this has never been directly examined. Here, combining new optical clearing techniques with staining for markers of DCT segments, state‐of‐the‐art high‐speed volumetric imaging and analysis tools we quantitatively assess DCT morphology in male and female mice, and study DCT remodeling response to furosemide.MethodsMale and Female (3‐month‐old) mice were treated with vehicle or Furosemide administered in the food (100 mg/kg per day) for seven days. Total NCC and phospho‐active NCC abundance (pNCC) was evaluated by Western Blot in one kidney, the other kidney was perfused and fixed for imaging. Kidneys were cleared using the Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC) pipeline, co‐stained with antibodies that label the early DCT (DCT1, parvalbumin) and the entire DCT (DCT1 & 2; NCC), imaged using a high‐speed spinning disc confocal microscope, and processed with 3D rendering and analysis software (IMARIS).ResultsWe confirmed previous studies that females have greater NCC expression in the basal state. Surprisingly, the length of the DCT was longer in male (~620 μm) than female mice (~560 μm). Furosemide treatment significantly increased the abundance of NCC and pNCC in both sexes, and this was paralleled by an expansion of the DCT. The remodeling response to furosemide was more profound in females (~20% increase in DCT length) than males (~8%). The DCT elongation effect of furosemide treatment in females stemmed largely from an increase in DCT1 length rather than a change in DCT2. By contrast, furosemide expanded the DCT1 and DCT2 to the same extent in males.SummaryOur data indicate that sexual dimorphism in NCC does not originate from differences in the DCT length. In fact, male have a longer DCT than female mice, which suggests that the density of NCC per unit of length is higher in females. Altogether, the differences in the remodeling response suggests that females have more “plastic” DCTs compared to males.Support or Funding InformationThis study was supported by NIH and Ben J. Lipps postdoctoral Fellowship award.
Spotlight on Tendon: Its Novel Role in Long Bone Growth and Rickets‐Caused Osteosclerosis
Authors: Li Hui , Jing Yan , Ma Chi , Wang Zheng , Feng Jian Q. 
Published: April 2020

INTRODUCTIONIt has been known for many years that tendon‐attached bone is rough and bump, and that patients with hypophosphatemic rickets (such as DMP1 mutations) develop osteosclerosis in the tendon‐attached area. The goal of this study was to test whether tendon directly forms bone and how hypophosphatemia initiates the onset of osteosclerosis in a Dmp1 KO mouse (a hypophosphatemic rickets model) using cell lineage tracing and multiple imaging techniques.METHODSWe generated a chondrocyte tracing mouse line (Col 10a1‐Cre;R26R tomato) and a tendon tracing mouse line (Scx‐CreERT2;R26R tomato) with and without a Dmp1 KO background, plus a high Pi rescue diet treatment for seven weeks.RESULTSOur key findings were: 1) the non‐tendon attached bone cells were negative in both tracing lines; 2) cells in the tendon‐attached bone were Scx+ but Col X−; 3). In the Dmp1 KO mice, tendon‐attached bone volume sharply increased, in which there were more EdU+ bone cells (i.e., more cell proliferation) with high expression levels of bone markers such as Runx2 and β‐catenin; and 4) a high Pi diet greatly improved the above phenotypes.CONCLUSIONTendon (an avascular tissue) directly forms bone cells in the tendon‐bone joint area, which is rough and porous compared to non‐tendon attached bones. Osteosclerosis in Dmp1 KO (a hypophosphatemia rickets model) is caused by accelerated cell transdifferentiation from tendon to bone due to hypophosphatemia.Support or Funding InformationU.S. National Institutes of Health grants to J.Q.F. (R01DE025014 and DE025659)
The Effect of Diabetic Conditions on the Delivery of a Pancreatic Hormone
Authors: Jacobs Monica , Pinger Cody , Gilmore Abigail , Spence Dana 
Published: April 2020

C‐peptide, a hormone that is found in the pancreatic β‐cell, is co‐secreted in equimolar amounts to insulin, and has been shown to alleviate microvascular complications found in diabetes. Prior research has indicated that C‐peptide requires a complex of human serum albumin (HSA) and zinc (Zn2+) to be transported in the bloodstream and elicit its effects. HSA is a prominent plasma protein and is involved in transporting many different molecules throughout the body. Due to the physiological conditions attributed to type 1 diabetes (T1D) there is both an increase in glycated HSA and circulation of free fatty acids; which are commonly bound to HSA. These alterations in homeostasis can inhibit HSA from binding and delivering C‐peptide to elicit its therapeutic effects. HSA was isolated from control (n=3) and diabetic (n=4) samples using antibody‐coated magnetic beads, and characterized through electrospray ionization time‐of‐flight mass spectrometry. A PolyJet 3D‐printer was used to fabricate ultrafiltration devices to measure C‐peptide and Zn2+ binding to HSA in diabetic conditions. Free C‐peptide was quantified using enzyme‐linked immunosorbent assay (ELISA), and free radioisotopic 65Zn2+ was measured on a 2480 WIZARD2 automatic gamma counter. Results indicate disruptions in C‐peptide’s binding affinity for HSA when free fatty acids are in solution (2.92 nM, n=12) as opposed to control conditions (3.68 nM, n=12). In addition, there is an inverse linear correlation with Zn2+ binding affinity and glycation of HSA (R2 = 0.7227, p<0.05). In discovering these correlations, we are better able to understand how diabetic conditions alter HSA’s binding abilities and thus homeostasis of Zn2+ and C‐peptide. Additionally, this has opened an unprecedented area of study beginning to look at the modifications in the diabetic bloodstream that can possibly disrupt the binding and delivery of C‐peptide.Support or Funding InformationNational Institutes of Health
The Complete Molecular Analysis of PTP1B Function
Authors: Pelletier Kristiane Torgeson , Page Rebecca , Peti Wolfgang 
Published: April 2020

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales and allostery. Recently, we showed that PTP1B, a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways, uses both conformational and dynamic allostery to communicate between its allosteric and active sites. Further, we also discovered that the rigidity of the catalytically essential WPD loop is indispensable for PTP1B activity, demonstrating that both conformational rigidity and dynamics are essential for controlling protein activity. To determine if additional pockets distal from the active site may also influence PTP1B activity, we integrated bioinformatics analysis, activity assays, X‐ray crystallography and a broad variety of different NMR dynamics measurements. Our data revealed the presence of additional sites, distinct from the established allosteric pocket, which can potently alter PTP1B activity when perturbed. Our data suggest that that are a variety of ways to modulate the catalytic activity of PTP1B, and, in turn, may mitigate disorders due to PTP1B dysregulation, such as cancer.Support or Funding InformationT32 GM008804