bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microvascular permeability in the absence of Epac1 activity
Authors: Karlsen Tine Veronica , Kopperud Reidun , Lidén Åsa , Rygh Cecilie Brekke , Reed Rolf K , Døskeland Stein Ove 
Published: June 2021

Epac1 is a cAMP binding protein which through intrinsic guanine nucleotide exchange factor activity couples cAMP production to activation of Rap. Epac1 is abundant in blood vessel endothelium, and may contribute in the cAMP mediated attenuation of inflammatory leakage by stabilizing the endothelial barrier. To establish how basal vascular permeability is affected in the absence of Epac1 in vivo we measured blood‐to‐tissue albumin clearance and plasma volumes with a dual‐label isotope technique in isofluorane anesthetized Epac1 knockout (KO) and wt mice. Albumin permeability, calculated as the ratio of albumin clearance to local vascular volume, was significantly increased in back skin, quadriceps muscle, inguinal fat and brown fat of Epac1 KO compared to wt mice. Furthermore, there was a significant increase in water content in inguinal fat of Epac1 KO mice and with a similar tendency observed in back skin. We conclude that intact signaling through Epac1 may be of importance to maintain the basal endothelial barrier integrity in vivo and thus prevent excessive protein leakage into the interstitium of skin, muscle and fat under steady‐state conditions.The work was supported by The Research Council of Norway and The Western Norway Health Authority. The following have contributed to produce and characterize the Epac1 KO mouse: C. Krakstad, K. Kristiansen, and L. Madsen.
Sexual dimorphism of vascular mechanics in rats
Authors: Mattar Louis M , Zamir Mair , Shoemaker J. Kevin 
Published: June 2021

The purpose of this study was to test the hypotheses that male and female rats would exhibit dimorphic mechanical properties of the hindlimb vasculature and that these differences would persist in hypertension. Vascular resistance (R), compliance (C), and tissue viscoelasticity (K) of the hindlimb vascular bed were quantified from concurrent femoral artery blood flow and pressure waveforms (lumped Windkessel model) in age‐matched Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR) male (n=10 each) and female (n=6 each). Female WKY rats displayed greater R (15.4±5.7 vs 7.7±3.3mmHg/mL/min; P<0.05) and K (3.4±2.4 vs 1.1±2.7mmHg/mL/min; P<0.05) with lower C (3.4E‐3±0.8E‐3 vs 4.9E‐3±0.9E‐3mL/mmHg/g; P<0.05) compared to males, respectively. Similarly, female SHRs had higher R (31.5±8.5 vs 17.6±7.8mmHg/mL/min; P<0.05) and K (7.3±2.5 vs 2.9±1.0mmHg/mL/min; P<0.05) with lower C (4.8E‐3±2.0E‐3 vs 6.8E‐3±3.5E‐3mL/mmHg/g; P<0.05) than their male counterparts. Main effects of strain and sex were observed for each of R, K and C variables (all P<0.05). Therefore, sex differences are apparent in the indices of steady state (R) and oscillatory (K and C) hindlimb vascular mechanics and these persist in hypertension.Supported by CIHR.
Perivascular adipose tissue in age‐associated arterial stiffening: Role of transforming growth factor beta 1
Authors: Fleenor Bradley S , Snieder Hylke , Marshall Kurt D , Seals Douglas R 
Published: June 2021

Aging causes large elastic artery stiffening associated with adventitial myofibroblast‐related increases in transforming growth factor beta 1 (TGF‐β1), collagen I and advanced glycation end‐products (AGEs). We tested the hypothesis that perivascular adipose tissue (PVAT) from older animals induces these protein changes in adventitial fibroblasts (AFB) and that the increase in TGF‐β1 causes arterial stiffening. AFB cultured from old (O, 24 mo, n=3–6) vs. young (Y, 6 mo, n=4–6) male Sprague Dawley rats had greater (p<0.05) smooth muscle alpha actin (SMαA, 1.92 ± 0.21 vs. 1.00 ± 0.08), a marker of myofibroblasts, TGF‐β1 (15.77 ± 1.13 vs. 1.00 ± 0.21), collagen I (1.71 ±0.04 vs. 1.00 ± 0.14) and AGEs (4.49 ± 1.69 vs. 1.00 ± 0.20). In AFB from Y, PVAT from O rats increased (p < 0.05) SMαA (1.76 ± 0.18), TGF‐β1 (7.28 ± 1.98) and collagen I (1.58 ± 0.21), but not AGEs (1.12 ± 0.38), and TGF‐β1 treatment mimicked these changes (SMαA: 1.00 ± 0.14 to 3.00 ± 0.44 and collagen I: 1.00 ± 0.04 to 1.71 ± 0.14, both p < 0.05; AGEs: 1.00 ± 0.04 vs. 0.91 ± 0.22). Arterial stiffness (aortic pulse wave velocity, aPWV) was greater (p < 0.05) in O (26–27 mo, n=5) vs. Y (4–6 mo, n=5) male C57Bl/6N mice (469 ± 16 vs. 351 ± 13) and 6‐week direct arterial application of recombinant TGF‐β1 to the aorta in vivo increased (p<0.05) aPWV in Y (417 ± 6). These results suggest that PVAT may contribute to age‐associated arterial stiffening via TGF‐β1.NIH AG013038, HL007822
Peripheral macrophage depletion impairs phenylephrine mediated contraction in aorta from stroke prone spontaneously hypertensive rats, but does not alter the effect of perivascular fat
Authors: Dorrance Anne McLaren , Pires Paulo Wagner , McClain Jonathon Lee , Watts Stephanie W 
Published: June 2021

Perivascular fat limits contraction in isolated aortic rings. These studies have been largely conducted in normotensive rats. We proposed that perivascular fat would impair contraction in aorta from stroke prone spontaneously hypertensive rats (SHSRP) and that peripheral macrophage depletion would exacerbate this impairment. SHRSP were treated with liposome‐encapsulated clodronate (CLOD) or placebo for six weeks from six weeks of age to remove the peripheral macrophages. Aortae where then removed and mounted in a wire myograph to measure isometric force generation with and without perivascular fat. CLOD had no effect on blood pressure (201±7 vs 200±5 mmHg, control vs CLOD) or body weight (251±7 vs 248±3g, control vs CLOD). The phenylephrine (PE) induced contraction was impaired in the aorta + fat from control SHRSP. Interestingly, macrophage ablation reduced the PE mediated contraction in the clean aorta but not in the aorta + fat. The KCl induced contraction was also reduced in the presence of fat in both groups. These results suggest that macrophage deletion has direct effects on the vascular smooth muscle to reduce contraction.

Control (n=5)
Control+Fat
CLOD (n=6)
CLOD+Fat

Max PE contraction (% of KCl)
85.3±5.2
65.9±4.8*
71.3±2.4*
59.2±3.1

EC50
−7.7±0.2
−6.7±0.1*
−7.3±0.2
−6.7±0.2

KCl induced contraction (mN)
23.5±1.6
14.4±0.2*
30.0±1.8
16.1±1.7#

different from Control.
different from CLOD
Dysfunctional bone marrow‐derived endothelial progenitor cells in chronic Ang II infusion rat model of hypertension
Authors: Jun Joo Yun , Zubcevic Jasenka , Afzal Aqeela , Carvajal Jessica Marulanda , Shan Zhiying , Mocco J , Raizada Mohan K 
Published: June 2021

Bone marrow (BM)‐derived endothelial progenitor cell (EPC) contribute to the maintenance of vascular homeostasis and EPC dysfunction is implicated in the vascular pathophysiology in hypertension. Thus, we propose Angiotensin II (Ang II)‐induced neurogenic hypertension is associated with dysfunctional EPC and elevated inflammatory cells (ICs). Ang II infused SD rats (200ng/kg/min, SC, 4–12 weeks) were used to induce hypertension. CD90+/CD4−/5−/8cells were isolated from femur and blood and their functions were assessed. BM and blood EPC decreased by 35% and 70% after 4 and 12 weeks of Ang II infusion, respectively. This was associated with significant increases in BM and blood IC (CD4+/8+/25+, CD3+/45+; CD68+) measured by FACS, leading to significant reduction in EPC/IC. BM EPC migration and proliferation ability toward SDF was reduced by 70±5% at 6 wks, 75±4% at 12 wks, and by 40±3% at 6 wks, 44±5% at 12 wks of Ang II infusion, respectively. Tube formation assay showed endothelial cells from Ang II rats decreased tube length (~25%) and branch numbers (~23%) compared to control. RT‐PCR also showed significant decrease in CXCR4 (~30%) and CXCL12 (~40%) mRNA from 12 weeks Ang II rats. In summary, these data demonstrate EPC/IC is significantly decreased as a result of decreased EPC/increased IC in Ang II induced hypertension. EPC from hypertensive rats are dysfunctional with decreased SDF‐1 and its receptor mRNA.HL33610
Regulation of the Na, HCO3‐cotransporter NBCn1 (SLC4A7) by a constitutively active ErbB2 receptor in MCF‐7 breast cancer cells
Authors: Gorbatenko Andrej , Olesen Christina W , Lauritzen Gitte , Valen Evind , Pedersen Stine Falsig 
Published: June 2021

We recently showed that the Na+, HCO3− cotransporter NBCn1 (SLC4A7) is strongly upregulated at the mRNA and protein levels in MCF‐7 breast cancer cells upon expression of a truncated constitutively active ErbB2 receptor (ΔNErbB2) common in breast cancer. Here, we explore the mechanisms involved in this upregulation. NBCn1 expression was also increased by stimulation of full‐length ErbB receptors. NBCn1 mRNA and protein levels in ΔNErbB2‐, but not in vector‐expressing MCF‐7 cells were reduced by inhibition of ERK or Src. PI3K‐Akt inhibition reduced NBCn1 expression in both cell types, suggesting a general rather than a ΔNErbB2‐specific role of this pathway. The putative promoter region was identified bioinformatically, and reporter assays using a 1.2 kb genomic region upstream of the slc4a7 gene showed that ΔNErbB2 expression increased luciferase activity 2.5 fold, demonstrating regulation of the NBCn1 promoter by ErbB2 signaling. The possible involvement of STAT3‐ and SP1‐dependent pathways is under investigation.In conclusion, downstream from ΔNErbB2, NBCn1 expression is strongly increased, at least in part through ERK‐ and Src‐dependent pathways impacting on the NBCn1 promoter region. Funding: Novo Foundation.
Neuronal Toxicity & Uptake of Cross‐Linked Copper/Zinc Superoxide Dismutase Nanozyme (cl‐SOD1 nano)
Authors: Savalia Krupa K , Manickam Devika S , Rosenbaugh Erin G , Kabanov Alexander V. , Zimmerman Matthew C 
Published: June 2021

Excessive production of superoxide in the central nervous system (CNS) has been implicated in many brain‐related cardiovascular diseases, including hypertension. However, therapeutic potential of antioxidant therapies has been limited by restricted transport across the blood‐brain barrier and poor pharmacodynamics. To address these limitations, we developed cl‐SOD1 nano, a nanoformulated antioxidant polyion complex with SOD1 protein encaged in cross‐linked block copolymers. Here, we tested the hypothesis that cl‐SOD1 nano is more efficient in delivering SOD1 to central neurons than free SOD1 without inducing significant cellular toxicity. Central CATH.a neurons from mice were exposed (1, 3, 6, 12, 24hr) to cl‐SOD1 nano or free SOD1 to assess cell viability and neuronal uptake. Although confocal microscopy and western blot analysis suggest a time‐dependent increase in neuronal uptake of cl‐SOD1 nano as compared to free SOD1, exposure to cl‐SOD1 nano significantly (P<0.05) reduced cell survival vs. vehicle‐treated neurons 24, 48 and 72 hr (57±8%, 63±3%, 60±4% survival, respectively) after removal of cl‐SOD1 nano from neuronal culture. Further experiments must decipher which component of cl‐SOD1 nano or possible polymer degradation is inducing toxicity. These studies would provide insight for future SOD nanozyme formulations which may optimally counteract excessive superoxide in the CNS.NIH P20RR021937
CT‐scan of the René Descartes (1596–1650) cranium
Authors: Le Floch-Prigent Patrice Pierre , Verdeille Stéphane , Froment Alain 
Published: June 2021

The CT‐scan of the René DESCARTES cranium (collections du Musée National d'Histoire Naturelle à Paris) was performed on a Siemens sensation 64 apparatus. The acquisition was realized in the orbito‐meatal horizontal planes. Then the frontal and sagittal planes were reconstructed. The data were analyzed. The intracranial capacity was between 1413 and 1431 cc depending of the plane. The main interest of the CT‐scan was to perform a deep and accurate study without any damage to this rare sample.Grant Funding Source : DEPARTMENT OF ANATOMY
UP‐REGULATION OF TFAP2C BY FGF DURING SHH INDUCTION IN LIMB BUDS
Authors: Kim Diana Jie Hea , Feenstra Jennifer M. , Pira Charmaine U. , Oberg Kerby C. 
Published: June 2021

During development, limb outgrowth has been linked to a reciprocal feedback loop between Fibroblast growth factors (Fgfs) and Sonic Hedgehog (Shh). Fgfs from the apical ectodermal ridge (AER) at the distal tip of the limb bud promote secretion of Shh from the zone of polarizing activity (ZPA), a cluster of cells in the distal posterior aspect of limb bud mesoderm. In turn, Shh induces Fgf from the posterior AER. This reciprocal loop is also critical to limb regeneration, and previous studies have shown that Shh up‐regulation correlates with the capacity of Fgfs to accomplish regenerative limb outgrowth. Using gene arrays, we have identified a number of potential Fgf downstream targets in the Fgf‐Shh pathway. In this report, we examined the expression of Transcription factor AP‐2 gamma (Tfap2c).To determine the time course of Fgf‐induced Tfap2c up‐regulation, we implanted Fgf‐soaked beads into stage 23 chick wing buds and evaluated Tfap2c expression by in situ hybridization at several time points.Tfap2c was up‐regulated within 3 hours of Fgf exposure; however, treatment with the protein synthesis inhibitor cycloheximide abolished induction.We conclude that Tfap2c is a close, but indirect, downstream target of Fgf during limb development and regeneration. Further studies are needed to determine whether Tfap2c participates in the Fgf‐Shh pathway or plays an alternative role in limb outgrowth.Grant Funding Source: departmental
Determination of mesenchymal stem cell; Roles of BMP4 and Scara5
Authors: Lee Hyemin , Kim Hyo Jung , Lee Yoo Jeong , Choi Hyeonjin , Kim Jae-woo 
Published: June 2021

The mesenchymal cell is a multipotent stem cell with the capacity to give rise to multiple cell types.Recently, a number of studies have demonstrated a role of BMP4 in the commitment of mesenchymal stem cells (C3H10T1/2) into the adipocyte lineage. Previously we investigated the genetic events underlying adipocyte commitment, for which C3H10T1/2 stem cells were treated with 5‐azaC and selected preadipocyte clonal lines, named A33 line. In this study, we analyzed the BMP4 promoter region in 10T1/2 and A33 cell. The activity was decreased dramatically to the level measured for the constructs between −197 and −60 bp promoter sequence. And then, we searched that global changes in gene expression of 10T1/2 and A33 cells were examined by microarray. We found that gene expression of scavenger receptor class A member 5 was significantly increased A33 compared 10T1/2. Further studies are underway to identify a transcription factor bound to this region, which will reveal the BMP4 upstream pathway essential for the adipocyte commitment of stem cells. And we will investigate the role of scara5 in mesenchymal stem cell development.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011‐0030711 and 2011‐0015665) and by Korean Research WCU grant (R31‐2008‐000‐10086‐0) from the Korean Ministry of Education, Science, and Technology.
Identification and sequence characterization of DNA double strand break hotspots
Authors: Roh Tae-Young , Shin KyungJu , Park Jihwan 
Published: June 2021

DNA double strand break (DSB) can be caused by many causes such as replication stress, reactive oxygen species from metabolic oxidative stress, ionizing radiation or chemicals in a cell. The proper repairing of DSBs is important to keep the cell integrity and maintain the genome stability. Non‐homologous end joining is a major predominant repair process to join DSB ends. Improper DNA repair and accumulation of defective DNA can induce genome instability which is linked to development of various cancers.To understand mechanism underlying spontaneous DSB occurrence, we mapped all DSB sites by high throughput sequencing after chromatin immunoprecipitation (ChIP) using antibodies against Ku70/80 dimer, DNAPKcs, and gamma H2AX in intestinal and diffuse cell types of gastric cancer. We defined DSB hot spots and identified their distinct features like levels of GC content, distribution of SNPs, overlapping of known common fragile hot spots. Our genome‐wide analyses of DSB hot spots could give an informative view of how tumors could be induced gradually.
Nanoclusters of biosynthetic enzymes define the supramolecular organization of leukotriene synthesis on nuclear membranes
Authors: Turman Melissa , Soberman Roy 
Published: June 2021

Leukotrienes (LTs) initiate and amplify immune responses. The assembly of macromolecular complexes containing 5‐lipoxygenase (5‐LO) and 5‐lipoxygenase‐activating protein (FLAP) on nuclear membranes initiates LT synthesis. We combined fluorescence lifetime imaging microscopy (FLIM) to probe 5‐LO/FLAP interactions, with stochastic optical reconstruction microscopy (STORM) to delineate the distribution of 5‐LO and FLAP on the nuclear envelope. LT synthesis in RBL‐2H3 cells was triggered via the high‐affinity IgE receptor FcεRI. Though cells primed with IgE did not produce LTs, a fraction of 5‐LO translocated to nuclear membranes and interacted with FLAP. Crosslinking FcεRI with IgE and antigen initiated LT synthesis and enhanced 5‐LO translocation and its interaction with FLAP. 5‐LO formed very small clusters on nuclear membranes of IgE‐primed cells, but addition of antigen induced redistribution into larger nanoclusters. In control and IgE‐loaded cells, FLAP was uniformly distributed along nuclear membranes but reorganized into discrete clusters upon FcεRI crosslinking. Coordinated assembly of FLAP and 5‐LO into large supramolecular complexes accompanies and may control LT synthesis.This work was supported by T32 DK7540 (MT) and R01 AI068871 (MT, RJS).
Design and Synthesis of Peptide Tertiary Amides‐A Fusion of Peptides and Peptoids
Authors: Qi Xin , Fang Pengfei , Guo Min , Kodadek Thomas 
Published: June 2021

Combinatorial libraries of peptides and peptoids (N‐alkylated oligoglycines) have been shown to be a good source of protein ligands. However, each of these compound classes has significant limitations with regard to the development of metabolically stable, high affinity and selectivity drug leads or tool compounds. Peptides are sensitive to enzymatic cleavage and are poorly bioavailable, while peptoids are relatively floppy molecules, which limits the affinity with which they recognize a target protein. To facilitate our screening effort, we describe here a synthetic route to generate large libraries of Peptide Tertiary Amides (PTAs). These compounds merge many of the favorable properties of peptides and peptoids. Large Libraries of PTAs are anticipated to be a superior source of leads for the development of useful pharmacological reagents.This work was supported by the NHLBI (NO1‐HV‐00242) and a transformational RO1 grant from the NIH GM090294.
ANTIOXIDANT AND NUTRITIVE VALUE OF MISTLETOE (Viscum Album)
Authors: NWOKE STELLA C , MAGBAGBEOLA ABIOLA O , EBUEHI OSARETIN A , IWALOKUN BAMIDELE A 
Published: June 2021

Mistletoe (Viscum album L. subsp. album) is known as an obligatory hemiparasite of trees and is consumed by herbivores in some places, especially during periods of forage shortage and drought. This experiment was conducted to determine nutrient composition and nutritive value of mistletoe with respect to its fertility enhancing potential. Mistletoe samples were collected from the wild orange tree in the thick forest of Egbeda, Lagos, Nigeria. In this study, Mistletoe was analyzed for its antioxidant and mineral content using spectrophotometric methods. The results revealed; Mean total antioxidant capacity was 81.77mg/g Ascobic acid equivalence, Mean total Phenolic content was 55.04mg/g Garlic equivalence Mean total Flavonoid is 68.985mg/g Rutin equivalence. % Inhibition DPPH Scavenging activity at 25ug/ml, 50ug/ml, 75ug/ml, 100ug/ml, concentrations were 50.21%, 56.09%,61.18% and 70.23% respectively. Also, the Reducing Power Ability recorded were 0.282ug/ml, 0.335ug/ml, 0.529ug/ml, 0.663ug/ml. A very high level of magnesium (240.8mg/100g), potassium (122.4mg/100g), moderate levels of calcium (55.2mg/100g), sodium (42.2mg/100g), and a fair amount of iron (16.4mg/100g). The proximate analysis for percentage Moisture, Ash, Fat, Protein, Crude fibre and carbohydrate, were 22.3, 5.785, 3.26, 5.475, 17.42, 47.54 respectively. Also the phytochemical screening shows the presence of alkaloids, tannins, saponinns, steroids, flavonoids, phenols, and lectins.Currently Viscum album has shown promising results in its use as a fertility enhancing plant, the above shows that it is of high nutritive value of health importance.
GPR179 interacts with TRPM1 in retinal depolarizing bipolar cells
Authors: Hasan Nazarul , Ray Thomas A. , Gregg Ronald G. 
Published: June 2021

BackgroundComplete congenital stationary night blindness (cCSNB) is a heterogeneous disorder of the retina characterized by impairment of low light vision and loss of the b‐wave of the electroretinogram. Mutations in GRM6, TRPM1and NYX, which encode proteins critical to signal transmission in one class of retinal interneurons called depolarizing bipolar cells (DBCs) cause cCSNB. We have shown that an orphan G‐protein coupled receptor, GPR179, also plays a vital role in DBC function.Methods/ResultsImmunochemical staining of retinal cross‐sections demonstrates that TRPM1 and GPR179 co‐localize on the dendritic tips of DBCs. Further, TRPM1 is lost from GPR179 mutant mice (Nob5
) indicating they may interact. We used a yeast two‐hybrid analyses and co‐immunoprecipitation (co‐IP) assays after expression in HEK293T cells to examine interactions. Both methods show that GPR179 and TRPM1 interact. Furthermore, antibodies to GPR179 could co‐IP TRPM1 from retinal lysates, confirming the interaction.ConclusionsThis is the first study to demonstrate the interaction of TRPM1 and a previously uncharacterized orphan G protein receptor, GPR179. This result was confirmed by three independent methods suggesting that the interaction of GPR179 and TRPM1 is a critical component of the G protein signal transduction cascade in DBCs.
Expression and Characterization of Rice (Oryza sativa) Monolignol β‐Glucosidase
Authors: baiya supaporn , Okita Thomas W , Cairns James Ketudat 
Published: June 2021

Phylogenetic analysis of Oryza sativa GH1 β‐glucosidases indicated that Os4BGlu14, Os4BGlu16, and Os4BGlu18 are closely related to known monolignol β‐glucosidases, leading to the hypothesis that they may be involved in lignification by hydrolysing monolignol glucosides. The cDNAs for each of these rice GH1 β‐glucosidase genes, Os4BGlu14, Os4BGlu16, and Os4bglu18 were cloned, sequenced, and ligated into pET32a, and the resulting recombinant plasmids were used to express the putative β‐glucosidases as fusion proteins with N‐terminal thioredoxin and His6 tags in E. coli Origami (DE3). No activity could be detected for proteins expressed from Os4BGlu14 and Os4BGlu16 in this system. Optimal expression of soluble Os4BGlu18 β‐glucosidase activity was obtained with induction at 18°C, 0.1 mM IPTG for 16 to 18 h. The recombinant protein was extracted and purified by immobilized metal affinity chromatography (IMAC). The optimum pH for Os4BGlu18 was found to be 5 and the enzyme was stable from pH 4 to pH 8. The temperature optimum of Os4BGlu18 was 55°C, but the enzyme was most stable at 20°C‐40°C. Os4BGlu18 hydrolyzed p‐nitrophenol (pNP)‐β‐D‐fucoside best, followed by pNP‐β‐D‐glucoside, pNP‐α‐L‐arabinoside, pNP‐β‐D‐galactoside and pNP‐β‐D‐xyloside, respectively. Moreover, Os4BGlu18 hydrozyed n‐octyl‐ β‐glucoside, n‐heptyl‐β‐glucoside, methyl‐β‐D‐glucopyranoside and daidzin. Among possible natural substrates, Os4BGlu18 slowly hydrolyzed the monolignol glycoside p‐coumaryl alcohol glucoside, along with salicin and arbutin. No activity was detected with coniferin and sinigrin, two other monolignol glucosides, suggesting another isoenzyme may be responsible for their hydrolysis in the plant.Financial support was provided by The Royal Golden Jubilee Ph.D Program of the Thailand Research Fund.