bluuebunny/crossref_metadata_2025 huggingface dataset:
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
Multilayered, Multi‐Component Anti‐infective Nanocoatings for Biomedicine
Authors: Mills David , Elfer Kate , McNamara Kaitlin , Manuel Morganne , Lvov Yuri
Published: June 2021

Nebulization was used to build multilayered polyelectrolyte nanocoatings (PMNs) onto poly‐e‐caprolactone (PCL) scaffolds. Nebulization creates surface nanocoatings through the conversion of polyelectrolyte solutions that can be sprayed directly onto a surface. This technique permits assembly of multilayer scaffolds with tunable architectures with the potential for diverse biomedical applications. PMNs were created and tested as anti‐infective (gentamicin‐based) nanocoatings. PLL and PGA were used to synthesize a ten‐bilayer structure. Gentamicin was embedded within each PMN using two different antibiotic gradients: deep to superficial and superficial to deep. PCL/PMNs/Gentamicin scaffolds were then placed onto bacteria‐laded agar plates over a 14‐day period. Image analysis was used to assess bacterial response to scaffolds. Controls consisted of PCL/PMNs scaffolds without gentamicin. Results showed that both antibiotic architectures proved effective in killing bacteria and controlling bacterial growth over the experimental period with the deep to superficial structure having the most profound effect. A biodegradable bandage consisting of the multi‐component (growth factor/antibiotic) nanocoatings is currently being tested for its anti‐infective/ regenerative properties. Louisiana Tech's NSF REU program and the Governor's Biotechnology Initiative provided funding support.
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.
Depletion of MutSbeta slows GAA•TTC repeat expansion in a cellular model of Friedreich Ataxia
Authors: Halabi Anasheh , Ditch Scott , Wang Jeffrey , Grabczyk Ed
Published: June 2021

Friedreich Ataxia (FRDA) is caused by expansion of a GAA•TTC triplet repeat in the first intron of the frataxin gene. FRDA is a fatal, autosomal recessive disease characterized by rapidly progressing neurodegeneration. Disease severity directly correlates with GAA•TTC repeat number. We have developed a human cellular model of FRDA that recapitulates the incremental, continuous expansion found in post‐mitotic tissues of affected patients. Our hypothesis is that repeat expansion is mediated by DNA repair enzymes in the mismatch repair pathway that are attracted to unusual structures formed by transcription through the GAA•TTC repeat. DNA mismatches and small loop‐outs are recognized by MutS heterodimers MutSalpha or MutSbeta (composed of MutS Homologue 2 (MSH2) and MSH6 or MSH2 and MSH3, respectively). We found that shRNA depletion of MSH2 or MSH3, but not MSH6, slowed GAA•TTC expansion in our FRDA model. We conclude that although MSH2 depletion is associated with colorectal cancer, targeting MSH3 has promising therapeutic potential in slowing FRDA progression. Funded by grants from the Friedreich's Ataxia Research Alliance and the LSUHSC Research Enhancement Fund.
On the mechanism of Pasteurella multocida toxin (PMT) induced mitogenesis: mTOR mediated upregulation of survivin and aurora kinase B in cultured cells
Authors: Oubrahim Hammou , Wong Allison , Wilson Brenda , Chock P. Boon
Published: June 2021

PMT is a bacterial single polypeptide known for its mitogenic properties for certain types of cells. In addition to its proliferative properties, it is a strong inducer of anchorage‐independent growth. However, the mechanism by which PMT exerts its biological effects is not well understood. We showed that PMT‐induced protein synthesis in cultured cells proceeds, in part, via a Gαq‐dependent activation of mTORC1, a key Ser/Thr kinase conserved from yeast to mammals known to be involved in the regulation of protein synthesis, cell proliferation and autophagy in a nutrient‐and energy‐responsive manner. Our results also reveal that PMT treatment induces a prolonged activation of the mitogen‐activated protein kinases (MAPKs) in a time‐ and dose‐dependent manner. Interestingly PMT‐induced mTOR and ERK signaling pathways activation were also detected when serum‐starved Swiss 3T3 and MEF cells were treated with conditioned media taken from PMT‐treated cells, suggesting the presence of a diffusible factor(s) that mediated these activations. In addition, recent experiments revealed that PMT induced a prolonged upregulation of chromosomal passenger complex (CPC) proteins including survivin and aurora kinase B both at mRNA and protein levels in serum‐starved Swiss 3T3 cells. Furthermore, using immunohistochemistry, we showed that survivin is associated with the spindle midzone at anaphase in PMT‐treated cells but not in control non‐treated cells. Interestingly, PMT‐induced CPC upregulation was suppressed by mTOR and MEK specific inhibitors, rapamycin and U0126 respectively. Together, these observations indicate that the PMT‐induced upregulation of survivin and aurora kinase B is mediated by the mTOR signaling pathway.
MethCy: DNA Methylation Analysis Software
Authors: Muhie Seid , Bhattacharya Surajit , Hammamieh Rasha , Chakraborty Nabarun , Jett Marti
Published: June 2021

Our objective is to develop DNA methylation analysis software that can be used both as a standalone tool, and as a Cytoscape plugin (www.cytoscape.org). The Limma package of BioConductor (www.bioconductor.org) was used to implement the pre‐analysis normalization. Methylation (status) assessment parameters (log ratio, Z‐score and fold change) were implemented using BioConductor's R packages. The software's Graphics User Interface (GUI) was developed using the Swing framework in Java. An R/Java interface, Rserve, was used as an interface between Java and R. Java has been used for its suitability for expansion of the software and interfacing of our plugin into Cytoscape. Using our in house generated (Agilent's platform based ChIP‐on‐chip) DNA‐methylation data, we ran log ratio analysis followed by >= 2 fold filter. We have identified ~ 3700 hypo‐and hyper‐methylation regions representing ~ 640 differentially methylated genes. Our software is compatible with both Unix/Linux and Windows operating systems. Unlike other proprietary software, our software will be in the public domain as a Cytoscape plugin. Once in Cytoscape, users can do gene ontology enrichments, construct (molecular) interaction networks and many other downstream analyses using other Cytoscape plugins.
Characterization of a novel Aedes aegypti ferritin subunit identified utilizing proteomic techniques
Authors: Geiser Dawn L. , Li Wenzhou , Gucinski Ashley C. , Patel Naren , Patel Pritesh , Wysocki Vicki H. , Winzerling Joy J.
Published: June 2021

Mosquitoes must blood fed for egg development. The nutrient‐rich blood meal provides a potentially toxic concentration of iron that is sequestered and transported throughout the mosquito in ferritin. Ferritin is a 24‐subunit protein composed of heavy and light chains. We generated a 24 h ovary proteome from Aedes aegypti (yellow fever mosquito) fed an artificial blood meal with or without hemoglobin to identify proteins prominently regulated by iron. Of 165 proteins observed from LC‐MS/MS analysis, 5 proteins were described as up regulated by iron and 4 down regulated utilizing high stringency criteria. The ferritin heavy chain homologue (HCH) was shown to be up regulated in the ovary. A preliminary proteomic analysis of the 24 h ovary from mosquitoes fed an artificial hemoglobin‐rich diet identified an iron‐regulated, previously uncharacterized ferritin light chain homologue (LCH2). Subsequent molecular characterization demonstrated that the LCH2 transcript was present in all developmental stages and up regulated in males and in females 72 h post blood feeding (PBF). LCH2 mRNA is highly expressed 72 h PBF in ovaries. Immunoblot analysis showed LCH2 protein in eggs, 1st instar larvae, males and females 72 h PBF as well as significantly up regulation in ovaries 72 h PBF. Further characterization of the LCH2 subunit will provide insights into the unique regulation of iron metabolism in mosquitoes. Research support from the Agricultural Experiment Station and the College of Agriculture and Life Sciences, the Undergraduate Biology Research Program and the Honors College at the University of Arizona.
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).
Identification of three distinct glycosylation sites in the ATP binding cassette class A3 (ABCA3) transporter
Authors: Mulugeta Surafel , Zhao Ming , Hawkins Arie , Beers Michael F
Published: June 2021

Glycosylation plays a critical role in the proper maintenance of protein structure to promote stability and/or to mediate trafficking and function of various proteins. ABCA3 is a polytopic transmembrane transporter that functions as a lipid and phospholipid pump. Within the first luminal loop of ABCA3, three putative glycosylation sites exist containing NATI (a), NCSS (b), and NHSK (c) motifs. To determine if these sites undergo glycosylation, we used alanine mutagenesis to generate fusion constructs encoding EGFP‐tagged to either wild type ABCA3 (ABCA3WT), single motif mutants (ABCA3a, ABCA3b, ABCA3c), double mutants (ABCA3a+b, ABCA3b+c), or a triple mutant (ABCA3a+b+c). Fluorescence examination of transiently transfected A549 cells showed cytosolic vesicle distribution of the single motif mutants similar to ABCA3WT. By Western blot, removal of N‐linked glycosylation by PNGaseF resulted in the migration of the ABCA3WT primary translation product band at a faster rate. Moreover, all three single motif mutant bands also migrated in a similar manner. However, double and triple mutants resulted in ER retained, unprocessed forms of ABCA3. Although the exact role of glycosylation in ABCA3 is yet to be fully explained, these results suggest that ABCA3 transporter is glycosylated on at least three specific sites and amino acid alteration of these motifs leads to abnormal trafficking of the transporter.
Premature Osteoarthritis in the Temporomandibular Joint of Heterozygous Disproportionate Micromelia Mice
Authors: Rees M. B. , Ricks M. L. , Falk D. J. , Farrell J. T. , Carr J. M. , Williams D. T. , Bridgewater L. C. , Seegmiller R. E. , Kooyman D. L.
Published: June 2021

A Col2a1 gene mutation disrupts collagen assembly and causes premature degeneration of knee articular cartilage in disproportionate micromelia (Dmm) mice. This study analyzed the temporomandibular joint (TMJ) in Dmm/+ mice to provide an animal model of TMJ osteoarthritis (OA) in humans. Dmm/+ mice and age‐matched controls were compared histologically at 2, 6, 9 and 12 months of age. Craniums were fixed in 4% paraformaldehyde, processed to paraffin sections, stained with Safranin‐O/Fast Green, and analyzed by light microscopy. OA severity was quantified using a modified Mankin scoring procedure. Activation of the unfolded protein stress response (UPR) was assayed by western blot. The Dmm/+ TMJ showed classic signs of OA as early as 6 months of age, whereas controls showed normal, intact cartilage. Mutant chondrocytes were clustered, leaving acellular regions in the matrix with decreased proteoglycan staining while pericellular regions showed increased staining. Dmm/+ TMJ showed increased levels of the proteins Ddr2, Mmp‐13, and HtrA1, which are associated with cartilage degradation. The levels of BiP and phospho‐elF2α, two markers of the UPR, were elevated at birth, suggesting that the UPR stress response may contribute to subsequent cartilage degeneration. This study demonstrates the utility of the Dmm/+ mouse TMJ as a viable model for TMJ OA in humans.
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.
CD81 promotes degradation of transferrin receptor 2 (TfR2) in Hep3B cells
Authors: Chen Juxing , DeMaster Kristina , Wentz Annie , Enns Caroline
Published: June 2021

Mutations in transferrin receptor 2 (TfR2) cause an iron overload disease called type 3 hereditary hemochromatosis (HH). The pathology is characterized by deposition of iron in the liver, heart, pancreas, and pituitary gland, resulting in cirrhosis, arrhythmias, diabetes, hypogonadism and arthritis. TfR2 is primarily expressed in hepatocytes and is proposed to sense iron levels in the blood and positively regulate the expression of hepdicin, a peptide hormone produced by the liver, and a master regulator of iron homeostasis. In keeping with the proposed role of TfR2 in iron sensing, previous studies showed that TfR2 has a short half‐life, is stabilized by holo‐Tf, and the cytoplasmic domain of TfR2 is largely responsible for the stabilization of TfR2 by holo‐Tf. However, the mechanism of TfR2 degradation and regulation of TfR2 on hepcidin expression remains to be determined. A yeast two‐hybrid approach using TfR2 cytoplamic domain as the “bait” and human liver library as the “prey” identified CD81 as interacting with TfR2. Immunoprecipitation results also showed that CD81 interacts with TfR2 but not with TfR1. Knockdown of CD81 using siRNA significantly increased the protein levels of TfR2. Knockdown of CD81 decreased the surface/total ratio of TfR2 and increased the half‐life of TfR2. Over‐expression of CD81 downregulates the protein levels of TfR2. These results indicate that CD81 promotes degradation of TfR2.
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.
Prospective Study To Investigate The Prevalence Of The 90 kDa Isoform Of Angiotensin Converting Enzyme In Vitória ‐ Brazil – Preliminary Results
Authors: Febba Andreia Cristina Silva , da Barrinha Fernandes Fernan , Aragão Danielle Sanches , Franco Maria do Carmo , Mill Jose Geraldo , de Castro Cintra Sesso Ricardo , Casarini Dulce Elena
Published: June 2021

Essential hypertension is a multifactorial. We aimed to investigate the prevalence of the urinary 90 kDa N‐domain ACE in a cohort of the Brazilian population from Vitória, and to verify its association with essential hypertension. A prospective study was performed to verify the presence of the urinary 90 kDa N‐domain ACE, using the Western Blotting technique. The blood pressure was also measured. The groups were divided as 1(190/65kDa); 2(190/90/65kDa) and 3(90/65kDa) according to the presence of isoform. We separate the groups in relation to blood pressure, such as hypertensive and normotensive subjects. The urine were analyzed and compared to the first phase: We note that the first and second phase group 1 remained similar (3.3%); in the second phase the group 2 presented a decrease in the percentage of cases 33.3% vs 23.3% and the 3 group showed an increase in the percentage of cases in the second phase when compared to the first part of the study (63,3% vs 73,3%). There was an increase in cases of hypertensive subjetcs in the second part of the study from 50% to 63.3%. This analysis indicated more than 90% of subjects had 90kDa ACE in their urine;The second phase shows an increased number of subjects with isoforms of 90 and 65 kDa as well as increasing the number of hypertensive subjects; 3.3% were normotensive and not express the 90kDa isoform;These data suggest a relationship between hypertension and the presence of 90 kDa ACE.
Mice bearing a targeted mutation of nBmp2 display decreased memory capabilities
Authors: Dhungel Saroj , Rogers Aubrey C , Cordner Ryan D , Mayo Jaime L , Steed Kevin , Badgley Corinne , Wallmann Andrew , Barrow Jeffrey R , Edwards Jeffrey G , Bridgewater Laura C
Published: June 2021

A variant of the secreted growth factor BMP‐2, called nBmp2, is translated from a downstream alternative start codon and localized to the nucleus. To determine the function of this novel nuclear variant, we generated mice bearing a mutation that prevents nuclear localization of nBmp2 without altering production of secreted BMP‐2. Skeletal analysis of mutant compared to wild type mice demonstrated that skeletal structure is entirely normal in the mutant mice, indicating that the function of secreted BMP‐2 was not altered by the mutation and that any phenotypic differences, therefore, are due to exclusion of nBmp2 from the nucleus. Intracellular calcium handling is aberrant in muscle from these mice, so this study was undertaken to measure calcium‐dependent neurological function. Mutant mice displayed significantly impaired memory capabilities in both the Morris water maze and the novel object recognition test. Synaptic plasticity, which is dependent on calcium transport and is the main cellular mechanism mediating learning and memory, was tested by measuring long‐term potentiation in hippocampal slices and was found to be significantly reduced in the mutant mice. Our data suggest that nBmp2 plays an important role in the processes of memory and learning, perhaps by affecting calcium transport in neural tissue.
LGI3 prevents UVB‐induced cell death of human keratinocytes
Authors: Kim Dong-Seok , Lee Seung Hoon , Jeong Yun-Mi , Kim So-Young , Jeong Hyo-Soon , Park Kyoung-Chan , Kim Myo-Kyoung , Baek Kwang Jin , Kwon Nyoun Soo , Yun Hye-Young
Published: June 2021

Leucine‐rich glioma inactivated 3 (LGI3), a member of LGI/epitempin (EPTP) family, is known to be mainly expressed in brain. However, the expression and physiological roles of LGI3 in skin cells remain unknown. In the present study, it was found for the first time that LGI3 is expressed in human skin. LGI3 was mostly expressed by normal human keratinocytes, although normal human fibroblasts and melanocytes also produced LGI3. Furthermore, ELISA and Western blot analysis showed that HaCaT human keratinocytes secreted LGI3 protein after exposure to UVB in a time‐ and dose‐dependent manner. We next investigated the possible role of LGI3 in keratinocytes. Our results showed that LGI3 protected HaCaT cells from UVB‐induced cell death. It was also found that LGI3 potently stimulates the phosphorylation of Akt, which is involved in the cell survival‐signaling cascade. Furthermore, LGI3 led to the phosphorylation of MDM2 and subsequent p53 degradation. Taken together, these data suggest that LGI3 may regulate p53 levels, and that keratinocyte‐derived LGI3 may act as a novel cytokine for skin homeostasis. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011‐0026587).
The intracellular trafficking and distribution of VEGF165
Authors: Guzman-Hernandez Maria Luisa , Potter Gael , Kim Yeun J , Kiss Jozsef Z , Balla Tamas
Published: June 2021

Vascular endothelial growth factor (VEGF) is a major regulator of physiological and pathological angiogenesis. Native VEGF from numerous sources corresponds to VEGF165 isoform. While the functional roles of VEGF165 have been extensively studied, its intracellular trafficking and mode of secretion remain largely unknown. Here, we created VEGF165 fused with GFP to monitor the distribution during its trafficking in COS‐7 or HUVEC cells. In parallel studies, we analyzed the release of VEGF165 into the media using Western blot. We found that VEGF165‐GFP forms dimers and gets glycosylated similarly to wild‐type. The molecule follows the endoplasmic reticulum‐Golgi pathway and GDP‐ and GTP‐locked forms of Sar1 and Arf1 GTPases, completely block VEGF secretion. Less clear is the post‐Golgi trafficking of VEGF. We found that VEGF165 is secreted even at 19 °C and its secretion is stimulated both by Ca2+ increases and PKC activators. Also, a significant fraction of VEGF remains associated with the cell surface in discrete areas. Exocytosis of VEGF165 requires PIP2 at the plasma membrane. Further studies are in progress to find the mechanism by which VEGF is released from the plasma membrane and to clarify the exact intracellular site(s) of proteolytic processing of VEGF165.
PERK phosphorylation on tyrosine 561 delays ER stress‐induced PERK activation
Authors: Yamani Lama , Latreille Mathieu , Larose Louise
Published: June 2021

The PKR‐like endoplasmic reticulum (ER) kinase (PERK) is an ER transmembrane ser/thr protein kinase that plays an essential role in mediating the unfolded protein response (UPR) activated during stress conditions in the ER lumen. The importance of PERK in pancreatic beta‐cell homeostasis is demonstrated in Wolcott‐ Rallison syndrome, a neonatal or early infancy form of insulin‐dependent diabetes in humans with inactivating mutations in the PERK gene. In this study, we provide strong evidence that PERK is phosphorylated on tyrosine 561 (Y561) in pervanadate‐treated MIN6 cells, a mouse pancreatic beta cell line in culture. In addition, we demonstrate that increased phosphorylation of PERK on Y561 delays PERK activation in response to pharmacological agents that induce ER stress. Interestingly, we discovered that the adaptor protein Nck1 directly interacts with PERK phosphorylated on Y561 through its SH2 domain. Finally, using recombinant wild‐type and kinase dead PERK proteins, our data strongly suggest that PERK phosphorylation on Y561 is dependent on PERK kinase activity. All together, our results indicate that PERK phosphorylation on Y561 and binding to Nck1 play an important role in regulating PERK activity. Supported by the Canadian Diabetes Association.
GPR179 interacts with TRPM1 in retinal depolarizing bipolar cells
Authors: Hasan Nazarul , Ray Thomas A. , Gregg Ronald G.
Published: June 2021

Background

Complete 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/Results

Immunochemical 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.

Conclusions

This 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.
Bilateral vascular and ductal renal anomalies. Case report
Authors: Zahoi Delia Elena , Baderca Flavia , Alexa Aurora , Daescu Ecaterina
Published: June 2021

During the routine dissection of a 62 year‐old male within the laboratory of the Department of Anatomy, several vascular and ductal renal anomalies were discovered. The subject had not had any kidney‐related health issues during his lifetime, and no other anomalies were discovered.

For the left kidney, three arteries, two veins and two urethers were found. Of the three arteries, the main one originated on the lateral side of the aorta (L1), the superior one – 1.5cm above the main one and the inferior one, which was a polar artery, 3cm below the main one, on the anterior side of the aorta. The veins were also unequal, the smaller one (inferior) had a diameter of 0.2cm and spilled into the left testicular vein, 5.5cm before its’ joining of the main renal vein. The left testicular vein also crossed path with the testicular artery, 3cm below its’ joining the renal artery. The two urethers ran parallel to each other but joined at the point of entry into the bladder.

The right kidney had a signle artery, originating in the aorta above the left main artery. It had a single vein, which was joined by the right testicular artery at 0.4cm before the inferior vena cava.

The presence of such vascular anomalies cannot be ignored. Knowledge of such anomalies is a necessity for the success of urological surgery and can serve as a guide in endovascular procedures.
A dot‐blot based assay to quantify collagen from large arteries
Authors: Condezo-Hoyos Luis A. , España-Caparros Gabriel , de Pablo Angel L López , Rodríguez-Rodríguez Pilar , Gutierrez-Arzapalo Perla Y. , Ancer Jesús , Gonzalez M. Carmen , Arribas Silvia M.
Published: June 2021

AIM

To develop a densitometric method based on dot blot and sirius red staining to quantify vascular collagen, using gelatin as standard, compatible with elastin purification.

METHODS

Aortae and carotid arteries from adult rats were dried 5 min, weighted and incubated in 0.1N boiling NaOH for 45min. NaOH extracts were tested by dot blot on nitrocellulose or PVDF membranes. We established the optimal conditions for: 1) Sirius red concentration, incubation time and temperature, 2) washing time and composition of washing solution, 3) linearity and specificity of the assay. The compatibility of this method with elastin purification and integrity was assessed by confocal microscopy (488 excitation, 520–600 emission nm, autofluorescent properties).

RESULTS

Optimal conditions for the densitometric method were: 1) Sirius red concentration 0.00125% w/v, 30 min at 4ºC, 2) tricloroacetic acid and acetone included in the washing solution increased signal/noise ratio, being compatible only with PVDF membranes and 3) gelatin concentration linearity range was 0–0.5 g. Collagen content was quantifiable in carotid and aortae NaOH extracts. Purified elastin has a similar organization to intact arteries.

CONCLUSION

We have developed a quick, simple, cheap and specific method to quantify collagen in vascular samples compatible with elastin purification. FINANCIAL SUPPORT: MICINN (FEM2009‐13434‐C02‐02).
Functional characterization of mitofusin‐like protein from Dictyostelium discoideum
Authors: Rai Amrita , Manstein Dietmar J
Published: June 2021

The balance between mitochondrial fission and fusion maintains mitochondrial morphology and function. In mammals, three large GTPases ‐ Opa1 and the mitofusins Mfn1 and Mfn2 ‐ are required for the fusion of inner and outer mitochondrial membranes. The Dictyostelium mitofusin MfnA GTPase domain shares only 24% identity with the corresponding region of human Mfn1 and lacks a typical Fzo‐mitofusin family region. To define the cellular function of the Dictyostelium protein, we used molecular genetic approaches to manipulate the production level of MfnA and to follow the localization of the fluorophore‐tagged protein. In cells producing MfnA with a C‐terminal eYFP‐tag, the protein is targeted to the outer mitochondrial membrane. Overproduction of wild type MfnA and GTPase deficient mutants K132A or S133N leads to peri‐nuclear mitochondrial clustering and the phenotype is not due to any cytoskeletal defect. Unlike mammalian Mfn1/2, both N‐terminal and C‐terminal MfnA domains are required for mitochondrial targeting. N‐terminal deleted construct shows partial co‐localization with the ER, while double deletion of N‐terminal and transmembrane domain induces association with plasma membrane and contractile vacuoles. MfnA null cells have fewer and more dispersed mitochondria but appear otherwise normal. Thus our results reveal that MfnA alters mitochondrial morphology by fusing outer mitochondrial membranes.
High Content Cell‐Based Screening for New Classes of Histone demethylases
Authors: Chen Degui , Cao Xiongwen , Qi Hongyun
Published: June 2021

Histone methylation can be removed by demethylases. Up to now, two classes of about 20 histone demethylases specific for different lysine residues were identified, but demethylases for several other lysine residues have not been found. Also, a demethylase activity against the arginine residue has not been convincingly demonstrated. To identify new histone demethylases, we performed high content cell‐based screening. In this screening, we transfected each cDNA encoding 2500 nuclear proteins with more than 300 amine‐acids in length into cells and obtained hits that could reduce the levels of several specific methylations in lysine and arginine residues in transfected cells by immunocytochemistry. Through western blot analysis on transfected and non‐transfected cells as the secondary screening, we narrowed down the number of hits as the candidates. The candidates were expressed and purified from bacteria or insect cells, and reacted with bulk histones with various methylations or histone peptides with specific methylations in the presence or absence of various set of cofactors. The reaction was subjected to western blot analysis or mass spectrometry analysis to examine if the candidates could remove the specific methylation from bulk histones or histone peptides. We are now in the process of confirming the demethylase activity by demethylation product characterization, domain mapping, and crossing out of cofactors.
The P2Y2 receptor regulates lipoprotein receptor‐related protein 1 expression in smooth muscle cells
Authors: Dissmore Tixieanna , Medeiros Denis M , Weisman Gary A , Bradford Barry , Mamedova Laman
Published: June 2021

Extracellular nucleotides exert various effects in the cardiovascular system. Uridine 5′‐triphosphate (UTP) regulates G‐protein‐coupled P2Y2 receptor (P2Y2R) activation which causes actin cytoskeletal reorganization. We isolated aortic smooth muscle cells (SMC) from wild‐type (WT) and P2Y2R−/− mice to investigate whether UTP and the P2Y2R modulate expression of low density lipoprotein receptor‐related protein 1 (LRP1) and low density lipoprotein receptor (LDLR). For LRP1 expression, cells were stimulated in the presence or absence of 100 μM UTP overnight. For LDLR mRNA expression, cells were transiently transfected for 24 h with cDNA encoding a hemagglutinin‐tagged WT P2Y2R or a mutant P2Y2R that does not bind filamin‐A (FLN‐A) and treated with 100 μM UTP overnight. Total RNA was isolated, reversed transcribed to cDNA, and mRNA abundance determined by RT‐PCR using the ΔCt method with GAPDH as a control gene. Results show SMC expressing the P2Y2R that lacks the FLN‐A binding domain exhibit 3‐fold lower LDLR expression than SMC expressing the WT P2Y2R. However, there was a 3‐fold increase in LRP1 mRNA expression in response to UTP in P2Y2R−/− SMC compared to WT. Actinomycin‐D (20 μg/ml) significantly reduced UTP‐induced LRP1 mRNA expression in P2Y2R−/− SMC (P < 0.05). These results might indicate the presence of a P2Y4R in P2Y2R−/− SMC as a possible cause for the effect of UTP, which is being further investigated.
CR‐108, a novel vitamin K derivative induces apoptosis through ROS and mitochondrial dysfunction in breast cancer
Authors: Chao Jui I , Yang Chun-Ru , Chen Chinpiao
Published: June 2021

Vitamin K derivatives exert anticancer activities. Here, we found a novel vitamin K derivative, CR‐108, which induced apoptosis in human breast cancer cells. CR‐108 reduced the cell viability in both the non‐HER‐2‐overexpressed MCF‐7 and HER‐2‐ overexpressed BT‐474 breast cancer cells. Interestingly, CR‐108 highly elicited the intracellular reactive oxygen species (ROS) in breast cancer cells. CR‐108 markedly induced the loss of mitochondrial membrane potential analyzed by fluorescence intensities of JC‐1 and DiOC6. Moreover, CR‐108 induced cytochrome c release from mitochondria to cytosol, elevated the levels of cleaved PARP proteins, and increased apoptotic induction. A ROS scavenger NAC completely blocked the CR‐ 108‐induced ROS, mitochondrial damage, and apoptosis. Meantime, CR‐108 increased the phosphorylated p38 kinase but conversely inhibited the survivin protein expression. NAC prevented the activation of p38 kinase and restored the survivin protein levels. In addition, a specific p38 kinase inhibitor SB202190 reduced the survivin protein level and attenuated the CR‐108‐induced cytotoxicity. Taken together, CR‐108 induces apoptosis through ROS and mitochondrial damage pathway associated with p38 kinase and survivin in the human breast cancer.
The unhappy triad: quest for a conclusive definition
Authors: Canby Craig A , Cassidy Keely M
Published: June 2021

The unhappy triad of the knee is classically defined as an acute injury to the anterior cruciate ligament (ACL) with concomitant damage to the medial collateral ligament and the medial meniscus. With the advent of advanced clinical and arthroscopic diagnosis, the classical definition may no longer be tenable. Consequently, a comprehensive review of the literature was performed to better define our understanding of the structures involved with acute injury to the ACL and chronic ACL deficiency. A search produced 32 peer‐reviewed references consistent with the review's aims. In a total of 598 knees with acute ACL and medial collateral ligament ruptures, there were 255 (43%) lateral meniscus tears, 233 (39%) medial meniscus tears, and 28 (4%) bilateral meniscus tears. In the setting of chronic ACL deficiency, with 1,780 ACL deficient knees examined, when combined with a rupture of the medial collateral ligament, there were 843 (47%) medial meniscus tears, 789 (44%) lateral meniscus tears and 52 (3%) bilateral tears. Our literature review suggests that the third member of the unhappy triad is the lateral meniscus, whereas the medial meniscus is more commonly injured in chronic ACL deficiency. These differences in injury patterns will aid in clinical diagnosis and management of patients with ACL injuries.
Effects of Tetra‐Alkyl Bis‐Phosphates on Butyrylcholinesterase
Authors: Feng Joshua J. , Turner Archie D. , Tu Kim , Acey Roger A. , Nakayama Kensaku
Published: June 2021

The progression of Alzheimer's Disease (AD) correlates positively with increasing levels of butyrylcholinesterase activity. As such, the inhibitors of this enzyme provide promise as potential therapeutics. Bisphosphates were evaluated for their inhibitory effects on the enzyme. The compounds were synthesized by the reaction of the corresponding dialkyl chlorophosphate with the appropriate diol in pyridine and dicholoromethane under inert conditions. The crude products were purified by flash chromatography to give the desired bisphosphates high purity. IC50's for the tetraethyl and tetrabutyl hexyl derivatives were determined to be 2.52 μM and 0.95 μM, respectively. The significance of the hexyl diphosphates’ inhibitory properties is highlighted when compared to the single phosphate derivative, diethyl hexyl phosphate, which showed little inhibitory activity. Moreover, analysis of the enzyme inhibition suggests that there are two binding sites, each with separate affinities. Modification of the carbon‐chain linker to produce shorter and longer bisphosphates produced an apparent increase in enzyme activity. The molecules used were tetraethyl‐3‐oxypentyl‐bisphosphate and 1,8‐bis(diethylphosphoryl)octane.
Simplified Apoptosis Analysis on a Novel Accessible Cell Analyzer
Authors: Khan Asima , Gillis Katherine , Clor Julie , Tyagarajan Kamala
Published: June 2021

Accurate detection and measurement of cellular apoptosis is critical and essential in the drug development and discovery process and in understanding mode of compound action. The assessment of cellular apoptosis has however been limited due to the requirement for expensive and complicated instrument platforms and expertise. Recently, a novel low‐cost cell analyzer, the Muse Cell Analyzer with guided touch screen interface was introduced for simplified multi‐dimensional cell health analysis. In this study, we present results from the application of the Muse Cell Analyzer for apoptosis studies using the Muse Annexin V & Dead Cell Assay, a rapid no‐wash assay for the identification of apoptotic cells. Jurkat and HeLa cells were treated with a range of cytotoxic and anti‐tumor compounds such as gambogic acid, camptothecin and staurosporine, and the results from percentage of live, early, late apoptotic, and dead cells were evaluated. Dose and time responses data were obtained for these inducers. Precision studies demonstrated that a high degree of precision (<10%) could be obtained on the system for multiple samples. The ability to obtain accurate and precise apoptosis information on an easy to use, accessible platform will enable researchers to monitor and study the mechanism of compound action in the drug development and discovery process with greater ease.
Cation‐Dependent Mannose 6‐Phosphate Receptor: Tag, You're it!
Authors: Bord Maxwell , Hermsen Carolyn , Heur Wongsai , Jones Christopher , Kennedy Erica , Keslin William , Khang May , Leedom Hannah , LeMense Andrew , McMurray David , Naber Evan , Parish Danielle , Rice Mary Elizabeth , Rodgers Alana , Rosio Charles , Schubert Jordan , Smith Alexandra , Vogt Gina , Olson Linda , Dahms Nancy
Published: June 2021

Approximately 50 different lysosomal storage disorders affect one in every 5000 births, limiting life expectancy and quality of life. Therapies are available for a handful of these disorders. Sixty different lysosomal enzymes are responsible for recycling macromolecules in cells. In normal functioning cells, lysosomal enzymes are synthesized in the endoplasmic reticulum and are transported to the lysosome. Transportation of lysosomal enzymes occurs when a mannose 6‐phosphate (Man‐6‐P) tag is placed on these enzymes, which is recognized by the cation‐dependent mannose‐6‐phophate receptor (CD‐MPR). The CD‐MPR binds to the Man‐6‐P tags through a recognition site (Y45, Q66, H105, R111, E133, R135, Y143) and transports the enzymes from the Golgi apparatus to an endosome where enzymes are released from CD‐MPR due to a more acidic environment. Enzymes are finally transported via vesicles to the lysosome to accomplish their function. Without the Man‐6‐P tags, CD‐MPR cannot bind to the enzymes, prohibiting transportation to an endosome and ultimately the lysosome. If lysosomal enzymes are not properly transported, they are secreted out of the cell, causing lysosomal storage disorders due to buildup of macromolecules in the lysosome. The Brown Deer SMART Team (Students Modeling A Research Topic) created a physical model of CD‐MPR using 3D printing technology. Supported by grants from NIH‐NCRR‐SEPA and NIH‐CTSA.