bluuebunny/crossref_metadata_2025 huggingface dataset:
Practical analysis of type 1 aortic arch anatomy – Towards a standardized arch endograft
Authors: Finlay Adrienne , Johnson Marjorie , Forbes Thomas
Published: June 2021

Endovascular repair of aortic arch pathologies currently requires custom‐made endografts to reflect an individual's anatomy, causing a treatment delay. This study's purpose is to identify common patterns in the anatomy of the type 1 aortic arch and its branches to aid in the development of an off‐the‐shelf endograft.

TeraRecon 3D imaging software was used for morphological assessment of CT scans from 45 patients (26 M, 19 F; mean age 68). A total of 13 measurements were made including diameter, distance and orientation of vessels. Statistical analysis indicated various trends. The diameter of the aorta was larger more proximally (closer to the coronary arteries), the distance between the inominate artery (IA) and the left common carotid artery (LCC) was approximately half the distance measured between the LCC and the left subclavian artery (LSC). Branch ostia diameters consistently ranked in the following order IA>LSC>LCC. Lastly, the LCC orientation, with respect to the other two vessels, was within a 50° range.

This study provides useful information for the production of an arch endograph prototype reflecting the anatomy of the majority of patients. Prospective validation of this design is required.

Grant Funding Source: Departmental
CASE REPORT: Cadaveric finding of mucocele appendix
Authors: Farias Anna C , Webber Christine A
Published: June 2021

INTRODUCTION

This case report describes an abnormally enlarged cadaveric appendix subsequently identified as a mucocele. An appendicular mucocele is defined as a general macroscopic dilation of the appendicular lumen caused by abnormal accumulation of mucous (Persaud, 2007). There has been no previous published report of an appendicular mucocele in the cadaver. Of the 608 cadavers dissected in the last 8 years at the University of Alberta this was our first observation.

CASE REPORT

The recorded cause of death for this 86 year old female patient was congestive heart failure. Routine dissection of the gastrointestinal system revealed an extremely large vermiform appendix. It was untethered and presented in the pelvic position i.e., directed toward the pelvic brim. Apart from of the usually large size, there were no overt signs of inflammation of the appendix or its surrounding tissue. The mesoappendix did not display any pathology and as expected, it contained the appendicular vessels.

CONCLUSION

This is the first reported case of an aberrantly enlarged mucocele appendix in the cadaver.

Grant Funding Source: Professional Expense Allowance
Soft Tissue Relationships of the Multifidus Muscle at the Lumbosacral Junction and the Sacroiliac Joint
Authors: Jones Beth Moody , Carey Fred
Published: June 2021

Impairment of the lumbosacral multifidus muscle has been identified as a significant factor contributing to low back pain. This report describes the anatomical relationships of the sacral portion of the multifidus to neighboring soft tissue structures in the lumbosacral (LS) and sacroiliac (SI) regions.

The LS regions of sixteen embalmed cadavers were examined by means of traditional anatomical dissection. Detailed dissections of the thoracolumbar fascia, erector spinae tendon (EST), gluteus maximus proximal attachments, multifidus and the sacral supraspinous ligaments were performed. The depth of the sacral supraspinous ligament at the LS junction was measured.

In all cadavers, the multifidus had attachments to the posterior sacroiliac ligament, to the sacrum, to the sacral supraspinous ligament and to the inferior aspect of the EST. There was a broad attachment of the gluteus maximus to the superior surface of the EST. The depth of the sacral supraspinous ligament from the erector spinae to the LS junction was grossly 2.5 cm (N=11).

The sacral component of the multifidus muscle was found to have complex relationships with muscles and connective tissue associated with the LS junction and the SI joint. An understanding of these soft tissue relationships is critical for the clinician who wishes to use anatomical evidence to guide treatment of injuries in this region of emerging clinical importance.

Grant Funding Source: none
Ouabain Induces Caspase‐2 Dependent Rock II Cleavage In HeLa Cells
Authors: özdemir aysun , bülbül döne , ark mustafa
Published: June 2021

We have previously showed that ouabain induced Rho kinase II (Rock II) cleavage via caspase‐2 dependent manner and produced ~140 kDa fragment of the enzyme in HUVEC during apoptosis. In the current work we evaluated that whether this phenomenon is unique for HUVEC or appears in different cell types especially in cancer cell lines. Subconfluent HeLa cells were exposed to ouabain (10 μM, 18 h), Rock inhibitor Y‐27632 (10 μM, 30 min before ouabain), caspase‐2 inhibitor z‐VDVAD‐fmk (25 μM, 2h before ouabain), caspase‐3 inhibitor Z‐DEVD‐fmk (25 μM, 2h before ouabain) and pan caspase inhibitor z‐VAD‐fmk (25μM, 2h before ouabain). After the treatments, pictures were taken through a phase‐contrast microscope at random sites and cells with blebs were counted. Rock II cleavage was evaluated by Western Blot and an xCelligence real time cell analyzer DP were used for cell detachment assay. Ouabain treatment resulted in membrane blebbing and cell‐substratum detachment. Y‐27632 did not prevent ouabain‐induced cell detachment. Neither Y‐27632 nor caspase inhibitors inhibited the formation of blebs. Therefore our results suggest that in contrast to previous works, Rock may not play a profound role in the formation of membrane blebs. Very little basal Rock II cleavage was detected in HeLa cells. Ouabain treatment enhanced this basal cleavage of Rock II. Although this cleavage was not inhibited by Y‐27632, all the caspase inhibitors prevented the proteolytic clevage of Rock II. It has been shown that activation of caspase‐3 results in cleavage and activation of caspase‐2. Therefore our results indicate that ouabain induces caspase‐3, which induces caspase‐2 activation causing proteolytic cleavage of Rock II in HeLa cells. As a result, ouabain‐induced Rock II cleavage by caspase‐2 may be a common process in different cell types.
An Archaeal B‐family DNA Polymerase Exists as a Trimer with Additional Annealing and Terminal Transferase Activities
Authors: Trakselis Michael
Published: June 2021

The primary DNA replication polymerase from Sulfolobus solfataricus (Dpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to cooperatively associate with itself to promote trimer formation. We have determined that these are specific polymerase‐polymerase interactions around DNA. This trimeric complex has unique thermodynamic binding parameters due to changes in the coupled equilibria of the various polymerase/DNA complexes. Interestingly, this unusual trimeric DNA polymerase complex has an increased kinetic rate of synthesis, extremely long processivity, increased DNA annealing activity and robust terminal transferase activity. Although the monomeric polymerase is fully functional, the trimeric polymerase affords additional activities that result in efficient activities at high temperatures in DNA replication or repair. Taken together, these results suggest the presence of a dynamic trimeric DNA polymerase complex that is able to process DNA substrates more efficiently than the monomeric form. This trimeric polymerase complex is implicated in the maintenance of the archaeal genome through coupled activities in DNA replication and repair.
Transcription alters Chromatin to Negatively Influence Replication Initiation
Authors: Carlile Candice Marie , Cook Jean Gowen
Published: June 2021

DNA replication begins at many defined sites in the genome called origins of replication. These origins must be spaced throughout the genome to complete DNA replication in a timely manner, but it's clear that origin activity is excluded from certain regions of the genome, such as gene bodies. Here, we show that chromatin modifications contribute to preventing replication initiation within gene bodies.

We recapitulated previous findings that transcription through an origin inhibits replication initiation from that origin. We examined if transcription machinery inhibits replication by: (1) physically displacing replication factors or (2) constructing a chromatin environment incompatible for replication initiation. Interestingly, we eliminate this inhibition of origin activity by deleting Set2, a histone methyltransferase that co‐transcriptionally adds methyl groups to histone H3 K36. These data suggest that methylation at H3K36 impedes replication initiation. In addition, artificially tethering Set2, but not catalytically dead Set2 near a plasmid‐born origin of replication, inhibits the ability of that origin to initiate DNA replication. These results suggest that transcription‐induced chromatin changes prevent replication initiation from an origin of replication. Mechanistic studies will elucidate how this methylation impacts the recruitment of replication proteins to prevent initiation.
DEVELOPMENTAL REGULATION OF THE MURINE β‐GLOBIN LOCUS
Authors: Oum Ji‐Hyun , Som Indrani , Eszterhas Susan , Little Jane , Fiering Steven , Dean Ann
Published: June 2021

The mouse β‐globin locus contains four genes sequentially expressed during development—εy and βH1 in the embryo and βmaj and βmin in the adult—under the influence of an upstream locus control region (LCR). 3C experiments have shown that the adult β‐globin genes establish proximity with the LCR to effect gene activation, but whether this is the case for the embryonic genes, which are closer to the LCR, is unclear. Using a knock‐in approach, we targeted a copy of the chicken β‐globin HS4 insulator between the LCR and εy, the closest downstream gene. Imposition of the insulator decreased recruitment of transcription activators and RNA pol II to εy and βH1 and repressed their expression. Instead, RNA pol II accumulated at the insulator accompanied by increased intergenic transcription. This result suggests that the insulator interrupts pol II tracking or spreading from the LCR to the downstream embryonic genes. We are breeding mice with this insertion to test its function during development. In addition, we are targeting HS4 to a position between the embryonic and adult globin genes. These experiments should provide insight into mechanisms underlying LCR/globin gene communication during development.
An analysis of the epigenetic basis of Argonaute 2 function at DNA repetitive sequences in human cells
Authors: Giles Keith E , Lefevre Gaelle , Felsenfeld Gary
Published: June 2021

To explore the role of RNAi –dependent mechanisms in maintaining heterochromatic structures in vertebrates, we have determined the distribution of binding sites for the protein Argonaute2 (hAgo2) in repeat sequences of the human cell line K562. Ago2 is associated with silent regions of the genome, and preferentially bound to silent satellite repeats. Peaks of hAgo2 binding in satellites were associated with roughly equal amounts of both sense and anti‐sense transcripts, were enriched for both histone H3 lysine 9 tri‐ and dimethylation, and depleted for acetylation of H3 lysine 9 and H4 lysine 16. Surprisingly, hAgo2 peaks and satellite repeats were enriched for nucleolar small RNAs and genomic regions known to associate with the nucleolus. Immunofluorescence indicated that hAgo2 and the remnants of the nucleolus co‐localized during mitosis in the vicinity of the chromosomes and the mitotic spindle. This work supports our previously published model of RNAi‐mediated maintenance of heterochromatin structure.
Exploring microRNA targets of angiogenin
Authors: Mir Aamir , Devaraj Tanuja , Dickson Kimberly Anne
Published: June 2021

Angiogenin (ANG) is a pancreatic‐type ribonuclease that induces neovascularization and facilitates tumor metastasis. In addition, mutations in ANG are associated with both familial and sporadic amyotrophic lateral sclerosis. Although relatively weak, the ribonucleolytic activity of ANG is essential for its function: catalytically inactive variants of ANG do not induce blood vessel growth, and impair neuronal health. ANG degrades tRNAs in cultured cells exposed to high levels of stress, yet the RNA targets relevant to angiogenesis have not been identified. In this study, we examined the effect of ANG on the relative abundance of microRNAs (miRNA) in HeLa and human umbilical vein endothelial (HUVE) cells. Using real‐time PCR, we measured the abundance of 4 miRNAs relative to a spliceosomal U6 RNA control. We observed significant increases and decreases in miRNA abundance in both HeLa and HUVE cells, suggesting that ANG might modulate miRNA expression, maturation, or degradation.

The Lawrence University Excellence in Science Fund supported this work.
Protective function of autophagy against oxidative stress in zebrafish
Authors: Imamura Shintaro , Yabu Takeshi , Yamashita Michiaki
Published: June 2021

Autophagy is a catabolic process by the lysosomal degradation of own components in cells. Molecular mechanisms involving in the cell damage and protective process under oxidative stress have not been characterized. To identify the autophagic pathway regulating the protective function under oxidative stress conditions in vivo, zebrafish models for macroautophagy can be used. To observe the induction of autophagy in zebrafish embryos, we generated the transgenic lines which expressed MAP1‐LC3B (LC3)‐GFP fusion protein as a marker of autophagy. When zebrafish embryos were immersed in hydrogen peroxide (0.1–1000 μM), tert‐butyl hydroperoxide (BHP, 0.1–10 μM) and methyl viologen (paraquat, 10–1000 μM), LC3 was induced. Morpholino antisense oligos for ATG5 and ATG7 were used to inhibit autophagic pathways. Oxidized proteins and DNA were induced in the 24 h embryo under oxidative stress conditions. Inhibition of the ATG5 and ATG7 functions caused developmental delay and embryonic lethal under oxidative stress conditions. Autophagy deficiency caused genomic instability under oxidative stress. Some chromosomes were broken and caused gene aneuploidy. These findings indicated that autophagy was essential to prevent the accumulation of oxidized proteins and the cells with a chromosome aberration to promote normal development.
Zymophagy, a novel mechanism for the inducible and selective autophagic degradation of secretory granules
Authors: Vaccaro Maria Ines , Grasso Daniel , Molejon Maria Ines , Calo Natalia Causada , Re Andrea Lo , Boggio Veronica , Ropolo Alejandro
Published: June 2021

Autophagy has recently elicited significant attention as a mechanism that either protects cells or promotes cell death. However, different autophagy pathways and cellular conditions remain to be elucidated. We report a thorough cellular and biochemical characterization of a novel selective autophagy that works as a protective cell response. This new selective VMP1‐mediated autophagy is activated in pancreatic acinar cells during pancreatitis‐induced vesicular transport alteration, in order to sequester and degrade potentially deleterious activated zymogen granules. We coined the term zymophagy to refer to this process. Zymophagy is mediated by VMP1, the ubiquitin‐protease USP9x and the ubiquitin binding protein p62. Moreover, VMP1 interacts with USP9x, indicating that there is a close cooperation between the autophagy pathway and the ubiquitin recognition machinery that is required for selective autophagosome formation. Zymophagy is activated by CCK‐R hyperstimulation in genetically engineered mice and cultured acinar cells, and by acute pancreatitis in humans. Furthermore, zymophagy has pathophysiological relevance by controlling pancreatitis‐induced intracellular zymogen activation and helping to prevent cell death. Together, this data reveals a novel selective form of autophagy mediated by the VMP1‐USP9x‐p62 pathway, playing a critical role in the protective cell response to disease.
Substrate recognition by Clp/Hsp100 proteins
Authors: Johnston Danielle , Doyle Shannon M , Hoskins Joel R , Wickner Sue
Published: June 2021

Clp/Hsp100 proteins are a major class of molecular chaperones belonging to the AAA+ family. Some members, such as ClpA, are involved in protein unfolding and degradation, while others, including E. coli ClpB and S. cerevisiae Hsp104, participate in the disaggregation and reactivation of aggregated proteins. Many proteins form disordered aggregates under severe stress but some proteins form ordered aggregates (amyloids) under normal growth conditions and are hallmarks of many devastating diseases, such as Alzheimer's and prion diseases. In yeast, Sup35 assembles into an amyloid that is dependent upon Hsp104 for propagation in vivo. Despite homology between the Clp proteins, only Hsp104 has been shown to have roles in both the assembly and disassembly pathways of a truncated form of Sup35 in vitro. Therefore, we have examined the substrate recognition for ClpB, Hsp104 and ClpA in order to better understand the seemingly unique function of Hsp104 in Sup35 remodeling. To do this, we monitored substrate binding and unfolding of native GFP fused to the amyloidogenic Sup35 NM polypeptide or to various nonamyloidogenic peptides of different lengths and sequences. The results show that ClpB, Hsp104 and ClpA differ in substrate binding preferences.
Colorectal cancer desmoplastic reaction affects tumor cell invasion
Authors: Coulson‐Thomas Vivien Jane , Coulson‐Thomas Yvette May , Gesteira Tarsis Ferreira , Pinhal Maria AS , Friedl Andreas , Toma Leny , Nader Helena Bonciani
Published: June 2021

During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread, therefore the study of desmoplasia is of vital importance. Myofibroblasts synthesize an amalgam of products including collagens and other ECM proteins, such as proteoglycans and are activated during a desmoplastic reaction. Small leucine rich proteoglycans have been characterized surrounding breast and pancreatic tumors and have the ability to suppress cell proliferation. In this study we have analyzed desmoplasia co‐cultivating colorectal cancer cells (Caco‐2 and HCT116) and myofibroblasts using various co‐culture systems. Our findings demonstrate that direct cell‐cell contact between myofibroblasts and colorectal cancer cells evokes an up‐regulation of the expression of ECM by myofibroblasts. The ECM accumulation produced when myofibroblasts are co‐cultivated with colorectal cancer cells appears unorganized and in bundles. This ECM accumulation slowed the migration and invasion of the colorectal tumor cells in both 2‐D and 3‐D co‐culture systems.
ATF3 Induction by Genistein is MAPK/ERK‐ and ATF4‐ independent in Human Cancer Cell DLD‐1
Authors: Zhang Yukun , Chen Hong , Pan Yuan‐Xiang
Published: June 2021

Activating transcription factor 3 (ATF3) is an adaptive response gene that plays important roles in tumorigenesis. The induction of ATF3 is mostly immediate and transient. Genistein, a soy‐derived isoflavone, has a growth inhibition effect on cancer cells. In the present study, we report that genistein persistently induces ATF3 and asparagine synthetase (ASNS) upregulation in the DLD‐1colon cancer cell line. Examination of the mitogen‐activated protein kinase (MAPK)/extra‐cellular signal regulated kinase (ERK) pathway and phospho‐eIF2α (p‐eIF2α)/activating transcription factor 4 (ATF4)/ASNS pathway shows that both can be activated by genistein treatment. Blockage of ERK phosphorylation did not abolish genistein‐induced ATF3 upregulation. ATF4 knockdown by siRNA transfection showed that induction of ATF3 occurred independently of ATF4. Furthermore, knockdown of ATF4 did not affect genistein‐induced growth inhibition; however, genistein‐triggered apoptosis was dependent of ATF4. Both of MAPK/ERK and ATF4 are required for genistein‐triggered ASNS induction. The difference we observed between ATF3 and ASNS induction by genistein treatment reveals a nonconventional stress‐induced ATF3 upregulation in colon cancer cells. These findings will facilitate future research on the mechanism of genistein effects on colon cancer development and treatment.
Folic acid supplementation inhibits NADPH oxidase‐mediated superoxide anion production in the kidney
Authors: Hwang Sun‐Young , Siow Yaw L , Au‐Yeung Kathy K.W. , House James , Karmin O
Published: June 2021

Hyperhomocysteinemia (HHcy), a condition of elevated blood homocysteine (Hcy) levels, is a metabolic disease. Our recent studies indicate that HHcy can lead to renal injury by inducing oxidative stress. Oxidative stress is one of the important mechanisms contributing to Hcy‐induced tissue injury. Folic acid supplementation is regarded as a promising approach for prevention and treatment of cardiovascular disease associated with HHcy due to its Hcy lowering effect. However, its effect on the kidney is not clear. The aim of this study was to examine the effect of folic acid supplementation on Hcy‐induced superoxide anion production via NADPH oxidase in the kidney. HHcy was induced in male SD rats fed a high‐methionine diet for 12 wk with or without folic acid supplementation. There was a significant increase in NADPH oxidase‐mediated superoxide production and lipid peroxidation in HHcy rats. Folic acid supplementation effectively antagonized HHcy‐induced oxidative stress. Inhibitory effect of folic acid on NADPH oxidase activation was mediated through inhibition of NOX4 and p22phox expression. In addition, folic acid supplementation attenuated xanthine oxidase activity and restored SOD activity in the kidney. These results suggest that folic acid supplementation may offer renal protective effect against oxidative stress. (This study was supported by NSERC)
Effect of adiponectin on human colorectal‐cancer cell proliferation
Authors: Copeland Matthew James , Branch Velora A , Parker Chasta L
Published: June 2021

Adiponectin (Acrp30) is a protein hormone released from adipocytes that has been shown to have an anti‐proliferative effect on cancer cells. Three cellular receptors from the progestin and AdipoQ Receptor (PAQR) family have been identified that can actively bind Acrp30: AdipoR1 and AdipoR2, located on the cell membrane; and AdipoR3 in the Golgi body. Currently, it is known that AdipoR1 and R2 allow Acrp30 to regulate fatty acid and glucose metabolism as well as cell growth and proliferation. Prior research has indicated that AdipoR1 and AdipoR2 may be involved in the Acrp30 anti‐proliferative effect but there has been minimal research on if or how AdipoR3 (PAQR3) may be involved. To determine a possible relationship between Acrp30, AdipoR3, and cell proliferation, the production of mRNA in HT‐29 human colo‐rectal cancer cells in the presence or absence of Acrp30 was quantified using quantitative real‐time PCR (qRT‐PCR). These mRNA levels were also compared to the levels of AdipoR1 and R2. Briefly, the mRNA of the cell was extracted, converted to cDNA, and evaluated by qRT‐PCR using fluorescent probe primers. Following this study, RNAi knockdown experiments of AdipoR3 will be performed after which the anti‐proliferative effect of Acrp30 on HT‐29 cells will be reanalyzed. Preliminary results confirm the anti‐proliferative nature of Acrp30 and also indicate altered AdipoR3 mRNA abundance in adiponectin treated colo‐rectal cancer cells. This work was supported by the Henson School of Science and Technology, Salisbury University, and the Cort Scholarship.
Identification of Trafficking Motifs Involved in Constitutive and Regulated Trafficking of System xc−
Authors: Georges Anne Elizabeth , Chase Leah Ann
Published: June 2021

System xc−, a membrane transport system that exchanges glutamate for cystine, has been shown to traffic to and from the plasma membrane, enabling a rapid response to oxidative stress. Trafficking is a common form of regulation among transport proteins, and various amino acid motifs have been shown to play integral roles in the trafficking of these transporters. This study sought to identify amino acids within xCT that are involved in its trafficking. Carboxyl‐terminal truncations and point mutants of xCT were constructed in a FLAG‐tagged cDNA vector and transfected into PC12 cells. The trafficking behavior of the mutant constructs was assayed using both fluorescence microscopy and a biotinylation protocol to separate cell surface proteins from intracellular proteins. Preliminary analysis of the data suggests 1) loss of the carboxyl‐terminus results in significantly reduced cell surface expression of xCT and 2) disruption of a consensus phosphorylation site leads to greater membrane localization of xCT, providing a mechanism for rapid regulation of transporter trafficking and activity. This research was supported by the Arnold and Mable Beckman Foundation and NSF RUI # 0843564.
Inhibition Of Cancer Cell Growth By Na/K‐ATPase Mimetic pNaKtide
Authors: Li Zhichuan , Lai Fangfang , Xie Joe X , Shapiro Joseph I , Xie Zijian
Published: June 2021

Cells contain a large pool of non‐pumping Na/K‐ATPase that participates in signal transduction. Here, we show that the surface expression of Na/K‐ATPase is significantly reduced in human prostate carcinoma and cancer cell lines because of increased endocytosis. This down‐regulation impairs the ability of Na/K‐ATPase to regulate key signaling processes. Supplement of the Na/K‐ATPase or pNaKtide, a mimetic peptide derived from Na/K‐ATPase, normalizes the defective signal transduction. Consequently, these treatments stimulates apoptosis, inhibits growth and migration in cultures of cancer cells. Moreover, administration of pNaKtide inhibits angiogenesis and growth of tumor xenograft. Thus, the new findings demonstrate the in vivo effectiveness of pNaKtide, and suggest that the defect in Na/K‐ATPase‐mediated signal transduction may be targeted for developing anti‐cancer therapeutics.
Computational Design of a Non‐lipid LPA2 Receptor Agonist With Antiapoptotic and Radioprotective Action
Authors: Tigyi Gabor , Nagyne‐Kiss Gyongyi , Fells James , Liu Dianna , Valentine William , Yue Junming , Emmons‐Thompson Karin , Yates Ryan , Shuyu E. , Lai Yun‐Ju , Lin Fang‐Tsyr , Parrill Abby L. , Deng Wenlin
Published: June 2021

Lysophosphatidic acid (LPA) is a growth factor‐like lysophospholipis that rescues apoptotically condemned cells. LPA protects IEC‐6 intestinal epithelial cells in vitro and intestinal crypts in vivo from radiation‐induced apoptosis. Using LPA receptor KO mice we established that the LPA2 receptor subtype plays an essential role in the antiapoptotic mechanism. Through its C‐terminal PDZ and LIM‐binding motifs LPA2 forms agonist‐dependent macromolecular signaling complexes. LPA2 interacts with the proapoptotic protein Siva‐1 and targets it for proteasomal degradation, which in turn arrests DNA‐damage‐activated apoptosis. Full activation of the NFkB and ERK1/2 prosurvival pathway requires formation of an LPA2, NHERF2, and TRIP‐6 ternary macromolecular complex. These signals inhibit the intrinsic mitochondrial apoptosis pathway. Furthermore, transfection of the LPA2 receptor into mouse embryonic fibroblasts derived from LPA1/2 double knockout mice restores LPA‐dependent radioprotection. Conditioned medium from gamma‐irradiated U937 cells induces bystander apoptosis in non‐irradiated IEC‐6 cultures and LPA2‐transfected MEFs. Using a validated computational model of the LPA2 receptor we developed a pharmacophore model and used it for in silico drug discovery of non‐lipid LPA2 agonists. We found a novel LPA2‐selective agonist GRI977143, which elicits an antiapoptotic effect and protects IEC‐6 cells from radiation‐ and chemotherapy‐induced apoptosis. GRI977143 also increases the survival of mice exposed to lethal levels of radiation. Supported by CA92160 and AI08405.
Effects of whey protein supplements and exercise training on body composition and dietary compensation in middle‐aged overweight/obese adults
Authors: Weinheimer Eileen M , Conley Travis B , Kobza Vanessa M , Sands Laura P , Lim Eunjung , Janle Elsa M , Campbell Wayne W
Published: June 2021

Purpose

This double‐blind, randomized, placebo‐controlled, community‐based 9‐month study assessed the effects of whey protein (WP) and exercise on body composition and dietary compensation in middle‐aged overweight/obese adults.

Methods

Men (n = 88, 48 ± 8 y, 30.6 ± 2.8 kg/m2) and women (n = 133, 48 ± 8 y, 29.8 ± 2.7 kg/m2; mean ± SE) consumed 200 kcal supplements with 0 (n = 84), 10 (n = 82), 20 (n = 25), or 30 (n = 30) g WP twice daily. All subjects performed resistance (2 d/wk) and aerobic (1 d/wk) exercise.

Results

24‐h urinary urea nitrogen output was progressively higher with greater WP, consistent with increasing total protein intake (group‐by‐time, p=0.009). Among all subjects, from pre to post strength (1RM) increased 15 ± 1 % (65.8 ± 3.8 kg, p<0.001), VO2max increased 9 ± 1 % (2.9 ± 0.4 ml·kg−1·min−1, p=<0.001), body weight was unchanged (0.1 ± 0.3 kg, p=0.8), lean body mass increased 1.9 ± 0.2 % (1.0 ± 0.1 kg, p<0.001) and fat mass decreased 2.6 ± 0.7 % (−0.9 ± 0.2 kg, p=0.001). Non‐supplement energy intake decreased 331 ± 49 kcal/d (p<0.001), thus the subjects maintained body weight by compensating for the 400 kcal/d supplements. WP amount did not influence these responses.

Conclusion

Whey protein supplementation did not promote exercise‐induced changes in strength, aerobic capacity and body composition.

Grant Funding Source: US Whey Protein Research Consortium; NIH T32AG025671 and UL1 RR025761
Analysis of intracellular localization of viral RNA sensor, RLR
Authors: Onomoto Koji , Yoneyama Mitsutoshi , Fujita Takashi
Published: June 2021

Type I interferons (IFNs) play a pivotal role for antiviral innate immunity and subsequent modulation of adaptive immunity. Normally IFN genes are not expressed, however upon viral infection, the genes are transiently activated, and the secreted IFNs inhibit viral replications through induction of a series of IFN stimulated genes.

Viral RNA appearing in cells as a result of viral infection is detected by cytoplasmic sensors, retinoic acid inducible gene‐I (RIG‐I)‐like receptors (RLRs). RLRs, which are DExD/H ‐ box containing RNA helicases, consist of RIG‐I, melanoma differentiation‐associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). All RLRs contain a conserved C‐terminal domain, which is responsible for specific recognition of the viral RNAs. Although the domain structure of RLRs for detection of non‐self RNA has been elucidated, it has been unclear how RLRs encounter viral RNA and trigger signaling.

To address this issue, we made specific antibodies against RLRs and examined their intracellular localization in virus‐infected cells by immunostaining using confocal fluorescence microscope. When cells were infected with Influenza A virus (IAV) deficient for its Non‐structural protein 1 (NS1), RIG‐I accumulated as granular‐like pattern in the cyotosol and colocalized with viral Nucleocapsid protein. Moreover, RIG‐I colocalized with MDA5, LGP2 and several antiviral proteins such as PKR and OAS. We also detected similar localization patterns of RIG‐I in cells‐infected with various RNA viruses such as HCV and NDV. We are going to discuss relationship between the subcellular localization of RLRs and viral detection in detail.
Kaposi¡s sarcoma‐associated herpesvirus degrades cellular TIR‐domain‐containing adaptor‐inducing beta interferon (TRIF)
Authors: Zhang Luwen , Ahmad Humera , Gubbels Rachel , Meyer Florencia , Waterbury Thomas , Lin Rongtuan
Published: June 2021

Kaposi's sarcoma‐associated herpesvirus (KSHV) is a human gamma herpesvirus associated with several human malignancies. The replication and transcription activator (RTA) is required for KSHV lytic replication. TIR‐domain‐containing adaptor‐inducing beta interferon (TRIF) is an adaptor molecule critically involved in the Toll‐like receptor 3 (TLR‐3) and TLR‐4 signaling pathways for type I interferon (IFN) production, a key component of innate immunity. We find that RTA specifically degrades TRIF by shortening the half‐life of TRIF protein. This RTA‐mediated‐degradation is mediated through the ubiquitin‐proteasome pathway because proteasome inhibitors as well as knockdown of cellular ubiquitin expression alleviate the degradation. RTA may use its ubiquitin ligase domain and targets multiple regions of TRIF for the degradation. In addition, physiological levels of TRIF protein are down‐regulated during KSHV lytic replication when RTA is expressed. Finally, RTA down‐regulates double‐stranded RNA‐initiated activation of TLR‐3 pathway, in the absence of degradation of IFN regulatory factor 7 (IRF‐7). This work suggests that KSHV employs novel mechanism to block the innate immunity by degrading TRIF protein, which may contribute to evasion of host immunity by the virus.
Differential apoptotic response in normal and virus‐transformed human cells
Authors: Maczis Melissa A , Birckbichler Paul J
Published: June 2021

Human fibroblasts (WI‐38) and their simian virus‐transformed counterparts (VA13a), a paired cell system, represent a unique model for experimentation. WI‐38 cells demonstrated contact inhibition of growth, whereas, the VA13a counterpart continued to proliferate forming multiple cell layers. The objective of the study was to treat WI‐38 and VA13a cells with sodium butyrate to produce a differential effect, namely the transformed cells would undergo apoptosis, while normal cells would not. A combined treatment of sodium butyrate and calcium ionophore (A23187) would produce apoptosis in both cell lines. Cell viability was determined by trypan blue exclusion and Hoechst/propidium iodide assay was used to monitor apoptosis. Sodium butyrate‐treated VA13a cells showed an altered “normal” morphology. Treatment with calcium ionophore primed the cells for apoptosis allowing for the formation of the isopeptide crosslinks. Applied clinically, this approach has the potential to produce a differential chemical response in the transformed (cancer) cells. Support for the study was provided by Slippery Rock University and the College of Health, Environment and Science student/faculty grants.
Simultaneous analysis of cell death mechanisms and oxidative stress using live cell fluorescence microscopy
Authors: Yan Michelle , Clarke Scott T , Huang Shih‐Jung , Singh Upinder , Janes Michael S , Dolman Nicholas J
Published: June 2021

Cell death can occur through multiple pathways, such as apoptosis, autophagy, and necrosis. Although necessary for proper growth and development, dysregulation of apoptosis has been associated with a variety of diseases including cancer and neurodegenerative disorders. Increased oxidative stress has also been associated with these diseases and has been shown to lead to cell death. Cell death can occur through a single pathway, or in concert involving multiple pathways. The goal of this study was to utilize multi‐parametric fluorescence microscopy to examine temporal characteristics of cell death after induction by various agonists. Using a fluorogenic caspase substrate in combination with a probe for oxidative stress, we observed that increased oxidative stress was followed by caspase activation after induction by several agonists. By simultaneously examining multiple parameters over time we were able to define the temporal progression of apoptosis relative to the onset of oxidative stress. Moreover, we were able to further characterize the mechanism of cell death by discriminating between cells which were apoptotic (active caspase‐3/7), autophagic (LC3B‐postive autophagosomes), or both. This multi‐parametric approach provided detailed information at the cellular level so that correlations and temporal resolution could be determined between oxidative stress and cell death mechanisms.
Effects of Cisplatin Chemotherapy Drug in Zebrafish
Authors: De Jesus Hector Jose , Podratz Jewel , Greenwood Tammy M , Ekker Stephen C , Windebank Anthony J
Published: June 2021

Chemotherapy‐induced peripheral neuropathy is a serious, lifelong side effect that impairs quality of life for long‐term cancer survivors. Cisplatin (a platinum based chemotherapy drug) is used to treat various cancers. However, platinum drug‐induced neuropathy in 20–30 percent of patients and it is a dose‐limiting side effect. The chemotherapeutic mechanism of cisplatin is through DNA binding, inducing DNA damage leading to apoptosis. Our study used zebrafish (Danio rerio), that have a nervous system shown to be regulated by shared death pathways including p53, to establish a model of cisplatin neurotoxicity. We show a survival curve of 72 hour post fertilization zebrafish treated with cisplatin that was dose and time‐dependent. Immunostaining with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) showed cisplatin induced cellular apoptosis. Moreover, to demonstrate if cisplatin causes neuron‐specific apoptosis we colocalized Elav antibody (neuron‐specific marker) with TUNEL assay. This new model of cisplatin neurotoxicity in zebrafish will allow further study of the p53 signaling pathway and its involvement in platinum drug‐induced neurotoxicity.
Gating of Pannexin‐1 Channels by P2X7R‐Induced Inflammation or Apoptosis in Macrophages
Authors: Antonopoulos Christina H. , Dubyak George R.
Published: June 2021

Pannexin‐1 hemichannels (Panx1) function as ATP efflux or dye influx channels and in inflammatory cascades initiated by P2X7 ATP receptors. Caspase‐mediated cleavage of the Panx1 C‐terminus is a gating mechanism for this channel in apoptotic cells. This study is testing whether Panx1 channel activity is similarly triggered by the delayed caspase‐3‐dependent apoptotic response to P2X7R activation in naïve macrophages versus the immediate caspase‐1‐dependent inflammatory response to P2X7R activation in endotoxin‐primed macrophages. Naïve or endotoxin‐treated J774 murine macrophages were transiently (10 min) stimulated with ATP to activate P2X7R and then incubated in ATP‐free medium for varying times prior to analysis of ethidium+ influx as an index of gated hemichannels. Increased ethidium+ influx was observed in endotoxin‐primed cells after 10 min of P2X7R stimulation followed by 10 min of ATP‐free incubation. In contrast, enhanced ethidium+ influx in naïve cells required more prolonged ATP‐free incubation following the 10 min P2X7R stimulation. Current experiments are comparing these responses in primary macrophages from control, Panx1‐knockout mice, and caspase‐1 knockout mice. Secondary activation of macrophage Panx1 hemichannels by P2X7R‐triggered apoptotic or inflammatory caspases may facilitate ATP‐induced ATP release as a feed forward loop for amplifying innate immune responses.

Support: R01‐GM36387
Luteolin inhibits Prevotella intermedia lipopolysaccharide‐induced production of nitric oxide and interleukin‐6 in murine macrophages by suppressing NF‐kappaB and STAT1 activity
Authors: Choi In Soon , Choi Eun‐Young , Jin Ji‐Young , Kim Sung‐Jo
Published: June 2021

Although a range of biological and pharmacological activities of luteolin have been reported, little is known about its potential anti‐inflammatory efficacy in periodontal disease. In the present study, we investigated the effects of luteolin on the production of inflammatory mediators by murine macrophage‐like RAW264.7 cells stimulated with LPS from P. intermedia, a major cause of inflammatory periodontal disease, and sought to determine underlying mechanisms of action. LPS was prepared from lyophilized P. intermedia ATCC 25611 cells by the standard hot phenol‐water method. Luteolin suppressed the production of nitric oxide (NO) and interleukin‐6 (IL‐6) at both gene transcription and translation levels in P. intermedia LPS‐activated RAW264.7 cells. Luteolin did not reduce NF‐κB transcriptional activity at the level of IκB‐α degradation. Luteolin blocked NF‐κB signaling through inhibition of nuclear translocation and DNA binding activity of NF‐κB p50 subunit and suppressed STAT1 signaling. Although further researches are required to clarify the detailed mechanism of action, we conclude that luteolin may contribute to blockade of the host‐destructive processes mediated by these two proinflammatory mediators, and could be a highly efficient modulator of host response in the treatment of inflammatory periodontal disease.
MAPK Response to X31 Influenza A Virus Infection in Murine Macrophage Cell Lines
Authors: Cannon Georgetta , Gronemus Jenny Q. , Williams Margaret S. , Callahan Michelle A. , Lowy R. Joel
Published: June 2021

RAW 264.7 and J774.1 murine macrophage cells are widely used to model responses to infectious diseases; therefore they were selected to explore early molecular responses to FLUA. NF‐kappa B is reported to be central to FLUA host‐response in other cell types. Previous data show that FLUA (X31/H3N2) does not activate the classical NF‐kappa B dependent pathway in these macrophages. Regulator proteins, I kappa B ‐alpha and ‐beta, are not degraded in response to FLUA infection, and p65 does not translocate from the cytoplasm to the nucleus. However, the cells display the expected increase in TNF‐alpha and other inflammatory cytokine and chemokine secretions. Mitogen Activated Phosphokinase (MAPK) signaling pathways are also reported to control production of inflammatory cytokines in response to FLUA. The activation of the MAPK pathway intermediates p38, Janus Kinases 1 and 2 (JNK 1/2), and Extracellular Regulated Kinases 1 and 2 (ERK 1/2) were investigated in both cell lines between 0.25 and 3 hours post‐infection. Each of these kinases showed increased phosphorylation post FLUA infection. JNK phosphorylation occurred early while p38 phosphorylation appeared late. Phosphorylation of ERK 1/2 occurred earlier in J774.1 cells as compared to RAW 264.7 cells. The results suggest that in these monocytic cells the MAPK pathways are important in the early pathogen activated molecular pattern (PAMP) response to FLUA.
Cloning of the autophagy‐related gene Beclin‐1 of the marine teleost Paralichthys olivaceus and its possible implication in viral replication
Authors: Kong Hee Jeong , Nam Bo‐Hye , Kim Young‐Ok , Kim Woo‐Jin , Lee Jeong‐Ho , Kim Dong Gyun , Kim Bong‐Suk , Kim Kyung‐Kil , Lee Sang‐Jun
Published: June 2021

Autophagy is essential for the maintenance of cellular homeostasis and for the orchestration of an efficient cellular response to stress. Beclin‐1, one of the central regulators of autophagy, participates in development and aging, and in protection against certain types of cancer, neurodegeneration and pathogen invasion. In the present study, we isolated the full‐length cDNA of PoBeclin‐1 from the marine teleost olive flounder Paralichthys olivaceus by screening of gill cDNA library and rapid amplification of cDNA ends (RACE). The PoBeclin‐1 cDNA is 2,179bp and encodes the polypeptides with 447 amino acids containing conserved Bcl‐2 binding and BH3 domains. PoBeclin‐1 transcripts were detected from early larva stage and ubiquitously in healthy flounder. The expression of PoBeclin‐1 mRNA was induced in the kidney and spleen challenged by viral hemorrhagic septicemia virus (VHSV), an economically important pathogen for aquaculture of flounder. Over‐expression of PoBeclin‐1 increased the expression of VHSV G protein and viral replication in HINAE cells. These results suggest that a potential role for PoBeclin‐1 in viral replication in flounder.
Identification of CNK3 in the Aldosterone‐regulated ENaC Regulatory Complex
Authors: Ziera Tim , Koo Eric , Soundararajan Rama , Pearce David
Published: June 2021

Members of the Connector Enhancer of KSR (CNK) family of proteins possess multiple protein interaction domains, and have been proposed to function as scaffolds possibly assisting various interactions in a multiple signaling cascade. Mammalian CNK3 is an aldosterone‐induced protein that is essential for the activity of the epithelial sodium channel (ENaC). We have shown earlier that ENaC surface expression and activity are regulated by the ENaC‐regulatory complex, GILZ1 and SGK1 are important aldosterone‐induced components thereof. The present study examines the role of CNK3 in GILZ1/SGK1‐mediated ENaC stimulation. Co‐immunoprecipitation experiments revealed specific physical interactions of CNK3 with both GILZ1 and SGK1 in an aldosterone‐dependent manner. GILZ1 markedly enhances the interaction of CNK3 with SGK1, and also augments CNK3 multimerization, an attribute that has been shown to be critical for protein scaffolding of other larger PDZ domain‐containing proteins. The present study suggests that CNK3 is an important component of the aldosterone‐regulated ENaC regulatory complex.
Different Scaffoldings of β‐arrestin‐1 in the Mechanism for Cofilin Activation
Authors: Lin Alice , DeFea Kathryn
Published: June 2021

β‐arrestin‐1 (β1) is a key mediator that activates and terminates G‐protein‐coupled receptor (GPCR) pathways independent of G‐protein coupling. Our lab has shown that β‐arrestins promote dephosphorylation and activation of cofilin activity downstream of protease‐activated receptor 2 (PAR2), resulting in an increase in actin filament reorganization and cell migration. Here, we demonstrate that β1 directly binds to both cofilin and the catalytic domain of LIM kinase (LIMK). Sandwich immunoassays demonstrate that both LIMK and cofilin bind to both N and C‐terminal β1 truncations containing amino acids 1–163 and 164–418 but do not bind a fragment consisting of amino acids 1–99, leading us to hypothesize that the binding domain for both proteins either encompasses a central region of β1. LIMK, which phosphorylates cofilin, inhibiting its actin filament severing activity is directly inhibited by β1 and by the β1 fragment containing amino acids 164–418, suggesting that one mechanism by which β1 enhances cofilin dephosphorylation is by binding to and inhibiting the kinase activity of LIMK. This project is supported by 3R01GM066151‐05S1.
Amyloid beta peptide internalization induces Myosin Vb redistribution
Authors: de Oliveira Leandro Teixeira , Matos Priscila A. , Provance D. William , de Andrade Leonardo Rodrigues , Mello Fernando G. , Sorenson Martha M. , Salerno Veronica P.
Published: June 2021

The fundamental amyloid hypothesis suggests that extracellular senile plaques formation precedes and leads to the neuronal abnormalities that underlie dementia. Recently the accumulation of Aβ has been demonstrated to occur within neurons prior senile plaques formation and is associated with alterations in the axonal trafficking and morphological abnormalities. Furthermore, intraneuronal Aβ accumulation has been reported to be critical to observed synaptic and cognitive dysfunction in AD. Myosin Vb is an actin‐based molecular motor implicated mainly in organelle transport of cargos in actin‐rich cortical regions, dendritic spines and axon terminals. Here we show by confocal microscopy and immunohistochemistry the intracellular accumulation of Aβ. The pattern of Aβ during the time course of incubation is reminiscent of the endocytic pathway. After an extended period of time, their distribution pattern converges and redistribute from the axon to cell body. We verified that internalization and pathern of distribution after cytoskeleton suggests an actin‐dependent process and a change in Aβ distribution was related to microtubule disruption. These observations are consistent with internalized Aβ disrupting intracellular trafficking by impacting myosin Vb which could influence the balance between Aβ production and Aβ clearance.

Supported by FAPERJ, CNPq, CAPES and PRONEX.