bluuebunny/crossref_metadata_2025 huggingface dataset:
Voltage‐dependent anion channel promotes mitophagy to protect neuron from death in an early brain injury following a subarachnoid hemorrhage in rats (730.1)
Authors: Zhou Changman , Lu Jianfei , Li Li , Li Jian , Mi Yongjian , Shi Zhao
Published: June 2021

The term mitophagy is coined to describe the selective removal of mitochondria by autophagy but the process itself is still contentious, especially in the early brain injury following subarachnoid hemorrhage (SAH). In the present study, we investigated the role of mitophagy following 48hours after SAH injury in rats. Specifically evaluating whether through an Voltage‐dependent anion channels(VDACs) interacting with microtubule‐associated protein 1 light chain3(LC3), mitophagy could orchestrate the induction of apoptotic and necrotic cell death in neurons. A VDAC1siRNA and a activitor Rapamycian(RAPA) were engaged. One hundred and twelve male Sprague‐Dawley rats were randomly divided into 4 groups: Sham, SAH, SAH+ VDAC1siRNA, and SAH+RAPA. Outcomes measured include mortality rate, brain edema, BBB disruption, and neurobehavioral testing. We also used western blotting techniques to measure the expressions of key mitophagic/autophagic proteins and pro‐apoptotic protein such as ROS, VDAC1, LC‐3II and Caspase‐3. Rapamycin treatment signiﬁcantly alleviated the mortality rate, cerebral edema, neurobehavioral deficits, and apoptotic and necrotic cell death in neurons as observed by histology following SAH injury. However, VDAC1siRNA deteriorated the brain injury on the basic injury of SAH. Immunohistochemical staining and western blot analysis demonstrated an increased expression of VDAC1, LC3, and a decrease of ROS and Caspase‐3 followed by rapamycin administration. In conclusion, this study showed that mitophagy following the SAH injury may in fact play a significant role in the neuroprotection following SAH and its mediation may be through attenuating the apoptosis and necrosis molecular pathway. This translates a preservation of functional integrity and an improvement in mortality.
Effects of maternal and post‐natal infection with porcine reproductive and respiratory syndrome virus on muscle growth and development in piglets (731.10)
Authors: Clark Diana , Clark Daniel , Rochell Samuel , Dilger Ryan , Johnson Rodney , Dilger Anna
Published: June 2021

Prenatal muscle development is altered by maternal nutrition, the effect of maternal infection during pregnancy on offspring muscle growth and development is less clear. We assessed muscle development in piglets born of dams infected with porcine reproductive and respiratory syndrome virus (PRRSV) during pregnancy and in postnatally infected PRRSV pigs. In study 1, pregnant gilts were inoculated with PRRSV or sterile culture medium (control) at d76 of gestation. Data collected from offspring at d0 and d28 of age included body and muscle weights and gene expression of longissumus dorsi muscle. In study 2, similar measurements were collected from pigs inoculated with PRRSV at d35 and sacrificed at d49 of age. Longissumus dorsi weight was reduced (P<0.01) at d28 in piglets from PRRSV inoculated gilts compared with control, but body or other muscle weights were unaffected at d0 and d28 of age. Furthermore, IGF1, IL‐6, and Pax7 expression tended to be reduced (P<0.10) at d28 in piglets from PRRSV inoculated gilts compared with controls, while myostatin and UCP3 expression did not differ. In addition, TNF‐α expression was increased (P<0.05) at d28 in piglets from PPRSV inoculated gilts compared with controls. Postnatal infection reduced (P<0.01) body and muscle weights in PRRSV inoculated‐piglets compared with controls. However, postnatal infection did not alter expression of myostatin, IGF1, Pax7, IL‐6, or TNF‐α.
Expression of tenomodulin in development mouse masseter muscle (731.2)
Authors: Sato Iwao , Miwa Yoko , Sunohara Masata , Shimada Kazuyuki
Published: June 2021

Tenomodulin (TNMD) is related to the type II transmembrane glycoprotein, which contains an angiogenesis inhibitor with an antiangiogenic cysteine‐rich C‐terminal domain. TNMD functions as an anti‐angiogenic molecule and is expressed primarily in hypovascular tissues, such as tendons and ligaments. The masseter muscle contains a tendinous skeletal element that is an important site of masticatory dysfunction. However, the relationship between the RNA transcript levels of TNMD and angiogenic factors, such as vascular endothelial growth factor (VEGF), in the connective tissue of developing mouse masseter muscle is unknown. Method: Crlj:CD1 (ICR) mice were fed and sacrificed at the age of day E12.5, E14.5 E17.5, E18.5, 0 ,5, 10,15 and 20 by an overdose of pentobarbital. Fresh samples of right masseter muscle and femoris rectus were removed for further investigation (n=3 in each group). The study followed the regulations of Nippon Dental University (i.e., Rules for the Care and Use of LaboratoryAnimals, no. 03‐34). A quantitative real‐time reverse transcription polymerase chain reaction (RT‐PCR) was used to measure the RNA expression of mouse TNMD and other endothelial markers. We also determined the protein expression of the TNMD gene and other endothelial markers using an enzyme‐linked immunosorbent assay (ELISA). Result: In the masseter muscle of developing mice aged E12.5 to 5 days, the levels of TNMD mRNAs is appeared and high from E17.5 (ANOVA, p<0.001) but gradually decreased in 5‐day‐old mice (ANOVA, p<0.001). In contrast, VEGF and CD31 are different expression pattern and decreased in mice aged 0 to 20 days (ANOVA,p<0.001). The different expression of TNMD indicates formation of tendon in the masseter muscle at embryonic stages in contrast to that of other angiogenesis markers.
Lower extremity skin temperature and intramuscular response to whole body vibration (731.6)
Authors: Crossley Kent , Denning W. , Rigby Justin , Feland J.
Published: June 2021

Previously established research has shown that whole body vibration (WBV) is effective at increasing intramuscular temperature (IMT) of the medial gastrocnemius (MG) at 26Hz. A “reaction” of itching and redness to WBV has been noted in prior research, but is not reported in all subjects. The purpose of this study was to determine if WBV affects skin and MG IMT differently in those exhibiting this reaction vs those with no reaction when performing a standard static semi‐squat WBV protocol. 60 subjects were screened, 10 exhibited a reaction; 6 had no reaction. Subjects completed two 5x60s bouts of WBV (26Hz, 4mm amplitude) on an oscillating platform with 15s rest between bouts. Skin and IMT were taken pre, mid (after 5 min vibration), and post 10 min of vibration. There was no difference in starting temperatures for skin or MG IMT. No significant change in skin or MG IMT of the non‐reactive group. Reactive group had significant increases in skin temperature (pre‐mid p<.0001, 2.32°C; pre‐post p<.0001, 2.57°C) and MG IMT (pre‐mid p<.0001, 1.5°C, mid‐post p<.004, 0.95°C, pre‐post p<.0001, 2.46°C). In conclusion, WBV exerts its effects upon reactive and non‐reactive groups differently. This difference could be due to histamine release due to WBV. Future research should be conducted to determine how WBV affects skin and muscle temperature or blood flow response.

Grant Funding Source: None
Plasma rich platelet use in experimental burn model of second and third degrees (734.4)
Authors: Monte‐Alto‐Costa Andrea , Venter Neil , Santos Jeanine , Marques Ruy
Published: June 2021

Plasma rick platelet (PRP) is a concentrated of platelets, that releases growth factors to stimulate healing. This study evaluates the effects of PRP on an experimental burn model. Wister rats were divided into 2 groups: second (SD) and third (TD) degrees burn; in each group, 8 animals were treated with PRP and 8 animals were not treated. Burn area was measured weekly until day 21, when animals were killed and samples collected. Sections were stained with hematoxylin and eosin and immunostained for CD68 and α‐smooth muscle actin. Hydroxyproline levels were determined by biochemical method. At day 21, wounds areas in SD where significantly smaller in the PRP treated animals. The density of inflammatory cells, CD68 positive macrophages, and blood vessels was greater in the control group of SD. In this study we showed that PRP treatment was able to improve the healing of second degree burns but not of the third degree burns.

Grant Funding Source: Supported by: FAPERJ, CNPq and CAPES
Blockade of the opioid growth factor receptor with naltrexone enhances wound repair in type 2 db/db mice (734.5)
Authors: Immonen Jessica , Zagon Ian , McLaughlin Patricia
Published: June 2021

Type 2 diabetes (T2D) is associated with impaired cutaneous wound healing, and can result in ulceration, infection and/or amputation. More than 26 million people in the U.S. have T2D and are vulnerable to epithelial‐related complications. New therapies regulating altered diabetic pathways are needed. Topical application of the opioid receptor antagonist naltrexone (NTX) dissolved in cream reverses delayed wound closure, and enhances angiogenesis and remodeling of full‐thickness wounds in T1D rats. NTX acts though the OGF‐OGFr axis to accelerate DNA synthesis and cell proliferation, and is a potential therapy for nonhealing wounds. In this study, the genetically obese mouse model db/db and normal C57Bl/6 mice received 6 mm diameter circular wounds on their dorsum. Wounds were treated topically 3 times daily with 10‐5 M NTX, or sterile saline, dissolved in cream, and photographed every 2 days. Db/db mice receiving saline had wounds that were 11‐92% larger than those in normal mice. NTX therapy reduced residual wound size by 13‐30% between days 8 and 14 compared to diabetic, vehicle‐treated wounds. Reepithelialization and DNA synthesis, as analyzed by epithelial thickness and BrdU labeling index, were accelerated in NTX treated wounds. These data indicate that a novel opioid receptor pathway is impaired under diabetic conditions, and modulation with NTX can regulate deleterious changes in healing seen in T2D.

Grant Funding Source: American Diabetes Association
Template homology determines the genetics and mechanisms of gross chromosomal rearrangements in S. cerevisiae (736.11)
Authors: Bell Sara , Putnam Christopher , Kolodner Richard
Published: June 2021

Double strand breaks (DSBs) can lead to genome instability and cell death, and must be faithfully repaired. In Saccharomyces cerevisiae, DSBs are primarily repaired via homologous recombination (HR), which uses homologous sequence as a template for repair. Improper repair can result in gross chromosomal rearrangements (GCRs) such as translocations. Aberrant HR may occur when a template sequence with imperfect homology is used instead of completely homologous sequence. It is not known how divergent homologous sequences are selected as targets for non‐allelic HR, or which pathways suppress the formation of such GCRs. To examine this, we devised 3 separate genetic assays to detect GCRs mediated by a distinct sequence with imperfect homology to the rest of the genome. The assays differ from each other in both percent homology to their target sequences and number of potential rearrangement targets. We find that the GCR rates for these assays differ from each other as well as from existing assays. Additionally, the GCRs detected by the 3 assays exhibit diverse genetic requirements, responding differently to mutations affecting the HR pathways, HR between divergent sequences, and the DNA replication checkpoint. Finally, we characterized the products of the GCRs observed in the 3 assays. These results suggest that the homology between the recombination substrate and its rearrangement targets influences the mechanisms and genetics of GCR formation and the GCR spectrum.

Grant Funding Source: NIH Grant GM26017
Reduced SHMT2 expression and serine/glycine ratio affect mtDNA (736.7)
Authors: Alonzo Judith , Stover Patrick
Published: June 2021

Maintaining proper nucleotide balance is required to avoid genome instability, for both mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA). Mitochondrial deoxynucleotide triphosphates (dNTPs) are obtained from either salvage or de novo biosynthesis pathways. De novo biosynthesis of thymidylate and purines is folate‐dependent. De novo thymidylate (dTMP) biosynthesis has been shown to occur within mitochondria for mtDNA synthesis and the nucleus for nuDNA synthesis. Disruption of nuclear de novo dTMP biosynthesis affects genome integrity and increases uracil levels in DNA.

In mitochondria, de novo dTMP biosynthesis requires serine hydroxymethyltransferase 2 (SHMT2), which transfers a one‐carbon unit from serine to tetrahydrofolate (THF), producing 5,10‐methyleneTHF and glycine; 5‐10‐methyleneTHF is then utilized by thymidylate synthase to generate dTMP from dUMP. In the nucleus, SHMT expression is limiting in de novo dTMP biosynthesis, and reduced SHMT expression elevates uracil content in nuDNA. We are investigating effects of reduced SHMT2 expression and alterations in the cellular serine/glycine ratio on mtDNA content, mitochondria mass, and uracil levels in mtDNA in HeLa cells. Preliminary data indicate that reduced SHMT2 expression and serine and glycine ratio affect mtDNA content and mtDNA integrity.

Grant Funding Source: Supported by NIH R37DK58144
Pathogen‐mediated regulation of small GTPases (739.9)
Authors: Neunuebel Maria , Chen Yang , Backlund Peter , Machner Matthias
Published: June 2021

Legionella pneumophila is an opportunistic pathogen that infects human alveolar macrophages causing a severe pneumonia known as Legionnaire’s disease. Upon phagocytosis into the host cell, L. pneumophila efficiently avoids endolysosomal fusion and degradation, and provides an environment that is suitable for bacterial replication by translocating a vast number of effector proteins into the host cell to subvert the function of key regulatory proteins such as small GTPases. Post‐translational modification of small GTPases is emerging as a critical strategy for virulence of intracellular pathogens, including L. pneumophila. My objective was to dissect how L. pneumophila exploits the ER‐to‐Golgi vesicle transport pathway through reversible AMPylation (covalent addition of adenosine 5‐monophosphate) of Rab1, a key regulator of endoplasmic reticulum (ER)‐to‐Golgi traffic. Rab1 is recruited to the Legionella‐containing vacuole (LCV), activated, and then AMPylated by SidM. Our results show that AMPylated Rab1 is locked in the active conformation until the Legionella effector protein SidD de‐AMPylates Rab1, causing the inactivation and subsequent release of Rab1 from the LCV. AMPylation is a novel post‐translational modification in the pathogen‐host context and it is an efficient strategy to control host cellular pathways during infection by L. pneumophila as well as other intracellular bacterial pathogens.

Grant Funding Source: Supported by the National Institute for Child Health nad Human Development
Autophagic degradation of CD81 by hepatitis C virus: a novel mechanism underlying viral superinfection exclusion (740.1)
Authors: Chen Steve , Ke Po‐Yuan
Published: June 2021

Autophagy, a lysosomal degradation pathway, is known to positively regulate HCV replication. However, it remains unknown how cells withstand HCV productive replication in the cell context of autophagy activation. Here we showed that HCV infection triggered superinfection exclusion through downregulating the cell surface and total expressions of CD81, an important HCV entry (co)receptor. HCV‐induced downregulation of CD81 was associated with the autophagy‐mediated, lysosomal degradation process as interference with autophagolysosome maturation with pharmacological inhibitors restored the level of CD81 in HCV‐infected cells. Moreover, confocal and electron microscopy analyses showed that HCV infection led to engulfment of CD81 within autophagic vacuoles matured at different stages of autophagy, and that these CD81‐coresiding autophagosomes were originated from endoplasmic reticulum (ER)‐derived preautophagosomal structure (PAS). Knockdown of autophagy‐related genes required for the assembly of PAS in HCV‐infected cells relieved cytoplasmic retention of CD81, restored CD81 surface expression, and re‐conferred cell permissiveness to HCV infection. Our findings reveal a novel role of autophagy in restricting HCV superinfection by sequestration and degradation of its entry (co)receptor through ER‐originated PAS and fusion with lysosome, thereby maintaining virus‐cell homeostasis.
Identification of differentially expressed transcripts in Tdrd7 null mutant mouse lens (747.1)
Authors: Patel Shaili , Barnum Carrie , Lachke Salil
Published: June 2021

Lens is a transparent tissue within the eye that focuses light on the retina and facilitates high‐resolution vision. Cataract is an eye disease that results due to the loss of lens transparency and causes severely impaired vision. It can be caused by genetic changes, aging or physiological conditions like diabetes. Treatment depends upon the patient's specific visual needs and often involves cataract surgery. Over 77 million individuals are affected worldwide, and in the United States alone, costs exceed $3 billion annually. Recently, a mutations in the human TDRD7 (Tudor domain containing protein 7) gene were shown to cause congenital/pediatric cataracts in patients. Moreover, it was also demonstrated that Tdrd7 targeted germline knockout or ENU‐induced knockout mouse mutants exhibit cataracts that closely resemble the human phenotype. My research goal is to understand the regulatory function of Tdrd7 in the lens. Specifically, I aim to investigate if Tdrd7 deficiency affects the mRNA profiles in the apical and the basal regions of differentiating lens fiber cells. To achieve this I have expanded the Tdrd7 germline knockout mouse mutant colony and collected Tdrd7 mutant and control (Tdrd7+/‐ mice that do not exhibit cataracts) embryonic lens tissue for analysis. To study the expression of mRNAs in different locations within fiber cells, I have undertaken standardization of an approach using Laser Capture Microdissection (LCM). LCM on embryonic lens sections from Tdrd7 null mutants and control will be used to isolate the apical and the basal fiber cell regions at embryonic stage E10.5, E11.5, and E12.5. Once the specific tissues are collected, expression of mRNAs will be tested by whole genome expression profiling using Illumina WG‐6 microarrays. Comparative analysis of gene expression profiles of the apical and basal tissues from mutant and control will allow the identification of transcripts that are misregulated as a result of Tdrd7 mutation.
Improved purification of glicentin and its use in a PCSK2 cleavage assay (753.3)
Authors: Mackin Robert
Published: June 2021

Glucagon is cleaved from a larger precursor, proglucagon, by the proprotein convertase PCSK2. This enzyme is believed to cut multiple sites within proglucagon, and it is also able to cleave at least one site within the insulin precursor, proinsulin. By using glicentin, an intermediate in the processing of proglucagon to glucagon, as a substrate, we hope to learn more about the specificity of prohormone maturation. Our previous procedure for bacterial expression and purification of glicentin was difficult to reproduce because of sample overloading and poor peak shape. These problems were solved by performing solid‐phase extraction on the bacterial extract, altering the anion‐exchange chromatography gradient, and changing the type of bead used during reversed‐phase chromatography. The purified glicentin was then incubated with PCSK2, as were proglucagon and proinsulin. Approximately 280 nM PCSK2 was incubated with 10 μM of each prohormone substrate at pH 5.0 for 2 hours at 37°C. Under the conditions used, about 50% of proglucagon (generating the intermediates glicentin and MPGF), 30% of proinsulin and 10% of glicentin were cleaved. These results suggest that there is a hierarchy of prohormone cleavage site susceptibility, and that differences in site‐specific cleavage rates may provide us with insight into the specificity of prohormone maturation.
Resveratrol prevents p53 core domain aggregation (754.1)
Authors: Costa Danielly , Campos Nathali , Rangel Luciana , Zanphorlin Letícia , Ramos Carlos , Silva Jerson
Published: June 2021

p53 has an essential role in preventing cancer development by inducing cell cycle arrest and/or apoptosis in response to cellular stress. Mutations in the p53 gene are described in over 50% of all human cancers. Besides the mutations, cellular aggregation of p53 can also inactivate the protein, leading to malignancy. Resveratrol, a natural polyphenol found in grapes and red wine, is able to induce p53‐dependent cell death in a variety of cell lines. Although several indirect mechanisms of p53 activation by resveratrol have been proposed, there is no evidence that this bioactive compound can interact with p53. Thus, we investigated a possible interaction between resveratrol and the p53 core domain (p53C). In addition, we tested the potential of resveratrol in preventing wild‐type and mutated p53 aggregation in vitro and in tumor cells. Experiments were performed by using fluorescence spectroscopy and fluorescence microscopy techniques. Our data suggest that an interaction between resveratrol and the wild‐type p53C does occur. We also found that resveratrol has the ability to inhibit the wild‐type p53 core domain as well as the R248Q p53 mutant to undergo in vitro aggregation. Additionally, resveratrol (50 and 100µM) reduces the formation of nuclear p53 aggregates in MDA‐MB231 human breast cancer cells. Our study provides evidence that resveratrol can directly modulate p53 and may pave the way for a better understanding of the mechanisms involved in p53 protein aggregation as a therapeutic strategy for cancer treatment.

Grant Funding Source: Supported by FAPERJ, CNPq, INCT/INBEB
The effects of curcumin, ellagic acid and methionine on post‐thawed Merino rams sperm parameters (759.1)
Authors: Omur Ali , Coyan Kenan , Ozturk Caner , Gungor Sukru , Bucak Mustafa
Published: June 2021

The aim of this study was to determine the effects of the antioxidants curcumin, ellagic acid and methionine on microscopic sperm parameters following the freeze‐thawing of Merino ram semen. Ejaculates were collected via artificial vagina from four Merino rams and microscopically evaluated and pooled at 37 ◦C. The pooled semen samples were diluted in a Tris‐based extender and separated to groups including curcumin (1, 2, 4 mM), ellagic acid (1, 2, 4 mM), methionine (1, 2, 4 mM) and no antioxidant (control). The diluted semen was slowly cooled to 5 ◦C and then the samples were frozen in liquid nitrogen vapour, plunged into liquid nitrogen (‐196ºC) for storage. Frozen straws were thawed individually at 37 ◦C for 30 s in a water bath for microscopic sperm evaluation. All of antioxidant groups led to higher percentage of sperm motility in comparison to the control group. The freezing extender supplemented with methionine (1 mM), curcumin (1 and 2 mM), ellagic acid (1 and 2 mM) led to higher percentage of sperm plasma membrane integrity when compared to the other groups (P < 0.05). Looking at the evaluation of sperm acrosome integrity, antioxidant groups led to higher percentace in comparison to the control. Methionine, curcumin and ellagic acid (1mM: 27.7±2.4, 28.0±2.1 and 26.8±2.0) groups provided more higher protective in terms of sperm mitochondrial activity when compared to the other groups (P < 0.05). Findings of this study showed that the different antioxidants (curcumin, methionine and ellagic acid) at different doses protective effects on the ram sperm parameters.

Grant Funding Source: Selcuk University SCIENTIFIC RESEARCH PROJECTS COORDINATION UNIT (Project no: 09102041)
Inhibition of TAK1 (MAP3K7) upregulates unfolded protein response and prevents high‐fat diet‐induced obesity (765.4)
Authors: Sai Kazuhito , Morioka Sho , Takaesu Giichi , Matsumoto Kunihiro , Ninomiya‐Tsuji Jun
Published: June 2021

High‐fat diet (HFD) ingestion induces endoplasmic reticulum (ER) stress in hypothalamic neurons leading to dysregulation of food intake and eventually obesity. Therefore, alleviating ER stress in the hypothalamus would be beneficial for treating obesity. Unfolded protein response (UPR) signaling pathways are activated to protect cells from ER stress. Several different pathways initiate induction of chaperones, attenuation of translation and protein degradation in UPR. However, it’s still elusive how each UPR pathway is regulated upon ER stress. TGF‐β‐activated kinase 1 (TAK1) is a member of MAP3K and known to be involved in various stress response signaling. In the present study, we investigated the role of TAK1 in ER stress response. We found that deletion of Tak1 protected cells from ER stress‐induced cell death. Intriguingly, induction of a molecular chaperon, BiP/GRP78, which is one of the major targets of UPR, was highly upregulated in Tak1‐deficient cells. These data suggest that TAK1 negatively regulates UPR and deletion of Tak1 alleviates ER stress and cell damage by upregulating BiP/GRP78. To examine the physiological relevance for the TAK1 regulation of ER stress, we generated mice harboring neuron‐specific deletion of Tak1 and tested the effect of Tak1 deletion in HFD‐induced obesity. We found that neuron‐specific deletion of Tak1 significantly reduced food intake and weight gain upon HFD feeding compared to wild type mice, suggesting that TAK1 may promote hypothalamic neuronal damage through ER stress. Our results revealed a new regulatory mechanism of ER stress which is involved in neuronal homeostasis.
Decreased sirt3 expression confers growth advantage in chronic lymphocytic leukemia cells (766.8)
Authors: Yu Wei , Denu John
Published: June 2021

Chronic lymphocytic leukemia (CLL), the most common leukemia in Western countries, is characterized by the expansion and accumulation of functionally defective B cells. NAD+‐dependent sirtuin 3 (SIRT3) is the major deacetylase within the mitochondrial matrix and has been shown to eliminate reactive oxygen species (ROS) by deacetylating and activating isocitrate dehydrogenase 2 (IDH2) and superoxide dismutase 2 (SOD2). Because SIRT3 levels decrease with age and cancer cells undergo aerobic glycolysis and have higher ROS, we hypothesized that CLL cells express lower levels of SIRT3. Indeed, we found that SIRT3 expression was significantly reduced in 12 primary samples from CLL patients and a number of B cell malignancy lines compared to primary B cells from healthy donors,which is correlated with higher levels of acetylated IDH2 and SOD2, lower IDH2 and SOD2 activities and higher ROS levels in CLL cells. Lastly we demonstrated that SIRT3 overexpression could partially reverse the malignant phenotype in a SIRT3‐deficient Raji cell line. In this first investigation of the role of SIRT3 in hematological malignancies, we have shown that loss of SIRT3 confers a growth advantage to both primary malignant CLL cells and B cell malignancy cell lines consistent with enhanced ROS production and the Warburg effect, whereby cancer cells undergo aerobic glycolysis. Our study suggests that activating the SIRT3 pathway could be considered a novel therapeutic approach for treating B cell malignancies, such as CLL.

Grant Funding Source: Supported by University of Wisconsin Carbone Comprehensive Cancer Center (UWCCC)
Manganese supplementation increases adiponectin secretion by upregulating disulfide bond A‐like protein in endothelial cells and Zucker diabetic fatty rats (767.13)
Authors: Burlet Elodie , Jain Sushil
Published: June 2021

Adiponectin is an insulin sensitizer that plays a role in the regulation of lipid and glucose homeostasis and has anti atherosclerotic effects. It is synthesized as a single polypeptide and is then assembled in the endoplasmic reticulum into different multimers. An adiponectin‐interacting protein named disulfide bond A‐like protein (DsbA‐L) was recently found to play an important role in multimerization of adiponectin; impairment in DsbA‐L is associated with diabetes and low adiponectin levels. Manganese (Mn) levels have been found to be low in atherosclerotic and diabetic patients. Previous studies in our lab have shown that Mn supplementation can lower blood cholesterol and ICAM‐1 levels in ZDF rats and reduce MCP‐1 secretion and monocyte adhesion to endothelial cells in HUVECs model (J. Biol. Chem, 2013 Mar 1; 288 (9):6409‐16). This study investigates the hypothesis that Mn supplementation affects DsbA‐L expression levels, and thereby adiponectin levels, in ZDF rats and endothelial cells (HUVECs). Results show that diabetic rats supplemented with Mn (gavaged daily with 16 mg/kg BW for seven weeks, n=5) have higher plasma adiponectin levels (41%, p=0.007) and higher DsbA‐L expression in the liver compared to control diabetic rats (n=6). Similarly, pre‐treatment with Mn (with 5 or 10 μM for 24 h) prevented the decrease in DsbAL expression and adiponectin levels in HUVECs and 3T3L1 adipocyte cells exposed to high glucose and MCP‐1. We further investigated the role of DsbA‐L protein by knocking down its expression in HUVECs. Preliminary studies with DsbA‐L siRNA show that beneficial effects of Mn supplementation on NOX4 expression and other biomarkers of vascular inflammation were abolished in DsbA‐L knocked down cells. Thus, this study demonstrates that DsbA‐L mediates the beneficial effects of Mn on adiponectin and provides a novel mechanism by which Mn supplementation reduces biomarkers of vascular inflammation in diabetes.
Effects of antioxidants on mouse diabetes caused by alloxan administration (767.2)
Authors: Masuoka Noriyoshi , Doi Wakana , Zukeran Ayumi , Kanaguchi Hiroki , Takemoto Kazunori , Ishihara Kohji
Published: June 2021

The reactive oxygen species generated with alloxan and reducing agents in the body selectively injure β‐cells in the pancreas so as to cause diabetes. As alloxan generated hydrogen peroxide with reduced glutathione, acatalasemic mice, having a quite low catalase activity in blood, became diabetic with a smaller dose than those having normal one. As the injury caused by oxidative stress is ameliorated by the intake of antioxidants, we chose vitamin E and a fermented burdock to serve as antioxidants. Effects of these diets on acatalasemic mouse diabetes caused by alloxan were examined. Mice were maintained on a control diet and one of these antioxidant diets, respectively, for 14 weeks. Alloxan was then intraperitoneally administered, and blood glucose, glucose tolerance and the insulin level in mouse blood were examined. The incidence of hyperglycemia in the mice maintained on the control diet was significantly higher than that in the mice maintained on the supplemented diet. The abnormal glucose metabolism caused by alloxan administration was ameliorated by both supplemented diets. It is deduced that these antioxidants can prevent a decrease of insulin concentration in the blood in this mouse model.
Exploring O‐GlcNAc modification of whole blood in healthy young adult males: relation to insulin sensitivity, anthropometric and metabolic markers (767.4)
Authors: Myslicki Jason , Shearer Jane , Hughey Curtis , Hittel Dustin , Belke Darrell
Published: June 2021

Abnormal O‐linked‐β‐N‐acetyl glucosamine glycosylation (O‐GlcNAc) has been increasingly published in relation to several chronic diseases including diabetes. Aims were to examine the relationship between anthropometric and metabolic markers and global protein O‐GlcNAc in whole blood from young adult male subjects participating in the Assessing Inherited Markers of Metabolic Syndrome in the Young (AIMMY) study. Subjects (n=24, 24±0.8y) were segregated by homeostatic model assessment score (HOMA‐IR); low (LH=0.60) or high (HH=1.61). No difference in body mass index (BMI), glucose or Hemoglobin a1c were found between groups. However, groups differed in % body fat, plasma triglycerides, and circulating insulin. Whole blood O‐GlcNAc levels and OGT modification were quantified by immunoblotting. Results demonstrated an increase in O‐GlcNAc modification in LH compared to HH (p=0.02) along with a strong positive correlation between HOMA‐IR and O‐GlcNAc (r=0.68, p<0.05). This effect was not related to differences in OGT expression. Quantifying differences in O‐GlcNAc levels of whole blood may detect metabolic disturbances present in a young healthy population free of any clinical pathological diagnosis.
Decrease in cystathionine‐gamma‐lyase (CSE) activity mediates the impaired H2S homeostasis in U937 monocytes, livers of type 1 diabetic rats and peripheral blood mononuclear cells of type 1 diabetic patients: role of hyperglycemia and hyperketo (767.5)
Authors: Manna Prasenjit , Jain Sushil
Published: June 2021

H2S is a novel signaling molecule. This study investigated the molecular mechanism that causes impaired H2S levels in diabetes using U937 monocytes, livers from streptozotocin‐treated type 1 (T1D) diabetic rats, and PBMC from T1D patients. T1D is associated with both hyperketonemia (acetoacetate, AA and β‐hydroxybutyrate, BHB) and hyperglycemia. Treatment of monocytes with high glucose (HG, 25 mM) and/or AA (4 mM) increased ROS (44%), and decreased CSE (a major enzyme that catalyzes H2S formation) protein expression (53%) and activity (39%), and reduced H2S levels (45%); however, BHB treatment had no effect (n=4). Signal silencing studies with CSE siRNA (71% knock down) showed a decrease in H2S levels (83%) in monocytes compared to controls. Liver plays a major role in the H2S homeostasis. Livers from T1D rats showed a significantly higher ROS production, lower CSE protein expression (48%) and activity (0.268±0.032 vs 0.175±0.014 nmol/min/mg protein), and lower H2S levels (46%) compared to controls (n=6). Further studies with T1D patients (n=17) also demonstrated a decrease in CSE protein expression (44%) and activity (0.099±0.023 vs 0.044±0.005 nmol/min/mg protein) in PBMC compared to those of age‐matched normals (n=18). This study demonstrates for the first time that both hyperglycemia and hyperketonemia mediate a reduction in CSE activity which can contribute to the impaired H2S signaling in diabetes.

Grant Funding Source: Supported by NIH RO1 AT007442 and Malcolm Feist Chair in Diabetes
Cocaine enhances HIV‐1 integration in CD4+ T cells by modulating the epigenetic DNA signatures of host genome (776.3)
Authors: Addai Amma , Pandhare Jui , Mantri Chinmay , Dash Chandravanu
Published: June 2021

Cocaine, a commonly abused drug among HIV patients, is known to increase HIV‐1 replication in cell cultures, peripheral blood mononuclear cells (PBMCs) and animal models. The goal of this study is to examine the effects of cocaine on HIV‐1 integration. During HIV‐1 replication, the viral genome is reverse transcribed into a dsDNA by HIV‐1 reverse transcriptase. This viral dsDNA is transported into the nucleus in the form of a pre‐integration complex (PICs) and integrates into the host genome. Our data demonstrates that cocaine treatment enhances HIV‐1 integration in primary CD4+ T cells. To understand the mechanism, we examined global DNA methylation in cocaine treated cells. Our data revealed that cocaine induced global DNA de‐methylation in CD4+ T cells. Since integration has been suggested to be disfavored in methylated DNA targets, we hypothesize that cocaine enhances integration by reducing the global DNA methylation signature of the host genome. Our in vitro studies using PICs from infected cells illustrate that integration is reduced in target DNA that are methylated compared to unmethylated target. To the best of our knowledge, this is the first report describing a role of cocaine on HIV‐1 integration in activated CD4+ T cells. We believe understanding the mechanism by which cocaine modulates HIV‐1 integration may aid in the discovery of novel targets to cure the co‐morbid condition of drug abuse and HIV/AIDS.

Grant Funding Source: *Amma Addai is supported by T32 grant #2T32H007735‐17 from NIH/NHLBI to Dr. S. E. Adunyah
Evaluation of mRNA extraction methods from murine skin (776.4)
Authors: Neumann Drexel , Atwood James , Nasti Tahseen , Tsuruta Yuko , Cochran Barry , Timares Laura
Published: June 2021

Intro: Transcriptome research has become increasingly popular to monitor global changes in gene expression. In particular, transcriptome differences between diseases versus healthy states can provide valuable insight into understanding immune response mechanisms. However, the isolation of high‐quality mRNA from tough tissues, such as skin, can represent a significant challenge in research. Herein, we compare two methods for mRNA extraction from two murine skin types with a specific focus on mRNA yields and integrity.

Methods: 1cm2 full‐thickness mouse skin (epidermal and dermal layers) samples were freshly harvested, separated from fat, and kept on ice until homogenization. Samples were cut into fragments and placed in 1ml Trizol, then homogenized using a manual Dounce homogenizer on ice, or an automated Omni Bead Ruptor 24 (BR24) bead mill homogenizer without refrigeration. Clarified sample supernatants were mixed with EtOH to precipitate nucleic acids, and sample yields and quality were determined by spectrophotometry. The mRNA purity and integrity of each sample was additionally determined using an Agilent BioAnalyzer.

Results: Spectrophotometric data indicated that, depending on skin tissue type and sample size, mRNA yield from samples processed by BR24 versus Dounce was increased 2‐6 fold. The 260/280 ratios were equivalent across both methods; however, the Alginet electropherograms revealed that Dounce homogenization produced samples with unacceptably high MW contaminant DNA and low MW degradation species, while the BR24 processing produced superior mRNA samples of outstanding purity and RNA integrity (RIN). (Omni BR24 RIN values 8.4 ‐ 8.8 ; Dounce RIN, 蠄 4.3). Thus, high quality mRNA can be efficiently isolated from difficult to disrupt tissues such as skin.
Gene expression alterations in patients with post‐traumatic stress disorder (776.6)
Authors: Muhie seid , Yang Ruoting , Hammamieh Rasha , Jett Marti
Published: June 2021

We profiled transcriptome changes in patients with post‐traumatic stress disorder (PTSD). Whole blood samples from 98 subjects (49 PTSD‐positives and 49 age, race and gender‐ matched controls) were assessed using microarray and real time polymerase chain reaction assays. About 1500 genes were differentially regulated in PTSD‐positive patients compared to the controls. Down‐regulated transcripts were involved in T‐cell immunity, cardiovascular activity, and synaptic transmissions. Suppressed pathways and functions were critical in maintaining one’s sense of well‐being, protective immunity, and cardiovascular health. Induced functions and pathways (significantly associated with up‐regulated transcripts) were largely related to traumatic stress responses such as endoplasmic reticulum stress, DNA repair, wound healing, and synaptic plasticity. Our data shows the pervasive negative impact of PTSD at the molecular level in regulation of key pathways and functions important to health.

Grant Funding Source: US Army and Veteran's Affairs
Comparison at the peptide level with post‐translational modification consideration reveals more differences between two unenriched samples (778.1)
Authors: Yin Jianrui , Shao Chen , Jia Lulu , Gao Youhe
Published: June 2021

In shotgun strategy, peptide sequences are first identified from MS/MS spectra, and the existence and abundance of the proteins are then inferred from the peptide information. However, the protein inference step can produce errors and a loss of information. To identify the information that is lost using the traditional approaches, this study compared the proteomic data of two leukemia cell lines (Jurkat and K562) at peptide level with consideration of post‐translational modifications (PTMs). After database search, the observed modification name was matched with the modification classification on the Unimod website. Only the peptides with ‘post‐translational’ modifications were compared between the two cell lines. As a result, a total of 44887 non‐redundant peptides were identified in both the Jurkat and K562 cell lines. Of these peptides, even without specific PTM enrichment, 11.43% of them (with at least two spectra in one cell line) existed in different PTM forms between the two cell lines, and 1.89% of the peptides were modified in both cell lines, but with different modifications or on different sites. The sensitivity of PTM identification will become greater and more differences will be found if PTMs are enriched or if the peptides can be compared in a quantitative manner. Therefore, comparing proteomic data at the peptide level with consideration of PTMs can reveal differences between two unenriched samples.

Grant Funding Source: Supported by National Basic Research Program of China (2012CB517606, 2013CB530805, 2011CB964901).
Identification of personal care, prescription, and other emerging contaminants in Lake Erie beach waters and an evaluation of their effects on aquatic life (779.6)
Authors: Parente Amy
Published: June 2021

Chemicals used extensively and intensively in our technological society are currently predicted to have surpassed the 100,000 mark in numbers and depending on their properties, modes, and quantity of use, a large number of these chemicals can reach the environment and have unpredictable but potentially harmful environmental and health impacts. Accurate methods of monitoring the levels of these chemicals, an understanding of their potential to damage natural inhabitants to these waters, and the ability to properly develop a risk assessment strategy are all critical pieces to the maintenance of a healthy aquatic ecosystem that does not pose a threat to human safety. Current research has identified several emerging contaminants in Lake Erie beach waters of Presque Isle State Park, including fluoxetine, triclosan, estradiol, and diuron, as well as the artificial sweetener sucralose. All of these chemicals have been shown to have negative health consequences in organisms from bacteria to humans. For most of these, the levels in the Park approach or exceed levels known to cause harm to aquatic species. We have also investigated the ability of these chemicals of concern to have genotoxic (and often synergistic) effects.
Pyroprinting: novel pyrosequencing‐based method for studying E. coli diversity and microbial source tracking (779.8)
Authors: Kent Jason , Alvarado Maria , VanderKelen Jennifer , Montana Aldrin , Soliman Jan , Dekhtyar Alex , Goodman Anya , Kitts Christopher , Black Michael
Published: June 2021

We have developed a novel genotypic fingerprinting method for typing Escherichia coli strains via simultaneous, multi‐locus pyrosequencing of the internal transcribed spacer (ITS) regions in ribosomal RNA operons. The data generated, called pyroprints, are unique to the polymorphic ITS alleles and their relative frequency among the seven rRNA operons in each E. coli isolate. Pyroprinting is a rapid, inexpensive, and highly reproducible method that produces readily digitized data for computational analysis and effectively discriminates closely related strains. A pyroprint database (www.cplop.org) and analytical tools for environmental forensics and microbial source tracking (MST) applications have been developed. Our pyroprint library currently contains over 5,000 E. coli isolates from avian and mammalian hosts. Pyroprinted isolates have been clustered into strains, some of which are shared by multiple hosts, while others appear host‐specific. To validate this method for MST, we have simulated environmental water samples by spiking them with fecal material from known hosts in pre‐determined ratios. Pyroprinting E. coli isolates from the simulated samples has allowed us to identify the origin of the isolates and ratios of different hosts that contributed to the mixed samples. This new MST method has been employed to identify sources of fecal contamination in San Luis Obispo Creek.

Grant Funding Source: Supported by NSF TUES # 1140828.
The α‐arrestin ARRDC3 regulates ubiquitin‐independent ALIX‐mediated GPCR lysosomal sorting (783.2)
Authors: Dores Michael , Lin Huilan , Buck Hannes , Mendez Francisco , Trejo JoAnn
Published: June 2021

Endosomal sorting and lysosomal degradation of G‐protein coupled receptors (GPCRs) maintains cell homeostasis by attenuating receptor signaling and desensitizing cells to persistent activation. GPCR lysosomal sorting defects cause aberrant cellular responses that contribute to many diseases, including chronic inflammation and cancer metastasis. Here, we demonstrate that the human α‐arrestin ARRDC3 regulates the lysosomal degradation of protease‐activated receptor‐1 (PAR1), a GPCR for thrombin. PAR1 belongs to a subset of GPCRs, including the purinergic receptor P2Y1, that are sorted to lysosomes independent of receptor ubiquitination. Instead, PAR1 and P2Y1 are targeted to lysosomes by YPXnL motifs that are bound by the ESCRT adaptor protein ALIX. ARRDC3 is required for PAR1 degradation, but not for the degradation of ubiquitinated receptors. ARRDC3 regulates ALIX interaction with PAR1 and the ESCRT‐III protein CHMP4. Intriguingly, depletion of ARRDC3 or ALIX blocks recovery of thrombin‐induced dephosphorylation of Yap1, an effector of the Hippo cell proliferation pathway, suggesting that GPCR degradation is important for regulation of Hippo signaling. This study reveals a novel role for α‐arrestins in the ubiquitin‐independent, ALIX‐mediated endosomal trafficking of GPCRs, and demonstrates the importance of lysosomal sorting in the regulation of GPCR‐mediated proliferative signaling.

Grant Funding Source: Supported by NIH NIGMS R01 GM090689
The anti‐diabetic drug metformin perturbs proliferation and cellular differentiation of pancreatic cancer cells: a key mechanism of its anticancer activity (784.6)
Authors: Das Amlan , De Archana , Maity Gargi , Banerjee Snigdha , Banerjee Sushanta
Published: June 2021

Background : Pancreatic cancer is the 5th most common cancer in the United States and is currently the fourth leading cause of cancer death, with a 5‐year overall survival of only 5%. It has been reported that Type 2 diabetes mellitus and associated hyperglycemia, hyperinsulinemia, and inflammation, are known to play important roles in the development of diabetes‐associated pancreatic cancer. Use of the anti‐diabetic drug metformin has been reported to reduce the risk of pancreatic cancer in diabetics and recognized as an antitumor agent with the potential to inhibit the progression of pancreatic tumors. However, the mode of action of Metformin on regulation of cellular differentiation, which are associated with are invasion and metastasis of pancreatic cancer, is uncertain.

Objective: The objective of the present study is to determine if Metformin plays any role cellular de‐differentiation through the reprogramming of EMT process that eventually prevents invasive phenotypes associated with CXCR‐4/SDF‐1 pathways.

Results: In our study we have observed that treatment of the pancreatic cancer cells such as Panc‐1 and AsPc‐1 resulted in a dose‐dependent inhibition of proliferation, accompanied by the induction of cytotoxicity through the regulation of the tumor suppressor gene p27. Moreover, Metformin treatment resulted in the morphological alteration as well as induction of EMT in pancreatic cancer cells. Metformin treatment prevents in vitro invasion of PC cells to words SDF‐1, a ligand for CXCR4 chemokine receptor actively associated with cancer metastasis.

Conclusion: The widely used anti‐diabetic drug metformin can suppress pancreatic tumor progression by inhibiting the chemokine‐induced invasiveness and motility.