bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective Role Of Antioxidant Supplementation In Tobacco Smoke Toxicity At The Blood‐Brain Barrier
Authors: Naik Pooja Shankar , Smith Quentin , Cucullo Luca
Published: June 2021

Tobacco smoke (TS) is enriched in highly reactive oxidative species (e.g., hydrogen peroxide, peroxynitrate) which can damage the blood‐brain barrier (BBB) and trigger inflammatory events leading to the pathogenesis and progression of various neurological disorders. Physiological levels of antioxidants vitamin C and E are significantly reduced in smokers in fate of oxidative stress caused by TS exposure. We hypothesize that antioxidants can reduce the harmful effects of ROS‐dependent TS toxicity at the BBB.

We exposed various in vitro BBB models to TS levels comparable to that found in chronic heavy smokers. Antioxidants supplementation of vitamin C and/or E at physiological concentration reduced TS‐induced vascular inflammatory response: release of proinflammatory cytokines from BBB endothelial cells and monocytes, release and activation of matrix metalloproteinases (MMPs), increased expression of vascular adhesion molecules. Furthermore, antioxidants reduced TS‐driven loss of BBB integrity and viability. Intravascular increase of nitric oxide (NO) by TS was also offset by antioxidants.

Our results indicate that antioxidants can avert TS‐induced vascular pro‐inflammatory stimuli in vitro thus protecting the BBB. However, while antioxidants can reduce vascular oxidative stress, it might not be effective to counteract other teratogenic and pathophysiological stimuli induced by smoking.

This work was supported by NIH/NIDA R01‐DA029121‐01A1 and Alternative Research Development Foundation (ARDF) to Dr. Luca Cucullo.
Behavior and memory evaluation in Wistar rats with hypothalamus arcuate nucleus lesion
Authors: Guimarães Ernesto da Silveira Goulart , de Caires Luiz Carlos , Musso Camila Manso , Vasconcellos Rebecca , Assis Diego , Andreazzi Ana Eliza , Mourão-Júnior Carlos Alberto , Garcia Raúl Marcel González
Published: June 2021

The administration of L‐monosodium glutamate (MSG) in neonatal rats causes lesions in the arcuate nucleus of the hypothalamus leading to obesity development. Because of the many connections between these regions with important neuronal regulating centers, the aim of this study was to evaluate the behavioral and memory patterns of these animals.

At the first 5 days of life, male Wistar rats received subcutaneous doses of MSG. At 90 days of life the animals was submit to the open field arena test regarding episodes of rearing, freezing, number of traversed squares and the time spent in the arena's center. We evaluated the animals’ memory using the object recognition test regarding the working memory (WM) and the long‐term memory (LTM). The Lee Index (LI) was calculated and the perigonadal fat pad was weighed to check the obesity and hypertrophy of adipocytes. The MSG rats showed a significant increase in the episodes of rearing, freezing and decrease in the number of squares traversed and in the time spent in the arena's center. In the LTM test MSG animals showed a reduced discrimination index, the WM test showed no significant difference. The MSG group had an increased LI and perigonadal fat pad mass.

MSG obese rats showed a behavior that indicates a stress and anxiety profile, which may be due to the arcuate nucleus lesion. The impaired LTM can be explained by the some neural connections between the hypothalamus and hippocampus.

Financial Support: Capes, CNPq, Fapemig and UFJF
Evidence of age‐related neuronal aberrations and dysfunction of sensory neurons in a polyglutamine model of Huntington's disease
Authors: Vayndorf Elena , Driscoll Monica , Taylor Barbara
Published: June 2021

Huntington's disease is a neurodegenerative disorder that impairs motor movements and cognitive ability. It is caused by expansion of the CAG trinucleotide repeat within the first exon of the huntingtin (HTT) gene. The resulting polyglutamine (polyQ) chain gives rise to a mutated huntingtin protein, which is associated with aggregate formation, neuronal dystrophy and severe dysfunction. C. elegans is a genetically tractable model whose intact nervous system possesses major hallmarks of mammalian neurological function. Our model of Huntington's disease expresses the toxic (Q128) and non‐toxic (Q19) forms of the polyglutamine chain in the mechanosensory neurons. We hypothesized that, compared to animals expressing non‐toxic huntingtin, animals expressing the toxic protein would exhibit significant morphological aberrations of mechanosensory neurons and the degree of aberrancy would correlate with age‐related reduced functionality and short‐term associative memory. Sensory neurons were imaged throughout the lifespan using fluorescent microscopy and standard nematode culturing techniques. Mechanosensory assays were performed by provoking animal response to gentle touch. Short‐term memory assays were conducted using an associative chemotaxis assay. Results indicate that expression of toxic huntingtin correlates with neuronal aberrations and defects in mechanosensory sensation with age.
Klotho Enhances Glucose‐induced Insulin Secretion by Upregulating Plasma Membrane Retention of TRPV2
Authors: Lin Yi , Sun Zhongjie
Published: June 2021

Background

Klotho is a recently discovered anti‐aging gene. Our recent studies indicated that Klotho gene is expressed in mouse pancreatic islets and in an insulinoma cell line (MIN6 β cells) as evidenced by expression of Klotho mRNA and protein. The purpose of this study was to investigate if Klotho is involved in the regulation of insulin secretion in β cells.

Results

Overexpression of mouse Klotho protein significantly increased plasma membrane retention of TRPV2 (transient receptor potential V2), calcium entry, the glucose‐induced increase in intracellular calcium, and the glucose‐induced insulin secretion in MIN6 β cells. On the other hand, knockdown of Klotho by siRNA significantly decreased plasma membrane retention of TRPV2 and attenuated glucose‐induced calcium entry and insulin secretion. Tranilast, a selective inhibitor of TRPV2, abolished the promoting effects of overexpression of Klotho on glucose‐induced calcium entry and insulin secretion in MIN6 cells. Thus, TRPV2 lies in the downstream of Klotho in the regulation of glucose‐induced insulin secretion.

Conclusion

The present study demonstrated, for the first time, that Klotho may increase glucose‐induced insulin secretion by up‐regulating membrane retention of TRVP2 and thus glucose‐induced calcium response. The findings reveal a previously unidentified role of Klotho in the regulation of insulin secretion in MIN6 β cells.
Large molecule delivery to the growth plate increases with limb temperature measured by in vivo multiphoton imaging
Authors: Serrat Maria A , Williams Rebecca M
Published: June 2021

Bone elongation occurs in cartilaginous growth plates, which rely on surrounding blood vessels for nutrition. New imaging modalities that enable real‐time study of vascular access to growth plates have potential to fundamentally advance understanding of physiological growth regulation. Our lab employs in vivo multiphoton microscopy to measure environmental effects on entrance of systemically‐introduced fluorescent tracers into tibial growth plates of 5 week old mice. We previously found temperature‐ and exercise‐related changes in small molecule delivery using the 332 Da tracer fluorescein. Here we test the hypothesis that warm peripheral temperature increases large molecule delivery to growth plates using 10 kDa dextrans that approximate the size of physiological factors such as IGF‐I, a potent growth stimulator. We immersed hindlimbs of mice in 22C or 37C Ringer's and kept core temperature constant. Large molecule entry into the growth plate was minimal at 22C and increased when the limb was warmed to 37C. Vessel diameter was increased at 37C, suggesting that heat enhances blood volume and bioavailability of large molecules around the growth plate. As our experimental temperatures were within ranges reported for human knee joints, these results are relevant to strategies for targeting delivery of therapeutic agents to growth plates of children. Supported by MU‐ADVANCE and NASA WV Space Grant Consortium.
How does Parkinson disease severity influence kinetics and kinematics during a rapid force production task?
Authors: Willis Ryan , Dibble Leland E. , Rockwell W. Tyler , Marcus Robin L. , LaStayo Paul C. , Foreman K. Bo
Published: June 2021

The cardinal signs of Parkinson disease (PD) include bradykinesia, hypokinesia, tremor, and rigidity. These symptoms worsen as the disease progresses, often resulting in postural instability. However, little attention has been given to the effects of disease severity on the kinetics and kinematics of individual lower extremity joints during rapid force production tasks necessary for balance responses. Therefore, we examined lower extremity joint moments and average angular velocity of 14 subjects with PD who were segregated into 2 groups (less severe [n=7, mean age=62.29±6.73] and more severe [n=7, mean age=73.00±6.19]) based on UPDRS motor subsection scores while ON medication (less severe=7.14±2.19, more severe=17.43±6.75). Subjects completed 4–5 counter movement jumps (CMJ) 1 hour after taking their medication. Kinetic and kinematic variables were recorded for the explosive phase of the CMJ. Subject's first 3 successful trials were averaged and examined using the Mann‐Whitney U Test (p≤0.05). Individuals with less severe involvement move significantly faster; however, their ability to produce torque around the hip, knee, and ankle is not significantly different compared to more severely affected individuals.

Grant Funding Source: NIH Grant #: 1 R15 HD056478‐01
A Novel Anti‐apoptotic Role of DMP1 in Normal and Pathological Conditions
Authors: Rangiani Afsaneh , Sun Yao , Nowar Nour , Sahfiey Alex , Yuan Baozhi , Qin Chunlin , Kuro-o Makoto , Feng Jian Q
Published: June 2021

Introduction

Deletion or mutations in Dmp1, expressed in both hard and soft tissues, leads to an increase of FGF23 and hypophosphatemic rickets. Deletion of FGF23 or Klotho (co‐receptor of FGF23) leads to a chronic kidney diseases like phenotype.

Purpose

To test the hypothesis that DMP1 plays a protective role in renal osteodystrophy caused by deletion of Klotho.

Methods

Dmp1 KO, Klotho KO, Dmp1‐Klotho double‐KO (dKO) mice were generated, sacrificed and compared to WT at 6 wk; x‐ray, uCT, SEM, serum biochemistry, histology, TRAP and TUNEL methods were used for phenotype analyses.

Results

dKO mice displayed an identical serum biochemical phenotype (high Pi/FGF23, low PTH) and aging as those in single Klotho KO, with unexpected massive ectopic calcification in the dental pulp/root canal, aorta and kidney, plus a substantial increase in apoptosis (P<0.001, compared to single Klotho KO). These pathological changes are completely different from either Dmp1 KO (low in apoptosis with normal life span and no ectopic ossification) or Klotho KO (a moderate increase in apoptosis and ectopic ossification in soft tissue).

Conclusion

DMP1 has a possible protective role in conditions such as the renal osteodystrophy. In addition, DMP1 appears to have a novel protective role for odontoblasts, osteocytes and endothelial cells of aorta and kidney in the pro‐apoptotic conditions such as the renal osteodystrophy.

Grant Funding Source: NIH grants to JQF for DEDE018486015209
Changes in semicircular canal morphology in response to selective breeding for high voluntary wheel running
Authors: Schutz Heidi , Jamniczky Heather A. , Hallgrímsson Benedikt , Garland Theodore
Published: June 2021

Variation in semicircular canal (SCC) radius of curvature (R) and shape show correlations with locomotor agility among species of mammals. We used laboratory mice from 4 replicate lines bred for high voluntary wheel running (HR) and 4 non‐selected control lines (C) to examine responses in the SCC to 8 weeks of access to wheels and 21 generations of selection. Mouse crania were μCT scanned at 21 μm resolution and linear measurements, 3D landmarks, and centroid sizes of the SCC were taken. Linear measures were used to calculate R for all three canals and their mean, whereas landmarks were used to generate multivariate descriptors of 3D canal shape.

HR mice weighed less than C and wheel access reduced body mass for both groups. ANCOVA showed that body mass was a significant positive predictor of R. However, when controlling for body mass we found no differences in R between HR and C, no effect of wheel access, and no interaction.

SCC shape was also significantly correlated with body mass; controlling for body mass or centroid size, there were significant differences in SCC shape between HR and C mice. These results demonstrate that semicircular canal morphology is responsive to selection for locomotor activity and that canal shape may be more sensitive than canal size.

Funding for this project provided by a U.C. Riverside Chancellor's Postdoctoral Fellowship to HS, Canadian Foundation for Innovation to BH and IOS‐1121273 to TG.
Effects of Mechanical Shear on Anisotropic Tissue Engineered Construct as Potential TMJ Repair
Authors: Mathema Anita , Mills David K
Published: June 2021

An intraarticular disc, separating the articulating bones of Temporomandibular joint (TMJ), is mainly composed of proteins called proteoglycans, collagen majority being collagen type I, II and elastin. Collagen fibers function to resist loads when the tissue experiences any kind of tension [Kim, 2003]. A TMJ disorder is a group of complex problems of TMJ and other tissue associated with it. The main goal of this research is to design and develop an anisotropic tissue engineered biopolymer scaffold to mimic the functional and structural aspect of TMJ disc using poly (ε‐caprolactone) (PCL) as an electrospun fiber embedded with halloysite nanotubes (HNTs) that has the potential for prolonged release of bioactive molecules. It is hypothesized that cellular exposure to the anisotropic PCL scaffold embedded with HNTs loaded bone morphogenetic protein‐2(BMP‐2) and shear stress will enhance ECM deposition, leading to more robust mechanical properties. ATCC pre‐osteoblast were differentiated to osteoblast for this study. Briefly, 5% w/v HNTs and 11% w/v PCL beads were added in chloroform and were sonicated to obtain the polymer solution for electrospinning. This study demonstrated that the BMP‐2 released from HNTs embedded in PCL scaffold had significant effect on osteoblast osteogenic activity. The results suggest that the scaffold has a potential for the development of biodegradable implant for tissue repair.
S. cerevisiae RFC Walker A and arginine finger mutants are defective in clamp opening and binding
Authors: Marzahn Melissa Rose , Bloom Linda B.
Published: June 2021

Though eukaryotic replication has been studied for over 30 years the exact mechanisms for each enzyme involved are largely unknown. The goal of this study is to kinetically evaluate the function of the clamp and clamp loader. The eukaryotic clamp, proliferating cell nuclear antigen (PCNA), tethers the polymerase to the template DNA and is loaded onto DNA by replication factor C (RFC), a heteropentamer complex belonging to the AAA+ family of ATPases. Several conserved ATPase motifs, the Walker A and B loops and arginine finger motif, coordinate ATP binding and hydrolysis. The Walker A loop participates in ATP binding while the arginine finger motif is involved in sensing bound ATP. Mutation of the Walker A Lys or arginine finger Arg residue dramatically reduces ATP binding or recognition of bound ATP, respectively. Conserved Lys or Arg residues were mutated in each of the 4 ATP sites, and RFC mutants containing a single mutation were characterized to determine the functional role each motif plays. Fluorescence‐based assays were used to determine how these mutations affect RFC's ability to bind DNA and bind and open PCNA in the steady‐state and pre‐steady‐state. Mutations at all sites except one, Rfc4 Arg, caused a dramatic loss of clamp binding and opening. This suggests that ATP binding is essential for clamp binding and opening but that recognition of bound ATP may be an ordered process within the clamp loading reaction.
An active site mutant allele of human Pol ε is a mutator in vitro and in vivo
Authors: Agbor Anderson Ayuk , Pursell Zachary F
Published: June 2021

DNA replication in eukaryotes is carried out primarily by three DNA polymerases (Pols): Pols α, δ and ε. While yeast Pol ε has been shown to function on the leading strand during DNA replication, the precise functions of human Pol ε in vivo remain relatively poorly understood.

Wild type pol ε from both yeast and humans is a highly accurate enzyme. Active site mutant alleles of yeast Pol ε were previously shown to be mutators with unique error signatures both in vitro and in vivo. We found that human Exo− M630G Pol ε is a 50‐fold mutator over the wild type enzyme in vitro, with a strong preference for T•dT and T•dG errors.

We introduced Exo− M630G Pol ε into human mismatch repair‐deficient HCT116 colorectal cancer cells. Mutations in this cell line arise primarily from errors made during replication. Cells transfected with Exo− M630G Pol ε exhibited an increase in HPRT1 mutant frequency over cells transfected with control vector. The results indicate that transfected Exo− M630G Pol ε is involved in replication, is a mutator during replication in vivo, and is at least partially dominant over the endogenous Exo+ Pol ε. Importantly, Exo− M630G Pol ε mutant lacking the C‐terminal tail, which is known to be essential in yeast, showed no increase in HPRT1 mutant frequency.

With the human Exo‐ M630G Pol ε mutator allele, we now have the ability to probe Pol ε function during human DNA replication and repair of damaged DNA.

NIH ES016780, NIH RR020152
Convergent Transcription at the Ataxin‐7 Locus Produces dsRNA Fragments that are Processed by Dicer‐1
Authors: Ladd Paula D. , Ward Jaqueline M. , Lomas Nicole S. , Pineda Victor V. , La Spada Albert R.
Published: June 2021

Spinocerebellar ataxia type 7 is a polyglutamine repeat disease characterized by retinal and cerebellar degeneration. The causative mutation is an expansion of a CAG repeat in the first coding exon of the ataxin‐7 gene. As with other unstable trinucleotide repeat loci, bidirectional transcription occurs at the repeat region, with the antisense transcript overlapping the first and second coding exons of the sense transcript. Interestingly, the antisense transcript is processed into multiple fragments. The 5’‐end of the antisense transcript is retained in the nucleus, while the remaining fragment is exported to the cytoplasm, where it is further processed. RNase A/T1 dsRNA duplex assays indicated the presence of dsRNAs, while northern blot analysis revealed small RNAs (20–60 nt), suggesting small processed RNA fragments are produced by convergent transcription at the ataxin‐7 locus. Dicer‐1 knockdown on primary mouse cerebellar astroctyes with an integrated ataxin‐7 genomic fragment, detected a significant increase in ataxin‐7 sense expression, suggesting evidence for Dicer‐1‐dependent processing. Funding from the NIH supported this research: R01 GM59356 and ARRA award GM59356‐09‐S1.
Abeta‐polyacrolein is neurotoxic in an amyloid‐independent manner
Authors: Theisen Christopher Shane , Seidler Norbert W
Published: June 2021

A previous study indicated that amyloid beta peptide (ABP) exhibits surfactant properties, suggesting that the toxicity of ABP in Alzheimer disease (AD) is attributed to the peptide's effect on the organization of water molecules. We previously proposed that ABP‐polyacrolein, which is derived from ABP‐induced polymerization of acrolein, may contribute to the neurotoxicity in AD. In this study, we examined the effect of ABP‐polyacrolein on gene expression in differentiated SH‐SY5Y cells using a PCR array composed of probes for 84 genes that are involved in neurotoxicity. We compared the results against the effects of ABP alone. We observed that ABP alone decreased the expression of eight different genes (criterion: greater than 2‐fold change). However, the downregulation of three of these genes (epiregulin, tryptophan hydroxylase‐1, and transient receptor potential cation channel‐M4) were appreciably attenuated by ABP‐polyacrolein presumably due to ABP‐polyacrolein's abrogation of ABP's surfactant‐like injury to the cell. Furthermore, we observed a unique downregulation of three genes with ABP‐polyacrolein (hephaestin, notch 4, and transient receptor potential cation channel‐M1). This suggests a novel mechanism of neurotoxicity not seen with ABP alone and likely due to the extensive polymeric arrangement of ABP‐polyacrolein, which is quite dissimilar from the typical amyloid structure.
Cell specific processing and release of the pro‐hormone candidate tumor suppressor, Ecrg4, from the epithelial cell surface
Authors: Dang Xitong , Podvin Sonia , Coimbra Raul , Eliceiri Brian , Baird Andrew
Published: June 2021

Objective

To study the processing of human Ecrg4 in epithelial cells.

Methods

Plasmids encoding human Ecrg4, Ecrg4 mutants and Ecrg4‐derived peptides fused with green fluorescent protein were transiently expressed in the prostate cancer (PC‐3) or human embryonic kidney (HEK) epithelial cells. The expression of Ecrg4 was analyzed by immunoblotting, immunohistochemistry and fluorescence and confocal microscopy. Cell surface‐bound Ecrg4 was detected by cell surface fractionation.

Results

Ecrg4 can be detected in the conditioned media of PC‐3 but not HEK cells 30 hours after transient transfection of pcDNA3‐hEcrg4. In both HEK and PC3 cells however, a “secreted” 14 kDa Ecrg4 product was found tethered to the outer cell surface and could not be released by washing cells with neutral (PBS), high salt (2M NaCl), acidic (Glycine, pH 2.8) or basic (Na2CO3, pH 11) buffers. Fusion of leader sequences with GFP established that Ecrg4 retention was mediated by amino acids 1–15 of the 30 amino acid leader sequence while Ecrg4(16–30) promoted retention. Mutagenesis studies further established that processing occurred at predicted furin and thrombin consensus sites at the cell surface.

Conclusion

Ecrg4 is cell membrane protein that can be shed into media by cell stimulation. This surface bound ECRG4 can be further processed at the furin site to release the ligand‐like peptides in conditioned media.
Complex Peptide Biosensors for Detection of Intracellular Kinase Biomarkers
Authors: Morris May C , Kurzawa Laetitia , Van Ngoc , Pellerano Morgan
Published: June 2021

Cyclin‐dependant kinases play a central role in coordinating cell cycle progression, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. We have developed a family of environmentally‐sensitive fluorescent peptide biosensors to probe the relative abundance and activities of CDK/cyclin kinases. CDKSENS and CDKACT sensors allow to probe CDK/Cyclin kinases in a sensitive and specific fashion in vitro. Complexation of these peptide biosensors with cell‐penetrating peptide carriers allows to form stable nanoparticles that can probe these kinases in living cells in a non‐invasive fashion (Kurzawa et al. PloS One 2011). This technology can be applied to monitor subtle alterations between healthy and cancer cell lines, to probe kinase levels and activities in tumour biopsies, and to monitor response to therapeutics in vitro and in vivo.

Source of Funding: Grants to MCM “Chercheuse d'Avenir” Région Languedoc‐Roussillon and INCA
Evidence for Activation of AMP Kinase (AMPK) in Nephropathic Cystinosis
Authors: Taub Mary , Cutuli Facundo
Published: June 2021

The objective of this study was to investigate the involvement of AMPK in the altered signaling that results in nephropathic cystinosis. Cystinosis is characterized by cysteine accumulation, due to mutations in CTNS, encoding for cystinosin, a lysosomal cystine transporter. Patients develop the Fanconi Syndrome, due to defective reabsorption by apical Na+/solute cotransport systems in the renal proximal tubule (RPT). In this study, cystinosin was knocked down >80% in primary rabbit RPT cells using cystinosin siRNA. This reduction in cystinosin strongly inhibited Na+ dependent Phosphate (Pi) uptake (70%). Intracellular Na+and Na,K‐ATPase were unaffected. However, ATP dropped 52%, the ADP/ATP ratio increased 300%, and apical NaPi2a transporters were reduced. Because increases in ADP result in AMPK phosphorylation and activation, the phosphorylated AMPKα subunit (pAMPKα) was examined. In primary RPT cells with a cystinosin knockdown the level of pAMPKα was elevated (8.0 +/− 4.2 fold relative to the control), and was unaffected by AICAR (5‐Aminoimidazole‐4‐Carboxamide‐1‐β‐D‐ribofuranoside), an AMPK activator (unlike control cells). Further studies are in progress to determine whether AMPK activation is responsible for the reduced growth, increased apoptotis, as well as reduced transport in primary RPT cells with a cystinosin knockdown. The funding was from the Cystinosis Research Foundation.
PIM1‐activated PRAS40 Regulates Radioresistance in Non‐small Cell Lung Cancer Cells through Interplay with FOXO3a, 14‐3‐3, and Protein Phosphatases
Authors: Kim Wanyeon , Youn HyeSook , Seong Ki Moon , Kang JiHoon , Youn BuHyun
Published: June 2021

Resistance of cancer cells to ionizing radiation (IR) plays an important role in the clinical setting of lung cancer treatment. To date, however, the exact molecular mechanism of radiosensitivity has not been well explained. In this study, we compared radioresistance in two types of non‐small cell lung cancer (NSCLC) cells, NCI‐H460 and A549, and investigated the signaling pathways that confer radioresistance. In radioresistant cells, IR led to overexpression of PIM1 and reduction of protein phosphatases (PP2A and PP5), which induced translocation of PIM1 into nucleus. Increased nuclear PIM1 phosphorylated PRAS40. Consequently, pPRAS40 made a trimeric complex with 14‐3‐3 and AKT‐activated pFOXO3a, which then moved rapidly to cytoplasm. Cytoplasmic retention of FOXO3a was associated with down‐regulation of pro‐apoptotic genes and possibly radioresistance. On the other hand, a suppressive effect of IR on protein phosphatases was not detected and, concomitantly, protein phosphatases down‐regulated PIM1 in radiosensitive cells. In this setting, PIM1‐activated pPRAS40, AKT‐activated pFOXO3a, and their complex formation with 14‐3‐3 could be key regulators of the IR‐induced radioresistance in NSCLC cells.
Nutlin‐3 activates MEK1/2‐ERK1/2 pathway via p53‐induced ROS accumulation
Authors: Lee Sun-Young , Ko Kyoung-Won , Choe Yun-Jeong , Kim Ho-Shik
Published: June 2021

Tumor suppressor p53 activates growth arrest or apoptosis in response to genotoxic stress. Along with its growth suppressive effect, p53 was also known to stimulate MAPK pathway, of which mechanisms still remains to elusive. In human osteosarcoma U2OS cells, nutlin‐3, a small molecular antagonist of MDM2, induced phosphorylation of MEK1/2 and ERK1/2 accompanied by p53 activation, which was completely inhibited by p53 siRNA. Nutlin‐ 3 induced accumulation of ROS and subsequently, TEMPO, a ROS scavenger, prevented the phosphorylation of MEK1/2 and ERK1/2. Moreover, pifithrin‐μwhich blocks mitochondrial localization of p53 prevented ROS accumulation and MEK1/2‐ ERK1/2 phosphorylation as well. Knockdown of MEK1/2 and ERK1/2 activity using chemical inhibitors or small interfering RNA enhanced cell death induced by nutlin‐3. Collectively, it can be concluded that nutlin‐3 activates MEK1/2‐ERK1/2 via ROS generated by mitochondrial localization of p53 activation and nutlin‐3‐induced ERK1/2 activation may play a role in protecting U20S cells from p53‐dependent apoptosis, constituting negative feedback loop of p53.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011‐ 0027115).
Effect of pathway‐interconnectors in SEB induced apoptosis related events in human PBMCs
Authors: Banwarth Emily Anne , Schneider Heather , Mendis Chanaka
Published: June 2021

Staphylococcus enterotoxin B (SEB) is produced by the bacterium Staphylococcus aureus and is the most common cause of food poisoning often accompanied with nausea, diarrhea, and intestinal cramping. The purpose of this study is to further investigate human peripheral blood mononuclear cells (PBMCs) in order to better understand the apoptosis related events induced by SEB. C‐Jun N‐Terminal Kinase (JNK) has been previously identified to be induced by SEB, and has demonstrated that it inter‐connects multiple signaling cascades. As a crucial pathway inter‐connector in SEB induced human PBMCs, we believe that JNK may possess inhibitory effects of SEB‐induced apoptosis. We will further evaluate the alterations induced by specific JNK inhibitor by the known SEB induced, apoptosis related (Caspases, BAX, RIPK2, etc.) gene expression profile using RT‐PCR. Protein expression patterns of some genes will be examined through ELISA. We believe that our work will allow us to better understand the complex interactions of multiple signal transduction pathways induced by SEB.
Construction of non‐visual arrestins with enhanced specificity for individual G protein‐coupled receptors
Authors: Gimenez Luis E , Gurevich Vsevolod V
Published: June 2021

We identified elements that drive arrestin‐3 (Arr3) binding to G protein coupled receptors. Based on the identification of residues that determine arrestin‐1 and ‐2 selectivity for cognate receptors and the analysis of the evolution in the arrestin family, we mutated “receptor discriminator” residues in Arr3. The recruitment of these mutants to different receptors was assessed using BRET‐based assays in cells. D260K and Q262P showed enhanced preference for M2 muscarinic (M2R), D1 (D1R), and D2 (D2R) dopamine receptors, over β2‐adrenergic receptors (β2AR). The specificity was further enhanced by combining D260K and Q262P. Conversely, Y239T enhanced Arr3 binding to β2AR, while reducing the binding to M2R, D1R, and D2R. Mutation Q257Y reduced arrestin recruitment to D2R. The Y239T+Q257Y combination virtually eliminated binding to β2AR, reduced D2R binding, and enhanced Arr3 recruitment to D1R. Thus, Y239T+Q257Y had opposite effects on Arr3 binding to D1R and D2R. Thus, manipulation of key residues on the receptor‐binding surface modifies receptor preference, allowing for the construction of non‐visual arrestins specific for particular GPCR subtypes. These findings pave the way to the construction of signaling‐biased arrestins targeting the receptor of choice for research or therapeutic purposes.

NIH grants GM081756, GM077561 and EY011500 (VVG).
Non‐visual arrestins function as simple scaffolds assembling the MKK4–JNK3a2 signaling complex
Authors: Zhan Xuanzhi , Kaoud Tamer S , Dalby Kevin N , Gurevich Vsevolod V
Published: June 2021

Arrestins are a small family of proteins with four mammalian members that play key roles in the regulation of multiple GPCR‐dependent and ‐independent signaling pathways. Although arrestins were reported to serve as scaffolds for MAP kinase cascades, promoting the activation of JNK3, ERK1/2, and p38, the molecular mechanisms involved were not elucidated and even the direct binding of arrestins with MAP kinases were never demonstrated. Here using purified proteins we show that both non‐visual arrestins directly bind JNK3α2 and its upstream activator MKK4, and that the affinity of arrestin‐3 for these kinases is higher than that of arrestin‐2. Reconstitution of the MKK4‐JNK3α2 signaling module from pure proteins in the presence of different arrestin‐3 concentrations showed that arrestin‐3 acts as a “true” scaffold, facilitating JNK3α2 phosphorylation by bringing the two kinases together. Both JNK3α2 phosphorylation by MKK4 and JNK3α2 activity towards its substrate ATF2 increase at low and then decrease at high arrestin‐3 levels, yielding bell‐shaped concentration dependence expected with true scaffolds that do not activate the upstream kinase or its substrate. Thus, direct binding of both kinases and true scaffolding is the molecular mechanism of arrestin‐3 action on the MKK4‐JNK3α2 signaling module.

NIH grants GM081756, GM077561, EY011500 (VVG), GM059802 and Welch Foundation (F‐1390) (KND).
Temporal and Spatial Patterns of Enteric Nervous System Regeneration
Authors: Huang Sunny Qi , Cuyar Eugenia , Tossas Karen , Arraras Jose E Garcia
Published: June 2021

Echinoderms are one of the most striking animal phyla due to their regeneration capabilities. We have studied the cellular and molecular basis of intestinal regeneration in the sea cucumber Holothuria glaberrima. These animals have the capacity to regenerate a completely functional intestine after undergoing autotomy. In the present study we characterize the regeneration of the enteric neural system using immunohistochemistry in normal (noneviscerated) and regenerated organisms at different stages of regeneration. In particular we used anti‐calbindin, anti‐GABA, anti‐GFSKLYFamide and RN1, a monoclonal antibody that labels holothurian neuronal structures, to determine the temporal and spatial pattern of enteric nervous system regeneration. Animals were left to regenerate for 7, 14, 21, 28 and 35 days and the density and distribution of cells and fibers at each stage was determined. Our results show that most of the enteric nervous system cells and fibers can be found after 35 days of regeneration, and that different cells and fiber populations appear at specific stages of regeneration. The study of enteric nervous system regeneration in the intestine of H. glaberrima will provide a better understanding of how this process occurs in echinoderms and of its limitations in vertebrate animals.
Interactions of Rat Embryonic Hippocampal Neuronal Stem Cell on Gold Nano‐Film Surface
Authors: Islam Asiful , Menon Latika
Published: June 2021

Hippocampal neuronal stem cell culture on the nano‐substrates has introduced new era in nano‐neuroscience research. Neurons were cultured on the gold nano‐film surfaces to see the viability and interactions between neurons and nanoscale surfaces. Embryonic day 18 rat hippocampus tissues were mechanically dissolved into neuronal cells and cultured on top of the gold nano‐flim (GNF) surfaces and preserved at 370 C constant temperatures in a 5% CO2 incubator. These neurons on the GNF surfaces started to grow from day 2 and survived up to 25 days or more. For adhesion and growth of neurons we used an external coating protein, Poly‐D‐Lysine (PDL) layer on the top of the GNF. The extracellular protein PDL acted as an intermediate substance for neurites growth on the gold nano films slips. Without a PDL coating layer showed no or very little growth of neurons on the GNF surfaces. This study is the initial result for implementing the gold nano‐substrates to record the electrical signals from the neurons through gold nano‐films or nano‐wires.
Phosphorylation of p57/coronin‐1 regulates its binding to actin cytoskeleton
Authors: Oku Teruaki , Nakano Mai , Kaneko Yutaka , Tsuiji Makoto , Toyoshima Satoshi , Tsuji Tsutomu
Published: June 2021

The actin‐binding protein p57/coronin‐1, a member of the coronin protein family, is selectively expressed in hematopoietic cells and plays a crucial role in the immune response through reorganization of actin filaments. We previously reported that p57/coronin‐1 and F‐actin transiently accumulated in phagocytic cups and phagosomal membranes during phagocytosis in neutrophils and neutrophil‐like HL60 cells, and dissociated from phagosomes accompanied by phosphorylation of p57/coronin‐1. In this study, we analyzed phosphorylation sites of p57/coronin‐1 and possible involvement of phosphorylation of p57/coronin‐1 in its binding to F‐actin. Analyses by two‐dimensional gel electrophoresis, MALDI‐TOF‐MS and site‐directed mutagenesis indicated that p57/coronin‐1 had two major phosphorylation sites. The phosphorylation was found to be inhibited by chelerythrine, an inhibitor of protein kinase C (PKC). We also found that phosphorylation of p57/coronin‐1 decreased its binding affinity to F‐actin. These results suggest that the phosphorylation of p57/coronin‐1 by PKC regulates the association of p57/coronin‐1 with actin cytoskeleton and that the regulation is implicated in phagosome maturation.
Structural and functional studies of plant glucan phosphatases
Authors: Husodo Satrio , Gentry Matthew S.
Published: June 2021

Glucan phosphatases (GPs) dephosphorylate starch to regulate degradation. Historically, GPs have been defined as proteins containing a dual specificity phosphatase (DSP) domain and a carbohydrate binding module (CBM). To date there are two published GPs: Starch‐Excess‐4 (SEX4) and Like‐SEX4‐1 (LSF1). Our lab recently determined the structure of SEX4. These data identified a previously unknown domain in the C‐terminus of GPs, the so‐called CT‐domain. The discovery of this domain allowed us to identify a third member of the GP family, LSF2. LSF2 contains both a DSP and CT domain, but lacks a CBM. We cloned LSF2, purified recombinant protein, and demonstrated that it has glucan phosphatase activity even though it lacks a CBM. Furthermore, SEX4 and LSF2 possess different specificities: SEX4 dephosphorylates starch preferentially at the C6 position of glucose, while LSF2 acts on the C3 position. The function of LSF1 is still unknown as it lacks phosphatase activity thus far. We are performing crystallization screenings on LSF1 and LSF2 to obtain their structures, and we are using molecular modeling of LSF1 and LSF2 to determine key residues involved in glucan binding. In addition, we aligned the known orthologs of each gene and mapped the degree of conservation on the structures to infer critical regions for GP activity. Cumulatively, these results allow us to determine the molecular mechanism of this class of enzymes.

Supported by NIH grants R00NS061803, P20RR020171, and R01NS070899, and University of Kentucky College of Medicine startup funds to M.S.G.
Proteomic profiling of mouse liver under conditions of Hepatitis C virus‐mediated oxidative damage and CuZnSOD deficiency
Authors: Ramachandran Prasanna , Chu Victor C , Huang Ting-Ting
Published: June 2021

Hepatitis C virus (HCV) infection is one of the leading causes of liver cancer. Chronic HCV infection increases reactive oxygen species (ROS) production and leads to hepatocarcinogenesis. However the mechanisms by which ROS leads to liver cancer is poorly understood. We aimed to identify proteins that could be implicated in HCV induced oxidative damage and carcinogenesis. We employed transgenic mice expressing the HCV polyprotein, as well as non‐transgenic controls, with various levels of the major antioxidant enzyme CuZn superoxide dismutase (CuZnSOD). Mice livers were collected at 3, 12, or 18 months of age and proteins underwent redox changes were tagged with fluorescent labels Bis‐ANS and iodoacetamide fluorescein. We used two‐dimensional gel (2D‐gel) electrophoresis and mass spectrometry to map the mouse liver proteome. The 2D‐gel analysis software Dymension was used to investigate differences in protein expression. Several proteins involved in redox homeostasis, sugar metabolism and apoptosis have been found to vary in expression. In summary, our mouse models and proteomic methodology shows great promise in yielding candidate proteins involved in HCV‐mediated hepatocarcinogenesis. Out research was funded by the National Cancer Institute (NCI); the Department of Veterans Affairs, Palo Alto, CA; the Digestive Disease Center (DDC) and Stanford University.
Ligand mediated transcriptional regulation using DNA aptamers in cell free systems
Authors: Iyer Sukanya , Doktycz Mitchel John
Published: June 2021

Engineering gene networks offers an opportunity to harness biological function for biotechnological and biomedical applications. Critical to these efforts is the availability of a large library of ligand sensitive gene regulatory systems and the ability to rapidly test and evaluate these networks. When compared to the use of live cells, designing such systems in a cell free context offers a potentially simpler and more flexible context. Here we describe a strategy to regulate T7 RNA polymerase using DNA aptamers in a cell free context. We test the hypothesis that a DNA aptamer, when placed near the transcription start site, interferes with transcription in the presence of the target molecule. We utilized a DNA aptamer that binds thrombin as a model system for demonstrating the feasibility of the approach. We show that for the hybrid T7‐aptamer promoter, thrombin addition results in up to a 5‐fold reduction in gene expression. We further demonstrate that gene expression is dependent on the thrombin concentration and can be tuned by altering the position of the aptamer relative to the transcription start site. These results pave the way for expanding the library of ligands that can be used for regulating gene expression and can enable facile engineering of gene networks in cell free systems. This research was supported by NIH Grant EB000657
History of the PA profession; from medical corpsmen to present day practitioners
Authors: Pagel Josanne Kissel
Published: June 2021

In 1965, Dr. Eugene Stead of the Duke University Medical Center, put together the first class of Physician Assistants (PA) in response to the shortage of primary care physicians. This first class consisted of medical corpsmen and graduated in October, 1968. All PAs upon graduation must sit for and pass the National Commission on Certification of PAs (NCCPA) exam. PAs recertify with this exam every six years. And all PAs are licensed by the State Governing body (usually the State Medical Board) to practice medicine with physician supervision. PAs work in all disciplines of medicine and surgery specialties. They conduct physical exams, diagnose and treat illnesses, order and interpret tests, counsel on preventive health care, assist in surgery, and prescribe medications and treatments. They teach, provide education for the patients and participate in research. The Physician/PA Team has been identified by the AAFP as a best practice for the future of Health Care Reform. There are presently over 80,000 PAs throughout the U.S., and the profession shows steady growth and demand due to decreased residents’ hours and a decrease in physicians going into primary care fields. In 2011, Forbes Magazine has selected the PA Degree the number one Master's degree to obtain for a profession.
Sphingosine 1‐phosphate regulates matrix metalloproteinase‐9 expression and breast cell invasion through S1P3‐Gαq coupling
Authors: Moon Aree , Kim Eun-Sook , Kim Jong-Sook , Kim Sang Geon , Hwang Sejin , Lee Chang Ho
Published: June 2021

Sphingosine 1‐phosphate (S1P) is known as a potent inflammatory mediator. In this study, the effect of the inflammatory lipid S1P on the regulation of invasive/migratory phenotypes of MCF10A human breast epithelial cells was investigated to elucidate a causal relationship between inflammation and the control of invasiveness of breast cells. We show that S1P causes induction of matrix metalloproteinase‐9 (MMP‐9) in vitro and in vivo, and thus enhances invasion and migration. We also show that fos plays a crucial role in the transcriptional activation of MMP‐9 by S1P. In addition, activation of extracellular‐signal‐regulated kinases 1 and 2 (ERK1/2), p38 and alpha serine/threonine‐protein kinase (Akt) are involved in the process of S1P‐mediated induction of MMP‐9 expression and invasion. Activation of the S1P receptor S1P3 and Gαq are required for S1P‐induced invasive/migratory responses, suggesting that the enhancement of S1P‐mediated invasiveness is triggered by the specific coupling of S1P3 to the heterotrimeric Gαq subunit. Activation of phospholipase C‐β4 and intracellular Ca2+ release are required for S1P‐induced MMP‐9 upregulation. Taken together, this study demonstrated that S1P regulates MMP‐9 induction and invasiveness through coupling of S1P3 and Gαq in MCF10A cells, thus providing a molecular basis for the crucial role of S1P in promoting breast cell invasion.
Lipogenesis by reductive carboxylation is regulated by Bcr‐Abl signaling
Authors: Leonardi Roberta , Jackowski Suzanne , Rock Charles O.
Published: June 2021

Lipogenesis in rapidly growing cells requires an efficient metabolism of nutrients to supply the acetyl‐CoA building blocks. Acetyl‐CoA is usually formed from glucose‐derived pyruvate via citrate. An alternate route to citrate is the reductive carboxylation of α‐ketoglutarate (αKG) by isocitrate dehydrogenase (IDH). Purified IDH catalyzed NADP+‐dependent decarboxylation of isocitrate proceeds until the substrates are nearly depleted; however, the NADPH‐dependent carboxylation reaction is stringently inhibited by NADP+ and isocitrate. Cell labeling with 13C‐precursors was used to measure the contribution of reductive carboxylation to lipogenesis by determining the isotopomer distribution in intracellular acetyl‐CoA. αKG carboxylation is responsible for forming only 10% of acetyl‐CoA in 293T cells; however, this pathway supports a majority of acetyl‐CoA synthesis when mitochondrial function is compromised. In contrast, reductive carboxylation is a major route to acetyl‐CoA in an Arf‐null, Bcr‐Abl‐positive pre‐B cell model of acute lymphoblastic leukemia. Inhibition of Bcr‐Abl kinase rapidly switches metabolism from αKG as a principal source for acetyl‐CoA to glucose‐derived pyruvate. Carbon acquisition by reductive carboxylation is an efficient and unappreciated mechanism to acquire carbon for lipid assembly that is regulated by Bcr‐Abl signaling. (Supported by CA21765 & ALSAC)
VLDL Selection into VLDL Transport Vesicle (VTV) is Regulated by CideB
Authors: Tiwari Samata , Siddiqi Shaila , Mani Arul , Siddiqi Shadab A.
Published: June 2021

Very low‐density lipoproteins (VLDLs) circulating in the plasma play a key role in the development of atherosclerosis. VLDLs are synthesized and secreted by the liver. The rate‐determining step in VLDL secretion is their transport from the endoplasmic reticulum (ER) to the Golgi, which is facilitated by a distinct vesicle, the VTV. We have shown that the VTV exports VLDL but not albumin from the same ER, however, the mechanism of VLDL selection into VTV is not known. Our proteomic analysis supported by immunoblotting data show that a 26 kDa protein, cideB which plays an important role in VLDL‐maturation, is present in the VTV. In current study, we investigated the role of cideB in VLDL selection into the VTV. Our co‐immunoprecipitation data revealed that cideB specifically interacts with VLDL structural protein, apolipoproteinB100 (apoB100), but not with albumin. Confocal microscopic data indicate that cideB co‐localizes with apoB100 in the ER. Additionally, cideB interacts with VTV‐budding initiator, Sar1. To test the role of cideB in VLDL selection into VTV, we performed an in vitro ER‐budding assay. We show that the blocking of cideB, inhibits VTV‐budding indicating a direct association between VLDL recruitment and VTV formation. These findings suggest that cideB regulates the selection of VLDL into VTV.