bluuebunny/crossref_metadata_2025 huggingface dataset:
GMPCPP and Paclitaxel Effect on Microtubule Stiffness
Authors: Cheng Sarah , Donhauser Zachary
Published: June 2021

Microtubules are dynamic polymers of tubulin that contribute to the structural framework of the eukaryotic cytoskeleton, pathways for intracellular transport, and are key players in cell division. In vivo, the majority of microtubules are hollow cylinders structured as 13 protofilaments; however depending on stabilization conditions when polymerized in vitro, the number of protofilaments in a microtubule can range from 8–17. To enable study of microtubules and avoid depolymerization, two chemicals are commonly used to stabilize microtubules from depolymerization: guanylyl‐α‐β‐methylene diphosphonate (GMPCPP), a slowly hydrolyzable GTP analog, and paclitaxel (taxol), an anti‐cancer drug. Analyzing physical properties of these microtubule variations can provide greater insight on the structure and stiffness of naturally occurring microtubule variations found in vivo. Optical bending measurements have found taxol reduces radial rigidity and contributes to microtubule flexibility while GMPCPP has an opposite, stiffening effect on microtubules. Atomic force microscopy (AFM) was used to make direct indentation stiffness measurements of microtubules polymerized under different stabilization techniques: GMPCPP only, taxol only, and combined GMPCPP with taxol. Greater information may also improve understanding of GTP hydrolysis and contribute to future in vitro studies of microtubules.
Mycobacterium smegmatis Ku binds zinc
Authors: Kushwaha Ambuj Kumar , Grove Anne
Published: June 2021

Ku protein is an important component of the non‐homologous end‐joining (NHEJ) pathway of DNA double strand break repair. Previously thought to be encoded only by eukaryotes, homologues of Ku protein have been identified in some bacterial species. The overall three dimensional topology of Ku is conserved from prokaryotes to eukaryotes and includes a central core consisting of a β‐barrel domain, pillar and bridge regions that together form a ring‐like structure. The bridge‐region is an extension of the β‐barrel core, which is well conserved and important for dimerization and DNA‐binding. Sequence analysis has revealed that the bridge‐region of Ku from several Gram‐positive bacteria and bacteriophages contains a potential zinc‐binding site with conventional HxxC and CxxC (where x is any residue) zinc binding motif, that belongs to a new family of zinc‐ribbon folding domain, suggested to be derived from a regular Zn‐ribbon by a segment‐swapping event. Interestingly, these sites have either deteriorated or been entirely lost in Ku from other organisms. Using an in vitro metal binding assay we have shown that M. smegmatis Ku binds zinc. Also, we observed that zinc binding stabilizes the Ku protein and protects it from oxidative damage. Based on our finding, we predict that retention of zinc binding site in Ku proteins from M. smegmatis and other related gram‐positive bacterial species might also provide protection against zinc toxicity.
The interaction of Hsp70s and lipids is conserved from bacteria to humans
Authors: Siracusa Matthew Conrad , Nikolaidis Nikolas
Published: June 2021

Hsp70s are molecular chaperones essential for protein homeostasis under both physiological and pathological conditions. Two mammalian Hsp70s, HSPA1A and HSPA8, have been found to associate with cellular membranes, where they bind to specific lipids. This binding localizes and anchors the chaperone to the plasma membrane, stabilizes the lysosomal membrane, regulates immune responses and cell signaling, and is required for microautophagy. Despite the essential role of membrane‐bound Hsp70s in immunogenic responses and cancer biology as well as cell signaling, the biological importance of Hsp70‐lipid interactions remains largely uncharacterized. Importantly, it remains unknown whether lipid‐binding is a function evolutionarily conserved in yeast and bacterial Hsp70s. In this study we examined the lipid‐binding properties of two yeast Hsp70s, the cytosolic SSA1 and the mitochondrial SSC1, and one bacterial Hsp70 and compared them to the mammalian Hsp70s. Our data demonstrate that although SSA1 and DnaK bind to several anionic lipids, SSC1 does not. Although further experimentation is required to determine the affinities and specificities of these interactions, our results suggest that lipid‐binding is an evolutionarily conserved property of Hsp70s and that they are functionally differentiated with respect to their lipid‐binding properties.

This project was supported by funds from CSUPERB and CSUF to NN
Role of arginine and lysine in the antimicrobial mechanism of histone‐derived peptides
Authors: Cutrona Kara , Elmore Donald
Published: June 2021

Antimicrobial peptides can kill microbes via various mechanisms including cell lysis or translocation and interaction with intracellular components. Understanding how particular residues affect peptide mechanisms would advance the development of improved drugs. Previous research has shown translocating ability to be promoted by increased arginine content and impaired by increased lysine content in cell‐pentetrating peptides. However, there is little research in this area on antimicrobial peptides. To this end, this study analyzed the antimicrobial mechanism of three histone‐derived peptides – buforin II, DesHDAP1 and parasin – in comparison to their R→K and K→R mutants that contained only lysine or arginine, respectfully, for their positive residues. Analogous to cell‐penetrating peptides, the antibacterial activity of these peptides increased with increased arginine content. These changes can be explained through differences in the translocating, membrane permeabilizing and DNA‐binding ability of peptides. Experimental results were reinforced with explicit molecular dynamics simulations using a 3:1 POPC:POPG membrane. This work was supported by National Institute of Allergy and Infectious Diseases (NIH/NIAID) award R15AI079685.
The role of cardiolipin in defining the mammalian ANT interactome
Authors: Lu Ya‐Wen , Baile Matthew Garrett , Huang Tai‐Chung , Pandey Akhilesh , Claypool Steven Michael
Published: June 2021

The major ADP/ATP carrier (AAC in yeast; ANT in mammals) in Saccharomyces cerevisiae, AAC2, mediates the 1:1 exchange of ADP into and ATP out of the mitochondrial matrix and is thus a required component of the oxidative phosphorylation machinery. Originally modeled to function in isolation, we recently demonstrated that yAAC2 associates with non‐yAAC2 partner proteins, including the respiratory supercomplex, in the context of cardiolipin (CL)‐containing membranes. As very little is known about the mammalian ANT interactome, this project aims to define the mammalian ANT interactome and determine if these interactions, like yAAC2, exhibit CL‐dependency. Using a combination of structural and mutational analyses, blue native‐PAGE, co‐immunoprecipitation, and stable isotope labeling by amino acids in cell culture (SILAC) on yeast, cell culture, and primary tissue models, we will determine the players that interact with mammalian ANT and the role CL plays in mediating these interactions. Results generated will allow for a more comprehensive understanding of the importance of the mammalian ANT interactome in health and disease. This work is supported by NHLBI grant R00HL089185 (SMC) and American Heart Association Predoctoral Fellowships 12PRE11910004 (YL) and 10PRE3280013 (MGB).
The ubiquitin conjugating enzyme UbcH10 competes with UbcH3 for binding to the SCF complex, a ubiquitin ligase involved in cell cycle progression
Authors: Le Feuvre Aurelie Yvette , Afful Derrick , Shang Fu , Taylor Allen
Published: June 2021

Ubiquitylation, which regulates most biological pathways, occurs through an enzymatic cascade involving a ubiquitin (ub) activating enzyme (E1), a ub conjugating enzyme (E2) and a ub ligase (E3). UbcH3 is the E2 that interacts with SCF (Skp1/Cul1/F‐box protein) complex and ubiquitylates many proteins (p27kip1, p21Cip1, cyclin E) that regulate the G1/S transition. Previously we demonstrated that UbcH3 interferes with UbcH10 functions, an E2 involved in the cell cycle regulation, at the G2/M transition, by competing for Anaphase‐promoting complex (APC). In this work, we aim to determine the effects of UbcH10 on the function of UbcH3. We showed by in vitro degradation assays that addition of UbcH10 inhibited UbcH3‐catalyzed degradation of a typical SCF substrate, p27kip1, while it had no effect on the degradation of a non SCF substrate, such as á‐lactalbumin. The competition for the binding to the SCF was confirmed by GST pull‐down assays using GST‐Roc1, the RING subunit of SCF that normally interacts with UbcH3. The results showed that UbcH10 was able to interact with Roc1 and decreased UbcH3/Roc1 interaction. These data suggest that a balance between UbcH3 and UbcH10 levels are critical for controlling the cell cycle progression and thus highlight a novel mechanism for regulating the progress through the cell cycle.
Functional analysis of the interaction between Kaposi sarcoma‐associated herpes virus anti‐apoptotic Bcl‐2 homolog and the human host pro‐apoptotic protein, BIK
Authors: Caruso Brittany , Walker Stephanie , Roecklein‐Canfield Jennifer
Published: June 2021

Kaposi sarcoma‐associated herpes virus (KSHV) is associated with various malignancies the most common being the skin cancer, Kaposi sarcoma. A common mechanism employed by tumor viruses is the inhibition of the apoptotic signaling pathway. The cellular BCL‐2 family of proteins are critical components of apoptotic signalling that converges at the mitochondria and is comprised of both pro‐and anti‐apoptotic proteins that share homology in four conserved trans‐membrane domains, BH1‐BH4. KSHV contains a gene, vBCL‐2, with functional and sequential homology to the anti‐apoptotic bcl‐2 family of proteins. This gene is expressed in the early stages of the viral lytic cycle and has been shown to affect the progression of apoptotic signaling in order to allow for proper viral replcation and virion production. Because apoptosis is highly linked with cell proliferation, growth, and development, problems with apoptosis can lead and are often linked to tumors and various cancers. We present here the characterization of the interaction between KSHV's vBcl‐2 protein and a human protein, Bik, a member of the BH3 only family of pro‐apoptotic proteins. Bik has significant antitumor activity, mediated by it's BH3 domain. Several mutant contructs of both the viral, vBcl‐2 protein and the human, Bik protein have been examined for loss of interaction and the effect on apoptosis. KSHV employs multiple mechanisms to manipulate the cell cycle and apoptosis leading to cell transformation. Understanding the role vBcl‐2 and the BH‐3 only family of proteins plays in the viral pathway to cancer will provide new information about the process of tumor development.
Nuclear localized BMP2 promotes cell cycle progression
Authors: Nichols Brandt A. , Goar Wesley A. , McCune Broc T. , Bridgewater Laura C.
Published: June 2021

Nuclear localized bone morphogenetic protein 2 (nBMP2) is a recently discovered protein produced by alternative translation of the BMP2 transcript. We have detected strong nBMP2 expression in numerous immortalized and tumor cell lines, but not in primary cells, suggesting that nBMP2 overexpression is correlated with cell cycle dysregulation. The objective of this study was to determine the impact of nBMP2 on the cell cycle. To accomplish this aim, we overexpressed nBMP2 in cultured cells, stained the cells with propidium iodide, and analyzed cell cycle distribution by flow cytometry. We found that overexpression of nBMP2 in the immortalized human cell line HEK293 and in the colon cancer cell line HT‐29 increased the percentage of cells in S phase. A yeast two‐hybrid screen identified the SCF E3 ligase ROC‐1 as an nBMP2 binding partner. ROC‐1 targets several cell cycle inhibitors (including p21, p27, and p57) for degradation by the proteasome, suggesting that overexpression of nBMP2 could promote the cell cycle by increasing degradation of one or more cell cycle inhibitors. Together, these data suggest that nBMP2 is a potential oncoprotein. Funding provided by NIH #AR048839.
Specific mutations on the receptor‐binding interface of non‐visual arrestins differently affect recruitment by the NPY receptor family
Authors: Gimenez Luis E. , Babilon Stefanie , Beck‐Sickinger Annette G. , Gurevich Vsevolod V.
Published: June 2021

Based on the identification of residues that determine receptor selectivity in arrestins and the phylogenetic analysis of the arrestin (arr) family, we introduced ten “discriminator” mutations in arr3. Using a BRET‐based live cell recruitment assay with M2 muscarinic (M2R), D1 (D1R), and D2 (D2R) dopamine and β2‐adrenergic receptors (β2AR), we showed that seven out of ten mutations differentially affect arr3 binding to individual receptors. D260K and Q262P reduced binding to β2AR much more than to other receptors. D260K+Q262P virtually eliminated β2AR binding, while preserving the interactions with M2R, D1R, and D2R. Conversely, Y239T enhanced arr3 binding to β2AR and reduced binding to M2R, D1R, and D2R, while Q256Y reduced recruitment to D2R. Y239T+Q256Y eliminated binding to D2R, but did not affect D1R, yielding a mutant with high selectivity for D1R over D2R. Using NPY receptors Y1, Y2 and Y5 we observed lack of recruitment of arr3 to the Y5R and equal recruitment of WT and all mutant arr3 to Y1. Most mutations significantly affected recruitment to Y2, with the largest effect for Y239T. Thus, manipulation of key residues on the receptor‐binding surface modifies receptor preference of arrestins, enabling construction of non‐visual arrestins specific for particular receptors.

Support: NIH GM081756, GM077561 and EY011500 to VVG.
Expression of 20[alpha] HSD in porcine ovary and placenta during early pregnancy
Authors: MIN Kwan‐Sik , Nanjidsuren Tsevelmaa , Park Chae‐Won
Published: June 2021

The aldo‐keto reductase (AKR) superfamily are monomeric oxidoreductases that catalyze the NADP(H)‐dependent reduction of a wide variety of substrates, including seroids, prostaglandins, bile acids, carbohydrates, and xenobiotics. A group of AKRs known as hydroxydteroid dehydrogenase (HSDs) play a pivotal role in the modulation and regulation of steroid hormones, such as androgen, estrogens, and progestins, and are thus considered important targets for drug design. Thus, to gain further insights into the expression and localization of 20 HSD in the porcine ovary and placenta during early pregnancy, we analyzed the mRNA and protein expressions and immunohistochemistry. The expression of mRNA and protein was particularly strong in the ovary on day 31 of pregnancy. We also reported that 20[alpha]‐HSD was localized in the villus of trophoblast and endometrium glands during early pregnancy. Further studies are needed to determine the functional significance of porcine 20[alpha]‐HSD during pregnancy.
Identification of binding proteins of the Xenopus morphogenetic factor, Tumorhead
Authors: Alicea‐Torres Kevin M. , Lopez Edgardo , Flores Noelia , Vega Irving E. , Traverso Edwin E.
Published: June 2021

Tumorhead (TH) is a protein that regulates cell proliferation during early embryogenesis in Xenopus laevis. To understand how TH functions at the molecular level, we have taken a mass spectrometry‐based proteomics approach to identify TH interacting proteins. TH immunoprecipitates (IPs) were prepared from cell extracts derived from Xenopus oocytes and embryos previously injected with mRNAs encoding epitope‐tagged versions of either wild type TH, or a membrane‐bound mutant of TH, which might serve as a more efficient bait. The TH IPs were subjected to polyacrylamide gel electrophoresis, followed by silver staining to visualize the various proteins that are present in the IPs. Protein bands present in TH IPs but absent in samples generated from control‐injected embryos are being processed for nanoelectrospray ionization tandem mass spectrometry to determine their identity. Once these proteins have been identified, we will perform traditional co‐immunoprecipitation and co‐localization experiments to confirm their interaction with TH. We will also perform gain and loss of function of the identified proteins to determine if any resulting phenotype phenocopies the corresponding TH gain and loss of function phenotypes. These experiments will allow us to start addressing if the interaction between TH and the identified proteins is important for TH's role in the control of cell proliferation.
Ras Activity Regulation by Monoubiquitination
Authors: Campbell Sharon L , Baker Rachael , Lewis Steven , Sasaki Atsuo , Wilkerson Emily M , Locasale Jason , Cantley Lewis C , Kuhlman Brian , Dohlman Henrik
Published: June 2021

Ras proteins are critical regulators of cellular growth. The population of activated, GTP‐bound Ras in normal cells is controlled by protein regulators. Oncogenic mutations promote activation by populating the active state of Ras, most frequently by impairing the action of proteins that downregulate its activity. Recently, a novel mechanism of Ras activation was identified, in which modification by ubiquitination was found to populate Ras in its active GTP‐bound state, enhance interactions with downstream effectors and promote Ras‐mediated tumorigenesis. These findings represent a paradigm shift in our understanding of the role monoubiquitination may play in protein regulation. By conducting NMR, modeling, biochemical and biological studies, we find that monoubiquitination of Ras at position 147 has little effect on intrinsic guanine nucleotide binding or hydrolysis, but impairs the ability of GTPase activating proteins to downregulate Ras activity, accounting for the accumulation of Ras‐GTP in cells. These findings reveal that a post‐translational modification can disrupt protein interactions that normally constrain cellular signaling and transformation.
PDZ‐RhoGEF and LARG are essential for embryo development, and provide a link between thrombin receptors and Rho activation
Authors: Mikelis Constantinos Marios , Simaan May , Li Wenling , Szabo Roman , Martin Daniel , Mukouyama Yosuke , Bugge Thomas H. , Gutkind Silvio J.
Published: June 2021

G protein coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation. Among them, PDZ‐RhoGEF, leukemia‐associated Rho GEF (LARG) and p115‐RhoGEF (p115) share the presence of a regulator of G protein signaling‐homology (RGS) domain. There is limited information on the biological roles of this RGS‐containing family of RhoGEFs in vivo. Here, we investigated the biological role of two structurally related RhoGEFs: PDZ‐RhoGEF and LARG, by generating knockout mice. Although these mice do not display any overt phenotype, compound deficiency in PDZ‐RhoGEF and LARG leads to early embryonic lethality and to complex developmental defects. Signaling from Gα11/q‐linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all 3 RGS‐containing RhoGEFs. However, combined lack of PDZ‐RhoGEF, LARG, and p115 expression abolished signaling through Gα12/13 to Rho, and thrombin‐induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS‐containing RhoGEF family in signaling to Rho by Gα12/13‐coupled GPCRs, which may likely play a critical role during embryonic development.
“...and we took the median of the three values”: Optimal treatment of replicate measurements in anthropometric studies
Authors: Villamor Eduardo , Bosch Ronald J
Published: June 2021

In an effort to ameliorate the effects of measurement error, anthropometric studies often include replicates of each measurement. There is marked inconsistency on the method used to combine these measures for analysis. We carried out 10,000 Monte Carlo simulations to explore the effect of 5 approaches to combine 3 measurements in a hypothetical 2‐group intervention study in which the outcome, length at age 1 y, was measured in triplicate. Each group had 100 subjects. One group had a true value with a normal distribution N(mean=76, SD=2.4 cm). We estimated statistical power to detect a 1 cm difference between the groups, based on a t‐test. The 5 combination methods simulated were the mean, median, median of pairwise averages, and the median or mean of only the first 2 measurements when they were close, or of 3 when the first 2 were far apart and a 3rd one (tiebreaker) was obtained. Under a realistic scenario with a measurement error distribution N(0, 0.8), highest power was reached by use of the median of pairwise averages, the mean, or the median. However, when a portion of the data (5%) were contaminated by greater error (e.g. due to data entry), the median of 3 measurements outperformed all other methods while the mean had the lowest performance. Using the median of 3 replicates is a safe and robust approach in the setting of measurement, recording, and data entry errors, which are not uncommon in anthropometric research.
Fructose, sweetened food and beverage intake and metabolic markers in children
Authors: County Christine , Thomas Alicia , Catalano Patrick M. , Nock Nora L.
Published: June 2021

Over the past three decades, high fructose corn syrup (HFCS) has replaced about one‐third of the sugar consumption in the U.S. and, interestingly, this increase parallels the rise in obesity. In contrast to glucose, fructose is not regulated by insulin or cellular energy status; and, in animals, fructose administration increases hyperuricemia, dyslipidemia, blood pressure, glucose intolerance and leptin resistance. However, the role of fructose on metabolic dysfunction in children has not been well explored and additional studies are needed to better inform policy. Thus, we examined associations between fructose and metabolic markers (fasting glucose, insulin, TG, HDL, LDL, leptin, TNF‐á, BP, weight, BMI) in 89 children, 6–11 years old, born from women with and without gestational diabetes mellitus (MGD). Previous results from this cohort have shown that children in the highest tertile of the CDC percentiles had significantly greater carbohydrate intake and fasting insulin and leptin levels. Analyses evaluating the intake of fructose, HFCS and sweetened foods and beverages on metabolic markers are underway and will be presented for the total sample and stratified by the presence or absence of MGD.

Grant Funding Source: NIH NCI K07129162
2‐Hydrazinoquinoline as a novel derivatization agent for LC‐MS‐Based metabolomic investigation of streptozotocin‐elicited ketoacidosis
Authors: Lu Yuwei , Yao Dan , Chen Chi
Published: June 2021

Aldehydes, ketones, and carboxylic acids are important intermediates and end products of many metabolic processes. In this study, the use of 2‐hydrazinoquinoline (2‐HQ) as a novel derivatization agent for LC‐MS analysis of aldehydes, ketones, and carboxylic acids in biological samples was explored, and the conditions for the derivatization reaction were optimized. The formation of carboxylic acid derivatives is attributed to the esterification reaction between 2‐HQ and carboxyl group, while the production of aldehyde and ketone derivatives is through the formation of Schiff bases between 2‐HQ and carbonyl group. Using this 2‐HQ‐based approach, the metabolic disorder induced by streptozotocin‐elicited diabetes was examined by the LC‐MS‐based metabolomics. The results showed the time‐dependent separation of mouse urine samples from STZ treatment in a multivariate model of urinary metabolites. Both known and novel small‐molecule biomarkers associated with STZ‐induced ketoacidosis were conveniently identified and subsequently elucidated, reflecting the dramatic changes in nutrient (glucose, amino acid, and lipid) and energy metabolism after STZ treatment.
Polyphenolic content of sweet dried cranberries compared to cranberry juice cocktail
Authors: Marais Jannie , Khoo Christina
Published: June 2021

Polyphenols such as flavonoids and phenolic acids are common constitutes in fresh fruit and fruit juices such as cranberry. Consumption of cranberry juice cocktail (CJC) can significantly contribute to total dietary polyphenol intake. Cranberry flavonoids, especially proanthocyanidins (PACs) play an important role in the inhibition of chronic diseases such as cardiovascular, periodontal, and gastrointestinal. Additionally, studies show that A‐type PACs found in cranberries could play a significant role in the prevention of urinary tract infections caused by pathogenic bacteria.

In this study, we compared the polyphenolic content of sweet dried cranberries (SDC's) with cranberry cocktail juice (CJC), a 27% cranberry juice drink. The total phenolic content of CJC determined by the Folin‐Ciocalteu method measured 190 mg gallic acid equivalent (GAE) per 240 ml serving. CJC also had an oxygen radical absorbance capacity (ORAC) of 2040 μmole Trolox equivalents (TE) and contained 35 mg of PACs per serving (measured by DMAC). A standard 40 gram serving of SDC's contained a total phenolic content of 177 mg GAE, delivered 38 mg PACs and had an ORAC value of 1596 μmole TEs. In addition to being a good source of fiber in the diet, SDC's are very comparable to CJC as another source of polyphenols in the diet. This research was supported by Ocean Spray Cranberries, Inc.
Cortricotropin‐releasing factor (CRF) may protect the gastric mucosa against injury through involvement CRF1 and CRF2 receptor types
Authors: Filaretova Ludmila , Morozova Olga , Myazina Marina , Bagaeva Tatiana
Published: June 2021

The stress response involves the activation of two corticotropin‐releasing factor (CRF) receptor types 1 and 2 (CRF1 and CRF2 receptors). In the present study we investigated whether exogenous CRF may protect the gastric mucosa against injury through involvement of CRF1 receptors or CRF2 receptors. Gastric injury was induced by indomethacin (35 mg/kg, s.c.) in conscious rats and by 3.5 h gastric ischemia‐reperfusion in anaesthetized rats. CRF at the doses, which markedly increased plasma corticosterone levels, significantly suppressed the occurrence of gastric erosion induced by each stimulus. The blockade of CRF action was investigated by the nonselective CRF1/CRF2 receptor antagonist, astressin, the CRF1 receptor antagonist NBI 27914, and the CRF2 receptor antagonist astressin2‐B. The protective effect of CRF on the gastric mucosa against indomethacin‐induced injury was prevented by astressin, NBI 27914 and also astressin2‐B. The protective effect of CRF on the gastric mucosa against ischemia‐reperfusion‐ induced lesion was prevented by astressin and astressin2‐B. The results obtained suggest that exogenous CRF may protect the gastric mucosa against injury through CRF1 and CRF2 receptors. Supported by Program Presidium RAS N 7 and RFBR grant 10–04‐00605.
Antiviral activity of Bifidobacterium adolescentis SPM1605 against Coxsackievirus B3
Authors: Kim Min Ji , Lee Do Kyung , Kang Joo Yeon , Park Jae Eun , Ha Nam Joo
Published: June 2021

Bifidobacteria are considered one of the most beneficial probiotics and have been widely studied for their effects against specific pathogenes. Coxsackievirus B3 (CVB3) is a member of the Picornaviridae and is responsible for a wide variety of mild or severe infections including acute and chronic inflammations. The present study investigated the antiviral activity of bifidobacteria isolated from healthy Koreans against CVB3. The plaque reduction assay has been accepted as the ‘gold standard’ for settling the antiviral activity of materials against these viruses. So the effect of bifidobacteria isolates against CVB3 was measured by the plaque reduction assay. And cellular toxicity of bifidobacteria in Hela cell was measured using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐ diphenyltetrazolium bromide (MTT) assay. Among 16 probiotics isolates (9 Bifidobacterium adolecentis, 3 B. longum, 2 B. pseudocatenulatum and 2 Lactobacillus ruminis), 4 B. adolescentis and 2 B. longum had the antiviral effect against CVB3, but the others did not show this effect. Furthermore, B. adolescentis SPM1605 showed the highest inhibitory effect against CVB3 among 6 probiotics possess antiviral activity. And this strain did not affect the viability of Hela cell. The results suggest that this B. adolescentis SPM1605 suppress CVB3 expression and could be potential application for alternative therapy of infectious disease by coxsackievirus.
FcRn Expression and Antibody Transcytosis in Adult Human and Non‐Human Primate Intestine
Authors: Hornby Pamela Janet , Cooper Philip R , Kliwinski Connie , Ragwan Edwin , Mabus John R , Harman Benjamin , Dorai Haimanti , Giles‐Komar Jill
Published: June 2021

The neonatal Fc receptor (FcRn) expressed in duodenal epithelial cells transports immunoglobulin G (IgG) in lactated milk into the newborn lamina propria, but its role in IgG transcytosis in adults is unknown. Therefore, we assessed the following: FcRn expression on human intestinal epithelial (hInEp) cells (flow cytometry and binding); transcytosis of IgG in human intestinal segments mounted in Ussing‐type flux chambers by ELISA; FcRn (mRNA and protein) expression in adjacent mucosa; serum IgG uptake after intestinal administration in isofluorane‐anesthetized cynomolgus monkeys. Surface expression and FcRn‐dependent binding of IgG was shown in caco‐2 and hInEp cells. Human intestinal villi enterocytes were FcRn‐immunostained; unexpectedly, FcRn +ve staining in crypt cells was colocalized with GLP‐1, GLP‐2 and CCK in enteroendocrine cells. Higher mucosal FcRn mRNA and protein expression was associated with greater luminal to serosal flux of mAb in human intestinal segments. In cynomolgus monkeys, serum levels of full‐length mAb (0.3% dose) were detected after direct infusion of mAb into ileum/proximal colon (2 mg/kg; n=3), but not after oral gavage. FcRn intestinal expression contributes to fractional uptake of mAb delivered intestinally; however epithelial cell mAb transcytosis is unlikely to be mediated solely by surface FcRn. The function of FcRn in enteroendocrine cells is unknown.
Role of kallikrein 6‐mediated PAR signaling in astrogliosis
Authors: Radulovic Maja , Yoon Hyesook , Vandell Alexander , Scarisbrick Isobel
Published: June 2021

A remarkable feature of the expression of kallikrein 6 (KLK6) in the CNS is its abundance in oligodendroglia and neurons, but relative lack of expression in astrocytes. We have shown in cases of CNS injury however, that KLK6 is induced in reactive astrocytes and exhibits prolonged expression in astroglial scar tissue in cases of spinal cord injury, multiple sclerosis and glioblastoma multiforme. In this study, we address the hypothesis that KLK6 plays a role in the promotion of astrogliosis and that this occurs by selective activation of protease activated receptors (PARs). Using primary murine astrocytes we show KLK6 promotes rapid transformation from an epithelioid to a stellate morphology and secretion of interleukin 6, each a hallmark of astrogliosis. The ability of KLK6 to promote stellation was shown to occur as early as 4 hours after a single pulse of KLK6, to be dose‐dependent, and to require activation of PKC and AKT signaling cascades. The stellation promoting activities of KLK6 were further shown to depend at least in part on activation of the thrombin receptor PAR1, since these effects were reduced in astrocytes derived from PAR1‐deficient mice and by a PAR1 specific inhibitor, SCH79797. These results point to novel roles for KLK6‐mediated PAR signaling in the spectrum of events that culminate in astrogliosis. This work was supported by NIH R01 NS052741.
Cannabidiol attenuates the long lasting cognitive deficits and anxiogenic‐like behaviors promoted by murine cerebral malaria
Authors: Campos Alline Cristina , Miranda Aline S , Brant Fatima , Rocha Natalia P , Machado Fabiana S , Rachid Milene A , Guimarães Francisco S , Teixeira Antonio L
Published: June 2021

Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection that causes permanent neurological and behavioral deficits after infection resolution by antimalarial drugs. Cannabidiol (CBD) is the major nonpsychotomimetic constituent of Cannabis sativa with neuroprotective properties. The present work aimed to determinate if CBD treatment could prevent behavioral changes found in mice infected by Plasmodium berghei‐ANKA (PbA). Female C57Bl6 mice were infected or not with PbA (10茐 parasitized/0.2 mL PBS i.p.). On day 3 after infection (dpi) all groups receive the first injection of CBD (30mg/Kg/day‐7 days) or vehicle. On 5dpi, infected animals started to be treated with artesunate (32mg/kg/day‐5 days i.p). Five days after completed clearance of parasitemia, all groups were submitted to the Object Recognition task (OR) and to the Elevated Plus Maze (EPM). After completed clearance of the paraesitemia by artesunate, PbA vehicle treated mice displayed memory deficits in the OR (represented by decrease in the % of new object index) and exhibited an increase in anxiety‐like behaviors in the EPM. Cannabidiol treatment was able to prevent long lasting anxiogenic and cognitive impairment found in PbA mice. Our results indicate that CBD could be useful as an adjunctive therapy to prevent brain damage during the course of CM.
Improvement in coronary heart disease risk factors during an intermittent fasting/calorie restriction regimen: Relationship to adipokine modulations
Authors: Kroeger Cynthia M. , Klempel Monica C. , Bhutani Surabhi , Trepanowski John F. , Varady Krista A.
Published: June 2021

Background

The ability of an intermittent fasting (IF)‐calorie restriction (CR) regimen (with or without liquid meals) to modulate adipokines in a way that is protective against coronary heart disease (CHD) has yet to be tested.

Objective

Accordingly, we examined the effects of an IFCR diet on adipokine profile, body composition, and markers of CHD risk in obese women.

Methods

Subjects (n = 54) were randomized to either the IFCR‐liquid (IFCR‐L) or IFCR‐food based (IFCR‐F) diet for 10 weeks.

Results

Greater decreases in body weight and waist circumference were noted in the IFCR‐L group (4 ± 1 kg; 6 ± 1 cm) versus the IFCR‐F group (3 ± 1 kg; 4 ± 1 cm). Similar reductions (P < 0.0001) in fat mass were demonstrated in the IFCR‐L (3 ± 1 kg) and IFCR‐F group (2 ± 1 kg). Reductions in total and LDL cholesterol levels were greater (P = 0.04) in the IFCR‐L (19 ± 10%; 20 ± 9%, respectively) versus the IFCR‐F group (8 ± 3%; 7 ± 4%, respectively). LDL peak particle size increased (P < 0.01) in the IFCR‐L group only. The proportion of small LDL particles decreased (P < 0.01) in both groups. Adipokines, such as leptin, interleukin‐6 (IL‐6), tumor necrosis factor‐alpha (TNF‐alpha), and insulin‐like growth factor‐1 (IGF‐1) decreased (P < 0.05), in the IFCR‐L group only.

Conclusion

These findings suggest that IFCR with a liquid diet favorably modulates visceral fat and adipokines in a way that may confer protection against CHD.
Mitochondria‐targeted antioxidant therapy with MitoQ ameliorates age‐related vascular endothelial dysfunction
Authors: Gioscia‐Ryan Rachel Anna , LaRocca Thomas J , Zigler Melanie C , Murphy Michael P , Seals Douglas R
Published: June 2021

Age‐related vascular endothelial dysfunction, characterized by a decline in endothelium‐dependent dilation (EDD), is mediated in large part by oxidative stress. Mitochondria are a major source of reactive oxygen species (ROS) and may be an important therapeutic target for reducing age‐related oxidative stress. We tested the hypothesis that MitoQ, a mitochondria‐targeted antioxidant, would improve vascular function in aging. Old mice (O, 26–28 mo) had elevated aortic whole‐cell (WC, O: 4964±127 AU p<0.05 vs. young [Y, 6–8 mo]: 1515±281) and mitochondria‐specific (MtS, O: 1370±170 p<0.05 vs. Y: 395±50) superoxide production (EPR), as well as ~2× greater (vs. Y) levels (Western blot) of nitrotyrosine (NT), a marker of oxidative protein modification. The age‐related increase in oxidative stress was associated with impaired maximal carotid artery EDD to acetylcholine (Ach; O: 67±8% p<0.05 vs. Y: 86±7%). Mitochondrial stress (0.5uM rotenone incubation) exacerbated endothelial dysfunction in arteries of O mice (~25% reduction in EDD, p<.05 vs. Ach alone), but not Y. MitoQ (250 uM in drinking water, 4 weeks) treatment in O mice (OMQ) normalized superoxide production (OMQ: WC, 1198±14; MtS, 658±112, both p<.05 vs. O) and NT. MitoQ treatment also restored EDD (OMQ: 92±4%, p<.05 vs. O) and conferred protection against acute mitochondrial stress (rotenone). Finally, ex‐vivo treatment of arteries with MitoQ abolished age‐related impairments in EDD. MitoQ may be a novel therapy for age‐related vascular dysfunction by reducing mitochondria‐derived oxidative stress.

NIH AG000279, AG039210, AG013038
MODEL PREDICTIONS OF ARTERIAL ADAPTATION TO STEADY‐FLOW LEFT VENTRICULAR ASSIST DEVICES
Authors: Nguyen Phuc H. , Tuzun Egemen , Knezek Sarah F. , Mohiuddin Mohammad W. , Quick Christopher M.
Published: June 2021

The pulsatile hemodynamics resulting from the interaction of a left ventricular assist device (LVAD) with a ventricle and an arterial system is quite complex. This complexity compounds with chronic adaptation. To simplify, investigators have focused on characterizing how altered arterial mechanical properties affect pulsatile pressure, or how altered pulsatile pressure affects ventricular mechanical properties. In a radically different approach, we modified a classical model of the arterial circulation to explore adaptation of the vasculature to decreased pulsatility induced by an LVAD. Each artery was assumed to adapt to circumferential wall stress and endothelial shear stress. The model resulted in values of radii, thicknesses, and stiffnesses as well as pulsatile pressures and flows consistent with reported values. Decreasing pulsatility of input flow to simulate LVAD implantation led to an acute decrease in regional pulse pressures. Arterial adaptation subsequently increased arterial stiffness and partially restored pulsatile pressures. These findings suggest that increases in arterial stiffness (and thus cardiac afterload) may negatively impact cardiac recovery and transplantation. More importantly, the current work reveals a new hemostatic principle: vascular adaptation to circumferential wall stress maintains pulsatility.
Contributions of cyclooxygenase (COX) and NO synthase (NOS) pathways to endothelium‐dependent dilatation in the finger of women
Authors: Marshall Janice Mary
Published: June 2021

We tested the roles of COX and NOS in finger vasodilatation evoked by iontophoresis of acetylcholine (ACh), using laser Doppler fluximetry. In 12 women in the low oestrogen (E2) phase of the menstrual cycle, aspirin (600mg p.o) decreased baseline finger red cell flux (RCF) from 55.0±7.8 to 30.6±5.5* perfusion units (pu; *: P<0.05), and the ACh‐evoked response from +296.8±23.9 to +251.4±24.4* pu. In the high E2 phase, COX inhibition decreased baseline from 68.0 ± 13.8 to 38.4 ± 8.1*, but had no effect on the ACh response (259.5±27.4 vs 251.2±23.3 pu). On different days, NOS inhibition with L‐NAME had no effect on baseline RCF in the low, or high E2 phase. In the low E2 phase, the ACh response was unchanged (+228.26±25.8 vs 225.6±21.3pu), but aspirin then decreased the response further to 175.9±17.6*pu. In the high E2 phase, L‐NAME increased the ACh response from +198.1±23.7 to +229.2±27.4*pu; COX inhibition restored it to +202.8±27.8pu. We propose that in the finger, NOS generates both NO and O2− and thereby, ONOO− which inhibits PGI2 synthase. E2 facilitates NOS and PGI2 synthase such that when E2 is high, PGI2 contributes to endothelium‐dependent finger dilatation only after NOS inhibition.
Immune cell mediated regulation of lymphatic contractility during inflammation
Authors: Chakraborty Sanjukta , Wang Wei , Zawieja David C , Muthuchamy Mariappan
Published: June 2021

The lymphatics are a pivotal player in inflammation and regulation of immune response. However, the interactions of the immune cells and lymphatics, and its effects on lymphatic contractility are not well understood. We hypothesize that during an inflammatory pathologic condition as induced by elevated levels of LPS, there is modulation of immune cell populations on or near the lymphatics, resulting in changes in cytokines/chemokines that impair lymphatic function. Rats were intraperitoneally injected with LPS, and immune cells (eosinophils, neutrophils, macrophages and monocytes) were detected by confocal microscopy after 3d in whole mount mesenteric lymphatics. In LPS treated animals, there was a significant reduction in eosinophils and neutrophils and an increase in macrophages and monocytes. Lymphatics from these rats also exhibited significant decrease in tonic and phasic contractions. Treatment of lymphatic muscle cells with some key cytokines released by these cells (IL4, IL13, IL5 and IL8) showed an increase in relative levels of phosphorylated myosin light chain 20 by activation of AKT and ERK1/2 and also mitigated LPS‐induced impairment of lymphatic contractility. Together, our data shows that LPS impairs lymphatic function by decreasing neutrophils and eosinophils, thereby reducing the availability of key cytokines and their effects on lymphatic muscle. AHA 2280005 to SC; KO2 HL86650 to MM
Changes in ionic currents of respiratory neurons produce sympathetic overactivity in chronic intermittent hypoxic rats
Authors: Moraes Davi J.A. , da Silva Melina P. , Zoccal Daniel B. , Varanda Wamberto A. , Machado Benedito H.
Published: June 2021

The coupling between respiratory and sympathetic neuronal activities is enhanced in rats submitted to chronic intermittent hypoxia (CIH) and contributes to the development of hypertension in this experimental model. We evaluated the mechanisms involved in the CIH‐induced changes in electrophysiological properties of medullary respiratory and pre‐sympathetic neurons. One day after the protocol of 10 days of CIH, using an in situ preparation we performed simultaneous recordings of respiratory and sympathetic nerves with blind whole cell path‐clamp of respiratory and pre‐sympathetic neurons of ventrolateral medulla. In CIH rats, the increased firing frequency of pre‐sympathetic neurons (n=27) and thoracic sympathetic activity (n=30), time‐locked with abdominal late‐expiratory activity (n=25), were dependent of synaptic inputs from respiratory neurons. Since medullary post‐ and pre‐inspiratory (pre‐I) neurons modulate the firing frequency of pre‐sympathetic neurons, we observed that the decreased and increased of intrinsic firing frequency of post‐I (n=28) and pre‐I neurons (n=28), respectively, were accompanied by changes in their input resistance and rheobase after CIH. These changes were due to increase in the inward riluzole‐sensitive persistent sodium current (iNaP; n=6) in pre‐I neurons and in the 4‐aminopyridine‐sensitive component of transient outward potassium current (TOC; n=9) in the post‐I neurons after CIH. These data describe the mechanisms underlying sympathetic overactivity, which critically depend on changes of ionic currents of medullary respiratory neurons of CIH rats.

Support:

FAPESP, CAPES and CNPQ.
Expression of dysfunctional connexin40 in vascular endothelium enhances myogenic constriction and reduces artery distensibility
Authors: Chaston Daniel J , Baillie Brett K , Grayson T Hilton , Courjaret Raphael J , Heisler Jillian M , Lau Katherine A , Machaca Khaled , Nicholson Bruce J , Ashton Anthony , Matthaei Klaus I , Hill Caryl E
Published: June 2021

Mutant forms of connexin40 (Cx40) are known to exist in the human population and increase the risk of cardiovascular diseases such as atrial fibrillation. We hypothesised that the existence of dysfunctional Cx40 proteins amongst the native Cx40 population in the vascular endothelium may also impair arterial function. To this end, we created a new transgenic mouse strain expressing a mutant Cx40 (Cx40T202S) specifically in the endothelium; leaving native Cx40 expression intact. The ability of this mutation to impair gap junction function was tested in Xenopus Oocytes and mouse coronary endothelial cells while its effect on arterial function was tested in isolated small mesenteric arteries using pressure myography. Cell coupling studies found that Cx40T202S could form electrically but not chemically patent gap junctions. Analysis of the myogenic response of arteries from Cx40T202S mice revealed increased sensitivity to intraluminal pressure compared to wild type (P<0.05). This effect coincided with a loss of basally activated endothelium dependent hyperpolarisation (EDH) while nitric oxide remained intact. Examination of passive arterial properties showed Cx40T202S arteries were significantly stiffer than wild type (P<0.05). We conclude that the presence of a mutant Cx40 in the vascular endothelium is sufficient to prevent basal EDH, and increase both myogenic sensitivity and arterial stiffness.
REDUCED NO BIOAVAILABILITY, OXIDATIVE STRESS AND ALTERATION OF CALCIUM HOMEOSTASIS IN VASCULAR ENDOTHELIUM FROM DIABETIC MICE
Authors: Banquet S , Bourguignon MP , Garry A , Royere E , Crespo C , Lapret I , Simonet S , Gosgnach W , Thollon C , Villeneuve N , Vilaine JP
Published: June 2021

Endothelial dysfunction is a major risk factor for vascular complications associated with diabetes. The purpose of this study was to evaluate the possible link between oxidative stress and alteration in calcium homeostasis in dysfunctional endothelial cells from db/db diabetic mice, 20 or 45 week old. Impaired flow‐induced dilation in mesenteric arteries and acetylcholine‐induced relaxation in aorta due to a decreased nitric oxide (NO) bioavailability were already shown in this model. For this study, mice aortic endothelial cells (MAEC) were isolated from vessel explants. Oxidative stress and Ca2+ homeostasis in MAEC were simultaneously recorded with DHE and Fura‐2 probes using an inverted epi‐fluorescence microscope. Compared to control mice, oxidative stress in MAEC from db/db mice increased with age leading to a progressive alteration of Ca2+ homeostasis, characterized by a rise in basal cytosolic calcium and a decrease in amplitude of acetylcholine‐induced calcium peak. Interestingly, a NO donor reduced oxidative stress and improved calcium signaling in endothelial cells from db/db mice. These results suggest that reduced NO bioavailability contributes to oxidative stress and abnormal calcium homeostasis in endothelial cells during progression of diabetes.
Resveratrol blunts the positive effects of exercise training in aged men; a double‐blind, randomized, placebo‐controlled training study
Authors: Gliemann Lasse , Nyberg Michael , Biensø Rasmus S , Iversen Ninna , Olesen Jesper , Mortensen Stefan P , Schmidt Jakob F , Pilegaard Henriette , Hellsten Ylva
Published: June 2021

Aging, as well as a physical inactive lifestyle, are well‐documented cardiovascular risk factors associated with decreased vascular function, increased tissue inflammation and increased presence of reactive oxygen species (ROS). Resveratrol is a polyphenol with antioxidant properties claimed to have anti‐aging effects and suggested to improve general health and cardiovascular parameters by scavenging ROS.

27 aged healthy inactive men (65 ± 1 years; BMI: 25.4 ± 0.7 kg 2m−1; MAP: 95.8 mmHg ± 2.2; VO2max: 2488 ± 72 mlO2 min−1) were randomized into 8 weeks of either 250 mg Resveratrol or Placebo concomitant with high intensity exercise training three times per week. Before and after the intervention period, leg hemodynamics during one‐leg knee‐extensor exercise were measured. In addition, biopsies and microdialysate were collected from the experimental leg.

Training increased VO2max more in the placebo group (19 vs. 13 %; P < 0.05) and decreased MAP in the placebo group only (P < 0.05). Protein expression of eNOS was unchanged and COX‐2 increased 2 fold in skeletal muscle of both groups. eNOS uncoupling was decreased by 40 % and COX‐1 was increased by 20 % in the placebo group only (P < 0.05).

These findings indicate that Resveratrol supplementation in humans blunts the positive effects of exercise training on aerobic power, blood pressure and vasoactive enzymes. Possible mechanisms include a reduction in ROS mediated signaling.