bluuebunny/crossref_metadata_2025 huggingface dataset:
Edwardsiella tarda Alter Their Protein Expression Profiles at Permissive Human Temperatures and Control the Host Cytoskeleton for their Benefit
Authors: Tenkink Byron Joel , Noort Rebecca , Siame Bupe Anthony , Guttman Julian Andrew , Leung Ka Yin
Published: June 2021

Edwardsiella tarda is an emerging enteric pathogen that infects both warm‐water fish and humans. While it is known that E. tarda utilizes syringe‐like type III and type VI secretion systems to deliver effector proteins into fish host cells during infection, little is known about E. tarda pathogenesis in humans at the sub‐cellular level. To investigate the pathogenic strategies employed by E. tarda we tested two hypotheses; (1) E. tarda alter the expression of secreted proteins at 37°C and (2) E. tarda hijack the host cytoskeletal system in human cells as part of their pathogenesis. To test these hypotheses we grew E. tarda at 30°C and at 37°C and compared type III and type VI secretion system protein expression profiles at the two temperatures. Some strains of E. tarda produced more secreted proteins at 37°C than at 30°C, which suggests greater activity from the pathogen's secretion systems at human body temperature. When E. tarda infections in HeLa cells were examined microscopically, we found cytoskeletal and organellar alterations in infected cells as compared to uninfected controls, demonstrating effector‐driven sub‐cellular alterations. Taken together our results indicate that E. tarda hone their protein expression machinery to their environment and control crucial aspects of their host cells during their infectious processes.

Grant Funding Source: Natural Science and Engineering Research Council of Canada
Magnetic resonance elastography (MRE) detects medullary renal fibrosis
Authors: Korsmo Michael James , Ebrahimi Behzad , Woollard John R. , Crane John A. , Eirin Alfonso , Krier James D. , Ehman Richard L. , Lerman Lilach O.
Published: June 2021

MRE is a noninvasive novel MRI modality that detects changes in tissue viscoelastic properties by imaging shear wave propagation. Renal artery stenosis (RAS) induces kidney fibrosis, yet we have previously shown that concomitant reductions in renal perfusion pressure mask an increase in cortical stiffness. We hypothesized that due to its lower vascular volume and turgor, change es in medullary elasticity in RAS would be detectable by MRE. MRE 3D elastograms were obtained in pigs after 10 weeks of RAS or matched controls (n=6 per group), and contrast‐enhanced computed tomography images obtained in the same pigs guided selection of MRE medullary regions of interest (Fig 1). Trichrome staining was used for computer‐aided quantification of medullary fibrosis ex‐vivo. The renal medulla was significantly more fibrotic (5.96±0.19 vs. 1.82±0.49%, p<0.001) and stiffer (10.9±0.84 vs. 7.88±0.26 kPa, p<0.05) in RAS compared to controls (Fig 1 and 2). The histological degree of medullary fibrosis directly correlated with MRE‐derived stiffness (R2=0.55, p<0.05, Fig 3). This study therefore suggests that renal medullary MRE may be useful for detection of chronic kidney fibrosis in RAS.

Grant Funding Source: NIH DK73608, HL77131, HL085307, and EB001981
FOXO Transcription Factors are Critical regulators of Neonatal Cardiomyocyte Proliferation
Authors: Sengupta Arunima , Yutzey katherine E
Published: June 2021

Cardiomyocytes (CM) are highly proliferative during embryogenesis and withdraw from the cell cycle soon after birth. Here, the role of FOXO in regulating CM proliferation in neonatal cardiomyocytes in mice is investigated. CM‐specific deficiency of FOXO1 and FOXO3 results in increased CM proliferation 1–3 days after birth. Inhibition of AMP‐activated protein kinase (AMPK), an upstream activator of FOXO, increases proliferation in cultured rat neonatal CM and this increase is attenuated with overexpression of FOXO1 supporting the requirement of an active AMPK/FOXO signaling to promote cell cycle withdrawal in neonatal CM. FOXM1 and IGF1 both promote CM proliferation, and CM‐specific loss of FOXOs results in increased expression of FOXM1 and IGF1. Furthermore, FOXO1 and FOXO3 directly bind to regulatory regions of FOXM1 in neonatal cardiomyocytes, and FOXO1 inhibits FOXM1 reporter gene expression in cotransfection assays. We identified IGF1 as a direct transcriptional target of FOXM1 by chromatin immunoprecipitation assay. These results suggest that FOXO activity inhibits FOXM1 thereby controlling IGF1 levels to promote cell cycle withdrawal in neonatal CM. Therefore, understanding the molecular mechanisms by which FOXO regulates CM proliferation could be harnessed in adults for the treatment of cardiac injury. This work is supported by an AHA Post‐Doctoral Fellowship to AS and NIH P01HL069779 to KEY.

Grant Funding Source : NIH P01HL069779 to KEY and AHA postdoctoral fellowship to AS
A Comparison of Commercial Anatomy Educational Software
Authors: Attardi Stefanie M , Rogers Kem A
Published: June 2021

An online version of an existing undergraduate, systemic human anatomy course is under development. To accommodate the laboratory component of the online section, commercially available anatomy educational software will be used to demonstrate 3D structures via application sharing within a virtual classroom. Eleven anatomy software programs were reviewed to determine their suitability, on the basis of: quality of digital models (resolution, comprehensiveness of anatomical structures and labels, inclusion of cross sectional anatomy), volumetric data used to create models, manipulation of models (virtual dissection, rotational axes, vantage points, rendering speed), program functionality (saving and sharing dissections, querying for structures, ease of use of the menu) and cost. The software we reviewed will not meet all of our online teaching needs. No program was found to have sufficient anatomical detail and visual clarity of digital models for the central nervous system (CNS). Supplementary online materials for the CNS component of the course will be developed to use in conjunction with one of the eleven existing software packages.

Grant Funding Source : Departmental Funding
An inhibitor of histone demethylases specifically blocks cancer growth in vitro and in vivo
Authors: Martinez Elisabeth D , Wang Lei , Best Anne , Varghese Diana , Chang Jianjun
Published: June 2021

Histone modifying enzymes are highly deregulated in cancers, leading to aberrant transcriptional programs and thus constitute key therapeutic targets. Using an unbiased cell‐based screen, we have identified a novel compound that modulates the activity of Jumonji histone demethylases. Here, we report the anti‐cancer properties and the mode of action of this small molecule called Elizib. We show, through viability measurements across a large panel of human cancer and normal epithelial cells, that this drug has remarkable specificity for cancer with IC50 values as low as 2 nM. In vivo, it effectively reduces tumor burden in mouse xenograft models. Elizib causes cancer‐cell specific changes in cell cycle progression, upregulates pro‐apoptotic and differentiation genes and downregulates proliferative genes, partially re‐establishing a normal transcriptional program in cancer cells, leading to their demise. Enzyme activity assays and chromatin immunoprecipitation studies demonstrate that our small molecule exerts these activities in cell culture and in vivo at least in part through inhibition of demethylases of the Jumonji family. Thus, we have identified a novel epigenetic inhibitor with specific anti‐cancer activity in cells and in vivo. Elizib opens up a new therapeutic venue and is a novel chemical tool to unravel aspects of epigenetic transcriptional regulation in cancer.
DNA damage‐induced NF‐kB activation promotes breast cancer metastasis via upregulation of microRNA‐21
Authors: Niu Jixiao , Tan Guangyun , Yang Chuan He , Fan Meiyun , Pfeffer Lawrence M. , Wu Zhao-Hui
Published: June 2021

OBJECTIVE

To investigate the role and mechanism of NF‐κB in cancer metastasis.

METHODS

DNA damage induced NF‐κB activity was determined by EMSA and microRNA‐21(miR‐21) expression was examined by Quantitative PCR separately. In addition, migration of MDA‐MB‐231 cells was investigated by Transwell invasion and wound healing assay in vitro.

RESULTS

We found that chemotherapeutic drug‐induced NF‐κB activation promotes breast cancer cell migration and invasion. The increased metastatic potential is dependent on IL‐6 induction mediated by genotoxic NF‐κB activation. Moreover, genotoxic treatment also upregulates oncogenic microRNA‐21 expression through eliciting NF‐κB recruitment to miR‐21 promoter region, where it cooperates with signal transducer and activator of transcription 3 (STAT3) to activate miR‐21 transcription. Induction of miR‐21 may enable cancer cells to elude DNA damage‐induced apoptosis and enhance the metastatic potential of breast cancer cells through repressing expression of PTEN and PDCD4.

CONCLUSIONS

Our data support a critical role of DNA damage‐induced NF‐κB activation in promoting cancer metastasis following genotoxic treatment, and NF‐κB‐dependent miR‐21 induction may contribute to both therapeutic resistance and metastasis in breast cancer. Financial support: This work was supported by NIH grants ‐ CA149251 (to Z.W.) and CA133322 (to L.M.P)
Physiological interaction of BRCA2 and PARP in the protozoan pathogen Trypanosoma brucei
Authors: Cooper Rachel Margaret , Misra Smita , Chaudhuri Minu , Chaudhuri Gautam
Published: June 2021

Many eukaryotes including the parasitic protozoan Trypanosoma brucei depend heavily on the BRCA2 protein, which is important for the mediation of DNA recombination and repair. An interesting aspect of BRCA2 deficiency in cancer cells is that these cells are ultra‐sensitive to the inhibitors of the chromatin modifying enzyme poly(ADP‐ribose) polymerase (PARP). In the absence of PARP, spontaneous single‐strand breaks collapse replication forks and trigger homologous recombination for repair. Thus, PARP activity is essential in homologous recombination‐deficient BRCA2‐mutant cancer cells. BRCA2 and PARP are not only expressed in the mammalian cells but also present in high levels in many other eukaryotic cells including the parasitic protozoan T. brucei. To evaluate the universality of the importance of these two proteins in the management of DNA damage, we determined the effect of PARP inhibitors such as 3‐AB, Veliparib, PF‐01367338, Olaparib and Iniparib in BRCA2 knocked down T. brucei cells treated with the DNA damaging agent cisplatin. Our in vitro data strongly suggest that a combination of a BRCA2 inhibitor (e.g. BRCA2 BRC peptide), a PARP inhibitor and a DNA damaging agent (e.g. cisplatin) will be a chemical regimen for the management of trypanosomiasis and similar protozoan infections. Supported by NIH grants 2SC1GM081146‐05 to MC, 1U54RR026140‐01 to SM and NIH grants R01AI042327, R21AI076757 to GC.
Defining the role of acylation in the cytoplasmic quality control of an engineered substrate
Authors: Guerriero Christopher J , Weiberth Kurt F , Nakatsukasa Kunio , Brodsky Jeffrey L
Published: June 2021

Newly synthesized proteins undergo chaperone‐assisted folding into a functional conformation. However, various insults can disrupt protein folding, including environmental stressors, defects in translation and/or post‐translational processing, or genetic mutations. Improperly folded proteins may either attain their proper conformation or undergo chaperone‐mediated selection and targeting for degradation by cytoplasmic quality control (cyto QC). Aberrant accumulation of misfolded proteins can be toxic to cells due to aggregation. Therefore, investigating how the inherent motifs within a substrate (degrons) influence degradation is critical for understanding protein homeostasis. Currently, it is unknown how acylation influences the machinery involved in degradation. To elucidate the chaperones required for the cytoQC of an acylated substrate, we employed a novel yeast degron with or without a motif to induce acylation. To compare and contrast the factors required for degradation, protein stability was measured in mutant yeast strains. These experiments revealed that degradation is dependent on the proteasome and two E3 ubiquitin ligases, San1p and Ubr1p. Furthermore, degradation relies heavily on the heat shock protein 70 (Hsp70), Ssa1p. Ongoing work is defining how this and other chaperones function to select these substrates. Funded by NIH F32‐GM090364 and GM75061.
A Cdc48p‐associated factor modulates endoplasmic reticulum‐associated degradation, cell stress, and ubiquitinated protein homeostasis
Authors: Tran Joseph R. , Brodsky Jeffrey L
Published: June 2021

The AAA‐ATPase, Cdc48p, catalyzes an array of cellular activities, including endoplasmic reticulum (ER)‐associated degradation (ERAD), ER/Golgi membrane dynamics, and DNA replication. Accumulating data suggest that unique Cdc48p‐partners target Cdc48p for these diverse functions. We report on a previously uncharacterized Cdc48p‐associated protein, Ydr049p, also known as Vms1p, which binds Cdc48p at the ER membrane and in the cytosol under non‐stressed conditions. Loss of VMS1 modestly slows the degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) but does not impair substrate ubiquitination. Instead, vms1 mutant cells accumulate Cdc48p‐bound ubiquitinated proteins at the ER membrane, suggesting a role after substrate ubiquitination. VMS1 also genetically interacts with select Ubiquitin regulatory X (UBX) and Ubiquitin fusion degradation (UFD) proteins, which are Cdc48p partners. Exacerbated growth defects are apparent in some mutant combinations and synergistic effects on the degradation of CFTR and CPY*, which is a soluble ERAD substrate, are evident in specific double mutants. These data suggest that Vms1p acts in parallel with Cdc48p partners to modulate ERAD and other cellular activities. To address this hypothesis, we have begun to analyze the spectrum of proteins that associate with Cdc48 in wild‐type and vms1 mutants. This work is supported by NIH GM75061.
FGD5 deficiency confers defective matrix adhesion and survival among endothelial cells
Authors: Nakhaei-Nejad Maryam , Haddad George , Murray Allan
Published: June 2021

FGD5 is a putative Rho family guanine nucleotide exchange factor enriched in vascular endothelial cells (EC). We observed FGD5 expression in human EC and seek to determine FGD5 function. Inhibition of FGD5 expression using RNAi decreased the protein by ~70%. In 3D angiogenesis in vitro, FGD5‐knockdown (kd) EC had 45 +/− 8% reduced sprout outgrowth and sprout length versus non‐silenced (NS) controls. We examined FGD5‐kd EC adhesion to matrix. FGD5‐kd cell adhesion to fibronectin was reduced by 28 +/− 4% vs. NS. Similarly, monolayer electrical impedance was decreased after seeding FGD5‐kd cells versus controls, and impedance increased at slower rate (4.6 +/− 0.6 siNS vs. 2.4 +/− 0.5 siFGD5 Ohm/min) reflecting decreased EC spreading. Apoptosis was examined by measurement of cleaved caspase‐3 expression and subdiploid DNA content. After pro‐apoptotic cyclohexamide and tumor necrosis factor‐Î± stimulus, FGD5‐kd markedly increased apoptosis (54 +/− 4% vs 34 +/− 8% EC display active caspase‐3 and 54 +/− 4% vs 29 +/− 3% EC have subdipolid DNA among siFGD5 vs siNS‐treated cells respectively). These results indicate that FGD5 in EC is involved in adhesion, survival, and angiogenesis.
The Role of Chromodomain in eukaryotic Elongation Factor 3 (eEF3)‐Ribosome Interaction
Authors: Sasikumar Arjun N , Kinzy Terri Goss
Published: June 2021

Translation elongation in eukaryotes is a conserved process catalyzed by eukaryotic Elongation Factor 1A (eEF1A) which delivers aminoacylated‐ tRNA to the ribosome and the ribosomal translocase eukaryotic Elongation Factor 2 (eEF2). In addition to these two G‐proteins, fungal translation is characterized by its unique and absolute requirement for an ATPase factor called eukaryotic Elongation Factor 3 (eEF3). eEF3 has two ATP binding cassette domains the second of which has a chromodomain insertion, predicted to play a key role in its function. Using Saccharomyces cerevisiae, we are characterizing the functional role of this domain in its interaction with the ribosomes. We have made a series of chromodomain mutants that adversely affect the translation elongation and the growth rate of the cells. The mutants also have severely compromised intrinsic as well as ribosome stimulated ATPase activity while the ribosome binding remains unaffected. Our work demonstrates that the chromodomain plays a significant role in the functioning of eEF3 as a translation elongation factor in fungi. Because it is an essential protein found across all fungal species, the characterization of its ribosomal interactions may help in utilizing eEF3 as a target for the development of a broad spectrum anti‐fungal with low host toxicity. This work is supported by the National Institutes of Health.
Investigating the Control of the Actin Cytoskeleton by EF1α
Authors: Colmer Sarah , Karas Christina , Scerbo Diego , Gomez Maria , Dunaway Stephen
Published: June 2021

Translation factor EF1α plays a critical role in protein synthesis, delivering aminoacyl tRNAs to the ribosome1. However, it has another less characterized function in regulating the actin cytoskeleton. EF1α binds actin directly or indirectly, as determined by co‐immunoprecipitation experiments2. Overexpression of EF1α in Schizosaccharomyces pombe results in aberrant cellular morphology2. EF1α homologs are potential cancer drug targets; the compound narciclasine, isolated from Amaryllidaceae, was shown to penetrate human melanoma cells, bind to eEF1A, and impede the proliferation of these cells3.

Three genes encoding EF1α are present in the fission yeast genome. Previously, we determined that antibodies against Saccharomyces cerevisiae elongation factor proteins EF1α, EF2, and EF3 were effective in detecting their homologous forms in S. pombe. We found that deletions of EF1α‐a and EF1α‐c resulted in a slight decrease in total EF1α protein levels by utilizing Western blot analysis. These deletions produced no noticeable defect in cell growth on plates or in liquid culture. Continuing these studies, we plan to knock out EF1α‐b and analyze protein production. We will conduct phalloidin staining on our deletion mutants to determine if gene deletion has any visible effect on the polymerization of actin. Furthermore, we will knock out all three genes encoding EF1α in a single strain to assess its essentiality.
A non‐radioactive high‐throughput assay for the detection of cellular protein synthesis
Authors: Abdelazim Suzanne Aly , Bachran Christopher , David Alexandre , Leppla Stephen H
Published: June 2021

Protein synthesis is an important feature of cell vitality and many drugs are targeting this process. In addition, several targeted tumor drugs, e.g. immunotoxins, contain protein toxins inhibiting protein synthesis. Up to date, no non‐radioactive assay exists for the direct detection of protein synthesis inhibition in cells. Here, puromycin was used to label naive proteins synthesized in cells in 96‐well plates. Puromycin is transferred onto the growing amino acid chain and terminates the protein synthesis. Subsequent fixation and permeabilization allowed for the immunological detection of puromycinylated proteins by an anti‐puromycin antibody. Analyses with several protein synthesis inhibitors as well as targeted toxins revealed a concentration‐dependent inhibition of cellular protein synthesis on four different cells lines. The kinetics of inhibition by the targeted toxins was studied to identify suitable time points for complete protein synthesis inhibition. The results demonstrate the successful development of a non‐radioactive and sensitive assay for the direct detection of protein synthesis inhibition in a 96‐well format. This assay should help to perform more immediate analyses of drugs targeted on protein synthesis and help to replace the commonly used radioactive leucine incorporation assay.
Role of SIRT1 in regulation of hepatic gene expression by thyroid hormone
Authors: Thakran Shalini , Sharma Pragya , Attia Ramy R , Hori Rod , Elam Marshall B , Cook George A , Park Edwards A
Published: June 2021

Thyroid hormone induces expression of hepatic genes such as pyruvate dehydrogenase kinase 4 (PDK4) and carnitine palmitoyltransferase1a (CPT1a) through the thyroid hormone receptor. Peroxisome proliferator‐activated receptor gamma coactivator 1 (PGC‐1α) enhances the thyroid hormone (T3) induction of these genes. The NAD‐dependent sirtuin 1 (SIRT1) deacetylase plays a critical role in cellular metabolism by deacetylating and activating transcription factors including PGC‐1α. The SIRT1 activator, resveratrol, improves mitochondrial function, increases fatty acid oxidation and decreases lipogenesis. Here, we investigated the role of SIRT1 in the T3 regulation of PDK4 and CPT1a genes. Our data indicate that resveratrol increases expression of PDK4 and CPT1a genes in the liver and enhances the T3 induction of these genes. Resveratrol enhanced the T3 induction through the gene promoters. Overexpression of SIRT1 has similar effects to resveratrol whereas knockdown of SIRT 1 reduces the T3 induction of PDK4 and CPT1a. Moreover, SIRT1 interacts with the thyroid hormone receptor β. Our data indicate that SIRT1 plays an important role in the thyroid hormone induction of hepatic genes such as PDK4 and CPT1a.

This work was supported by National Institute of Health and American Heart Association.
The Role of Tyk2 in Regulating Energy Expenditure and Preventing Obesity
Authors: Raje Vidisha B , Derecka Marta , Gornicka Agnieszka , Sczepanek Karol , Sisler Jennifer , Croniger Colleen M , Larner Andrew
Published: June 2021

Brown adipose tissue(BAT) and skeletal muscle(SKM) are the two major tissues involved in energy expenditure, mediated by thermogenesis.

We have observed that mice that do not express the JAK kinase‐ Tyk2 become spontaneously obese and exhibit the metabolic syndrome. Tyk2−/− mice show defects in BAT and SKM, which arise from the same lineage of Myf5+ progenitors of the mesenchymal lineage. Tyk2−/− mice exhibit decreased expression of BAT specific genes like UCP1 and PRDM16. The expression of SKM specific transcription factors is elevated in Tyk2−/− BAT. Tyk2−/− mice also have more muscle mass compared to the wild type controls. However, the skeletal muscle is functionally impaired as shown by decreased mitochondria respiration capacity, decreased expression of mRNAs involved in muscle contraction, exercise intolerence and a defect in maintaining the core‐body temperature upon cold exposure.

Taken together, these findings suggest a novel role for Tyk2 kinase in regulation of energy expenditure and preventing the obese phenotype.
Endothelial cell senescence suppresses argininosuccinate synthetase 1 expression by promoter methylation while laminar shear stress rescues it by a mechanism involving KLF4
Authors: Boo Yong Chool , Mun Gyeong In
Published: June 2021

Argininosuccinate synthetase 1 (ASS1) regulates the provision of L‐arginine to nitric oxide synthase 3 (NOS3). Previous studies demonstrated that ASS1 and NOS3 expressions were enhanced by laminar shear stress (LSS), and ASS1 but not NOS3 expression was suppressed by endothelial cell senescence, so that the impaired NO production in senescent cells could be attributed to the insufficient expression of ASS1, but not of NOS3. The regulation of ASS1 expression by LSS and cellular senescence was investigated in human umbilical vein endothelial cells (HUVECs), focusing on Kruppel‐like factors (KLFs) and promoter methylation. The basal and stimulated expression levels of KLF4 were similar in young versus senescent HUVECs. Regulatory roles of KLF4 in ASS1 expression were confirmed by siRNA‐mediated knockdown and over‐expression of KLF4. Bisulphite sequencing analysis revealed that cellular senescence caused ASS1 promoter hypermethylation. Treatment of senescent HUVECs with 5′‐aza‐2′‐deoxycytidine to inhibit DNA methylation led to increases of ASS1 expression levels under both static and LSS conditions. ASS1 promoter reporter activity was lowered by cellular senescence or DNA methylation and it was restored by LSS or KLF4 over‐expression. This study indicated that endothelial ASS1 expression levels were regulated not only by KLFs but also by promoter methylation in response to LSS and cellular senescence.
Detection of peptidyl‐4‐hydroxyproline in Bacillus anthracis
Authors: Rebecchi Kathryn , Xu Li , Williams Todd , Mure Minae
Published: June 2021

Human prolyl‐4‐hydroxylase (P4H) catalyzes hydroxylation of peptidyl proline to 4‐hydroxyproline (4‐Hyp) in collagen, essential for extracellular matrix stabilization. P4H overexpression is associated with fibrosis and cancer. Little structural information is available for the α2 catalytic domain of human‐P4H. We are studying a putative P4H from Bacillus anthracis (anthrax‐P4H), α2 dimer, as a model for human‐P4H. Since there is no precedent for peptidyl 4‐Hyp in bacteria, we conducted amino acid analysis of total protein extracts from B. anthracis. Under nutrient‐rich conditions, B. anthracis forms vegetative cells and, upon starvation, forms spores. We detected peptidyl 4‐Hyp in exosporium (outermost spore layer) and vegetative cells of B. anthracis by multiple reaction monitoring triple quadruple mass spectrometry. This is the first example of peptidyl 4‐Hyp in bacteria and anthrax‐P4H is most likely responsible for the post‐translational modification. Using immunostaining, we detected the most anthrax‐P4H in vegetative cells without endospores, less after forming endospores, a small amount in nude spores, and no anthrax‐P4H in the exosporium. Our results suggest that the native substrate for anthrax‐P4H (precursor peptidyl‐proline) exists in vegetative cells and the product (peptidyl 4‐Hyp) is secreted into the exosporium after post‐translational modifications.
Polymeric microparticle loaded with Atenolol as a drug delivery system to cardiovascular system
Authors: Oliveira Naiara A , Gomes Anderson J
Published: June 2021

This study aimed to investigate the effect of an established anti‐hypertensive drug such as atenolol (ATL) when encapsulated in polymeric microparticles (PLGA). ATL is a prominently drug on the cardioselective beta blockers list. It is a â1 blocker that has low lipid solubility. The new drug delivery system (ATL‐PLGA) was designed to protect the drug from environmental and to promote a sustained drug delivery profile. Parameters such as particle size, zeta potential, drug encapsulation efficiency, short‐term stability (at 37o ± 1 for 3 weeks) external morphology, and in vitro release behavior were evaluated at different preparation protocols. Scanning electron microscopy and dynamic light scattering revealed that particles loaded with ATL are spherical in shape, and they have a diameter between 92 and 512 nm and a low tendency to aggregate, with zeta potential −24.2 ± 3.4 mV. The encapsulation efficiency obtained in this procedure was 77.0± 4.6%. The particles are stable in the period of the study. The new drug delivery systems possess some features parameters that allow it to be tested in biological samples such as vascular smooth muscle cells. Supported by FAPDF, CAPES, CNPq, FINATEC and DPP‐UnB.
Using NTHi growth studies to demonstrate the biological significance of c‐heme covalent attachment
Authors: Kalmeta Breanna , Grimaldi Kyle , Iqbal Arooj , Weekes Danielle , Bren Kara L , Michel Lea Vacca
Published: June 2021

The property that defines c‐type heme proteins is the covalent attachment of the heme group to the polypeptide chain of the protein, such as in cytochrome c. The covalent attachment of the c‐heme (usually by thioether linkages donated by two Cysteine residues) is energetically expensive, and the biological motivation for the attachment is unknown. We propose that the biological significance of the covalently attached c‐heme is to prevent heme‐dependent bacteria, such as Nontypable Haemophilus influenzae (NTHi), from scavenging or stealing heme from c‐heme proteins. In our study, we prepared wild‐type, single and double mutant cytochromes c using recombinant DNA technology, protein expression in Escherichia coli, and purification via ion affinity column chromatography. The wild‐type, single, and double mutant proteins contain two, one, and zero covalent heme attachments, respectively. We then grew NTHi in the presence of all three proteins (as well as hemin and water as controls). The results of our results suggest that, indeed, cytochrome c can only act as a heme source for NTHi when one or both covalent heme attachments are removed. This study was funded by the Rochester Institute of Technology.
Glycoprotein Changes in the Bovine Sperm Plasma Membrane during Maturation in the Epididymis
Authors: Buchanan Teresa L , Mclean Eric , Raychoudhury Samir , Nagdas Subir K
Published: June 2021

The plasma membrane of spermatozoa is a mosaic of distinct domains corresponding to defined segments of the head and the flagellum. To attain fertilizing ability, sperm undergo several biochemical changes during transit in the epididymal duct. The objective of the present study is to analyze the maturation‐associated changes of bovine sperm surface glycoproteins during epididymal maturation. Among the several lectins tested, we found a 19kDa WGA binding polypeptide, termed as WGA19 polypeptide, only present in the plasma membranes of cauda sperm, not in the caput sperm plasma membrane. Both luminal fluid and spermatozoa of the cauda epididymis exhibited the presence of WGA19 polypeptide. No band was observed in epididymal fluid or spermatozoa of the caput epididymis. Phase separation analysis of cauda sperm plasma membrane fraction demonstrates that WGA19 polypeptide is an integral membrane protein. Proteomic identification of the WGA19 polypeptide yielded five peptides that matched the sequence of human calmodulin 3. The WGA19 polypeptide was retained within the hybrid membrane complex of bovine sperm following capacitation and the acrosome reaction. This study suggests that 19kDa WGA polypeptide secreted from the cauda epididymis, anchores or/ binds to the plasma membrane, and may have an important role in sperm‐egg interactions. Supported by NIH/NIGMS/1SC3GM096875‐01 and Graduate School Program.
Cardiac Mitochondrial Phenotype of the taz shRNA Mouse Model of Human Barth Syndrome
Authors: Le Catherine H , de Mooy Anthony Bret , Chicco Adam J
Published: June 2021

Barth syndrome (BTHS) is an X‐linked cardioskeletal myopathy that results from a mutation in the Tafazzin (taz) gene. Tafazzin is a phospholipid transacylase required for the optimal synthesis of cardiolipin (CL), a mitochondrial phospholipid that provides functional support to several mitochondrial proteins. In this study, the cardiac mitochondrial phenotype of a new mouse model of BTHS (taz shRNA; TAZ) was characterized. Cardiac mitochondria were isolated from male TAZ and wild type (WT) mice at 3–4 months of age for assessment of respiratory parameters, H2O2 production and Ca2+ tolerance. Mitochondria from TAZ mice had 50–60% lower rates of state 3 and state 4 respiration, with little change in respiratory efficiency vs. WT. Response to increasing levels of ADP was also severely blunted in TAZ, indicating impaired respiratory control. Absolute levels of H2O2 production were similar in TAZ and WT but were higher in TAZ when normalized for electron flow rate. Ca2+‐induced swelling was impaired in TAZ, as was sensitivity to cyclosporin A, indicating resistance to mitochondrial permeability transition. These findings demonstrate that taz deficiency impairs several aspects of mitochondrial function and provide the basis for further inquiry into the precise roles of taz and cardiolipin metabolism in mitochondrial (patho)physiology. Funding: AHA and Barth Syndrome Foundation
An improved procedure for isolation of functional synaptosomes for the transient generation of cybrids from frozen human brain
Authors: Castora Frank J. , Trevino Michelle B.
Published: June 2021

Cybrids are cell lines derived from the fusion of mtDNA‐depleted cells with cytoplasts or enucleated cells. To date, there are no reports of cybrid fusions from frozen human tissue. In this study, we developed a new method of synaptosome isolation, the “Frozen Brain Synaptosomes” or FBS method. Synaptosomes were isolated from fresh mouse, frozen mouse and frozen human brain by centrifugation in an iodixanol gradient. The effectiveness of our FBS method was compared with current synaptosome isolation procedures that have been utilized to make cybrid cell lines from fresh tissue. The FBS method yielded the most structurally intact synaptosome preparation from frozen tissue as judged by electron microscopy. We assessed cytochrome c oxidase and citrate synthase activity, phospho‐synapsin‐1 and total ATP levels. The FBS method yielded the greatest enrichment of intact synaptosomes, high protein, mitochondrial enzyme activity and ATP levels, and functional synaptosomes as defined by their indirect response to a phosphatase inhibitor okadaic acid (OKA). We expected that the synaptosomes isolated from frozen postmortem brain would be sufficient in quantity and quality to generate cybrid cells. From ten fusion experiments, we concluded that two putative cybrid cell lines contained the exogenous synaptosomal mitochondria based upon mtDNA sequence analysis. Research support was from the Virginia Center on Aging.
Biochemical Mechanism of FIT Proteins in Mediating Lipid Droplet Formation
Authors: Gross David A. , Zhan Chenyang , Silver David L.
Published: June 2021

The process of lipid droplet (LD) formation is an evolutionarily conserved process among all eukaryotes and plays an important role in both cellular physiology and disease. Recently, our lab discovered Fat storage‐Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2) as an evolutionarily‐conserved family of proteins involved in fat storage. These proteins have distinct tissue distributions, are both exclusively localized to the endoplasmic reticulum, mediate triglyceride (TAG)‐rich LD accumulation when overexpressed, but do not synthesize TAG. Our lab has shown that FIT proteins purified in detergent micelles directly bind to TAG with specificity and saturation binding kinetics in order to mediate LD formation. A FIT2 gain‐of‐function mutant that forms larger LDs, FLL(157–9)AAA, showed a significant increase in TAG binding, while FIT1 and a FIT2 partial loss‐of‐function mutant, N80A, displayed abrogated TAG binding, producing smaller LDs relative to wild‐type FIT2. Here, we present biochemical evidence supporting a unique two‐step mechanism for LD formation via FIT2. Attempts to reconstitute the activity of FIT proteins will also be presented. In summary, FIT proteins are the first transmembrane domain‐containing proteins shown to bind TAG, a function that is important for FIT‐mediated LD formation.
Chemical approaches to the investigation of protein‐membrane binding interactions using synthetic lipid probes
Authors: Best Michael D.
Published: June 2021

Signaling lipids present in cell membranes act as important regulators of biological processes and have been implicated in the onset of numerous diseases. A common role of these lipids is the recruitment of proteins to the cell membrane surface through binding events that generally regulate protein function and localization. While understanding these interactions at the molecular level is of great interest, such efforts are hindered by the complexity of the membrane environment in which binding occurs. Towards this end, chemical strategies will be presented by which protein–membrane recognition events can be efficiently characterized. First, the design and synthesis of lipid probes corresponding to signaling lipids including diacylglycerol, phosphatidic acid and the phosphoinositides will be described. Next, the application of these probes for elucidating binding details will be presented, including the development of multi‐format microarray analysis for investigating protein–lipid recognition in different contexts, and bifunctional probes that allow for collective labeling of target receptors in cell extracts, including those associated with disease.
The COPI vesicle associated protein Arf1 GTPase‐activation protein 1 (ARFGAP1) is required for lipid droplet biogenesis
Authors: Gannon Joan , Fernandez-Rodriguez Julia , Asp Lennart , Fazel Ali , Bergeron John JM , Nilsson Tommy
Published: June 2021

Excess free fatty acids are stored in liver as triglycerides in organelles called lipid droplets (LDs). They are not inert storage containers, but have proteins directed to their surface. This study identified ARFGAP1 on LDs derived from liver and cultured cells. ARFGAP1 regulates the dynamic cycling of ARF1 on Golgi membranes affecting COPI vesicle formation, SNARE priming and lipid metabolism. ARFGAP2 and 3 are also candidates for regulating ARF1 function on the Golgi membrane but have not been studied within the context of COPI function until recently. Previous studies on LDs showed ARFGAP1 responds to changes in diacylglycerol, implicating it in triglyceride synthesis the main component of LD. We show a requirement for ARFGAP1 in LD biogenesis using a combination of proteomics, siRNA, immunolocalisation techniques and GFP fusion proteins. We will discuss the possible machinery in which ARFGAP1 is involved in regards to LD production and/or fusion. This research was funded by the Research Institute of McGill University Health Centre, Montreal, The Swedish Research Council and a CIHR grant to JJMB. Research was partly conducted through the Centre for Cellular Imaging, Gothenburg, Sweden.
Live cell imaging of lipid droplet breakdown and growth in adipocytes
Authors: Wolinski Heimo , Paar Margret , Jüngst Christian , Steiner Noemi A. , Kolb Dagmar , Lass Achim , Zimmermann Robert , Zumbusch Andreas , Kohlwein Sepp D.
Published: June 2021

Lipid droplets (LDs) are the main lipid storage depots for neutral lipids (‘fat’) mainly triacylglycerols (TAGs) in all eukaryotic cells. Synthesis, storage, and mobilization of TAGs are critical cellular processes to maintain cellular lipid and energy homeostasis. Although many molecular and biochemical details of neutral lipid metabolism have been revealed basic cell biological aspects of LDs such as the mode of LD breakdown and of LD growth are still elusive. We have applied label‐free coherent anti‐Stokes Raman scattering (CARS) microscopy, long‐term four‐dimensional live cell imaging and three‐dimensional electron microscopy to study lipid droplet remodelling in intact murine and human adipocytes. We show that under selected nutritional conditions neutral lipid breakdown and synthesis can occur in parallel and suggest an underlying mechanism to prevent cellular lipid overflow and lipotoxicity. Furthermore, we demonstrate that LD of adipocytes grow by lipid transfer between closely associated organelles without physical interaction over large LD surface areas. In addition, we demonstrate the specific detection of isotope labeled fatty acids and their incorporation into LD using CARS microscopy. Advantages and limitations of chemical imaging versus fluorescence labeling for monitoring lipids in intact cells are discussed. FWF, LIPOTOX F3005‐B12; DK‐ME W901‐B05, GOLD (GEN‐AU); KoRS‐CB; BWS.
FSH can induce ERK activation in a cAMP independent manner
Authors: Curtis Chelsea L , Cohen Brian D
Published: June 2021

When human follicle stimulating hormone (hFSH) binds to its receptor (hFSHR) it signals through the Gs alpha protein leading to an increase in cyclic AMP (via adenylyl cyclase) which activates protein kinase A (PKA). In addition to this canonical pathway, activation of both the p38 and p44/42 (ERK1) MAP kinases has also been demonstrated in response to FSH. Previous research in our lab has demonstrated that when cells are treated with the cholesterol depleting agent methyl beta‐cyclodextrin it blocks FSH stimulation of adenylyl cyclase but not the activation of ERK1. Surprisingly, when cells were treated with H89, an inhibitor of PKA, FSH induced ERK1 activation was decreased. When these two treatments were combined, FSH induced ERK1 activation was restored. These seemingly inconsistent findings suggest an alternate pathway of activation for ERK1 that is regulated by cyclic AMP but PKA independent. The identification of the regulators in this alternate pathway could lead to new therapies for regulating reproduction.
Determination of the Intracellular, Surface, and Extracellular Localization of Hsp70s under Different Stress Conditions
Authors: Medina Rebeca , Rashedan Manely , Nikolaidis Nikolas
Published: June 2021

Members of the Hsp70 family are primarily localized in different subcellular compartments, including the cytosol, endoplasmic reticulum, and mitochondria. Apart from their primary localization, different Hsp70s have also been found in other places within the cell, as well as the cellular membrane and the extracellular milieu. However, it is unknown which stresses are responsible for their re‐localization, membrane translocation, and secretion. To this end we studied the localization of four members of the Hsp70 family under different stresses using sub‐cellular fractionation and confocal microscopy. These experiments revealed that under normal growth conditions all four Hsp70s were present in multiple fractions, as well as in the extracellular medium. In stressed cells, Hsp70 amounts in each fraction were significantly different from the untreated cells. These results suggest that stress causes re‐localization and differential membrane‐anchorage and secretion of the four Hsp70s. Additional experiments using pharmacological manipulation of intracellular trafficking pathways are currently being performed to identify the molecular mechanism used by Hsp70s to achieve their translocation, membrane anchorage, and subsequent secretion. This study is the first step towards elucidating the biological importance of the re‐localization and membrane occurrence of these chaperones.

This project was supported by funds from CSUPERB and CSUF to NN.
NogoB receptor is essential for extraembryonic vascular development and protein glycosylation
Authors: Park Eon Joo , Sessa William C.
Published: June 2021

Nogo‐B receptor (NgBR) was identified as an interacting protein necessary for NogoB stimulated chemotaxis and tube formation of endothelial cells in vitro. Recently, our lab has shown that NgBR interacts with dehydrodolichyl diphosphate synthase (DHDDS), enhances DHDDS protein stability, and promotes cis‐IPTase activity in vitro. To investigate NgBR function in vivo, we have generated conditional knockout mice. We have found that global deficiency of NgBR results in peri‐implantation embryonic lethality before e6.5. Furthermore, to specifically study the function of NgBR in the endothelial cells, we also generated NgBR cKO mice model using Tie2Cre. The NgBR cKO embryo died at E10.5 with vascular development defect in extraembryonic tissue. The yolk sac of the mutant embryo shows poorly organized and dilated primary capillaries. Moreover, most VEGFR2 in the mutant yolk sac was in the non‐glycosylated, non‐functional form rather than in the fully or semi‐glycosylated form of VEGFR2, which indicates that VEGFR2‐dependent signaling is down‐regulated in the mutant. The mutant also had decreased expression of endothelial cell markers. Taken together, these results suggest that during early embyronic development, NgBR has a role in extraembryonic vascular development by regulating protein N‐glycosylation. The NgBR cKO mice will provide a useful model system to investigate protein glycosylation in vivo.
Catalytic and Biological Features of Human Light Chains Suppressing of Infection of Influenza Virus A Type
Authors: Hifumi Emi , Fujimoto Naoko , Uda Taizo
Published: June 2021

By immunizing ground‐state peptides or proteins into mice, the authors have established murine catalytic antibody light chains (antigenases) that could destroy such as HIV‐1 gp41, H. pylori urease, TNFa etc. We proposed a new concept that the antigenase posseses a catalytic‐triad structure composed Ser, His and Asp.

Based on above concept, we cloned cDNAs (from a volunteer infected with influenza virus) encoding human antibody kappa light chain belonging to subgroup II of germline genes, because the group encodes a catalytic triad‐like structure with high possibility. The cDNAs were transformed into E. coli and each human light chain was expressed as the protein. The highly purified (>95%) human light chains were firstly submitted to the catalytic activity tests and to in vitro assays for examining the infectivity of influenza virus. Some clones showed the amidase and DNase activity. Surprisingly, #4 clone exhibited a strong activity to digest pBR322 plasmid. And the #4 clone suppressed the infection of influenza virus (H1N1) in vitro assay. In addition, we performed in vivo assays. The administered mice with the influenza virus treated with #4 antigenase enhanced the number of survival mice compared with non‐treated mice. Furthermore, the weigh of the antigenase‐treated mice did not decrease at all, while the antigenase‐not‐treated mice showed the marked decrease of the weigh.
Effects of chalcone on oxidatively stressed Caenorhabditis elegans
Authors: Duong Michelle , Susman Kathleen , Caruso Francesco , Rossi Miriam
Published: June 2021

Chalcones (1, 3‐diaryl‐2‐propene‐1‐ones) are natural compounds extracted from fruits and vegetables. Structural and in vitro studies of chalcones have shown they have a large range of biological activities. Previously we have shown that a trihydroxychalcone showed marked antioxidant activity using UV‐vis assays with DPPH, a stable free radical. Additionally, when two oxidatively stressed cell lines, L‐6 myoblasts and THP‐1 monocytes, were treated with chalcone, strong antioxidant activity was seen at nanomolar concentrations, making it an extremely effective free radical scavenger. We now describe the potency of the antioxidant chalcone on mitigating the harmful effects of ROS (reactive oxygen species) in vivo using the model organism Caenorhabditis elegans. The organisms are treated with UV light and paraquat (an intracellular superoxide radical generator) to mimic oxidative stress and then examine if the chalcones’ ability to scavenge the free radicals influences lifespan extension by systematic counting of specimens and evaluation of their behavior under the microscope. If these studies are promising, we will then examine whether chalcone increases thermal tolerance in C. elegans. Finally, we hope to identify genes through multiple genotypes of C. elegans in order to target genes that mediate lifespan and are up‐regulated as a result of chalcone treatment.