bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting the [CL/cyt c] complex to protect electron transport and prevent ROS formation prevents mitochondrial IR injury without ROS scavenging (LB169)
Authors: Chao Wesley , Birk Alex , Liu ShaoYi , Soong Yi , Bracken Clay , Szeto Hazel
Published: June 2021

SS‐31 (D‐Arg‐dimethylTyr‐Lys‐Phe‐NH2) is a ROS‐scavenging cardiolipin‐targeting tetrapeptide which is known to protect against oxidative stress‐induced ischemia‐reperfusion (IR) injury. The interaction of CL with cytochrome c (cyt c) is necessary for electron carrier function of cyt c but can also lead to a [CL/cyt c] complex formation, which prevents electron transfer and promotes superoxide and hydrogen peroxide generation. SS‐31 was shown to protect the carrier function of cyt c while preventing oxidative stress and optimizing bioenergetic efficiency. To distinguish between ROS scavenging and inhibition of ROS formation in mitochondrial protection during IR injury, we developed an analog of SS‐31 which lacks the ROS‐scavenging dimethylTyr moiety (SS‐20; Phe‐D‐Arg‐Phe‐Lys‐NH2). We showed that SS‐20 interacts specifically with CL in liposomes and mitochondria. In isolated mitochondria, SS‐20 increases oxygen respiration and reduces hydrogen peroxide formation. Although SS‐20 cannot scavenge ROS, it is as effective as SS‐31 at protecting cristae architecture during ischemia and accelerating ATP recovery upon reperfusion in a model of acute kidney ischemia. Thus we demonstrate that targeting the [CL/cyt c] complex to protect electron transport and prevent ROS formation prevents mitochondrial IR injury without ROS scavenging.
X‐ray structure of the human mitochondrial kinase ADCK3 elucidates the kinase fold of the ancient UbiB family (LB173)
Authors: Reidenbach Andrew , Stefely Jonathan , Saunders Jaclyn , Johnson Isabel , Oruganty Krishnadev , Barber Grant , Li Sheng , Kannan Natarajan , Bingman Craig , Pagliarini David
Published: June 2021

The UbiB family of kinases represents an estimated 25% of the protein kinase‐like (PKL) family members in the microbial kingdom, and members of this family are required for the biosynthesis of coenzyme Q from E. coli to humans. We present the first structure of a UbiB family member, the human mitochondrial kinase ADCK3, which is mutated in a human cerebellar ataxia (ARCA2) associated with coenzyme Q deficiency. The structure of ADCK3 reveals that the signature, invariant KxGQ motif of UbiB kinases interacts directly with the kinase active site, in close proximity to residues important for nucleotide binding. Differential scanning fluorimetry (DSF) and deuterium exchange mass spectrometry were used to confirm that this motif is important for proper nucleotide binding, and to establish that ADP binding has a global stabilizing effect on the kinase. Last, mapping ADCK3 patient mutations onto our structure suggests how these mutations can destabilize the kinase. We purified and analyzed the thermal stability of eight such ARCA2 patient mutations using DSF and found that all but three of these proteins are less stable than wild type ADCK3. Our findings form the foundation for studying the enzymatic activity of the UbiB family and for investigating how disruptions in this kinase can give rise to a human neurodegenerative disorder.

Grant Funding Source: A.G.R. was supported by NIH Chemistry‐Biology Interface Training Grant NIGMS T32 GM008505, D.J.P. was supported by NIH Grant U01 GM94622 (Protein Structure Initiative: Biology, Mitochondrial Protein Partnership) and a Searle Scholars Award
Hypoxia impairs maturation of the Na,K‐ATPase (LB180)
Authors: Capri Joseph , Tokhtaeva Elmira , Sun Haying , Angulo Martin , Dada Laura , Sznajder Jacob , Kaplan Jack , Whitelegge Julian , Vagin Olga
Published: June 2021

Renal tissue hypoxia is a major cause of acute renal failure. The goal of this study was to elucidate the mechanism(s) underlying role of the Na,K‐ATPase in the adaptive response of renal cells to a hypoxic stress. Kidney microsome membranes were isolated from mice exposed to 7% O2 (hypoxia) or to room air for three days, proteins were extracted by a non‐ionic detergent, immunoprecipitated using anti‐Na,K‐ATPase antibodies, and analyzed by nLC‐MS/MS and Western blot analyses. Hypoxia did not change the total amount of the Na,K‐ATPase, as was assessed by comparing the Mascot scores and confirmed by a Western blot analysis. In contrast, hypoxia significantly decreased the amount of the Na,K‐ATPase‐co‐immunoprecipitated ER chaperones and enzymes presumably involved in the maturation of the Na,K‐ATPase, including BiP, Hsp40, calnexin, PDI, glucosidase, mannosidase and DnaJ. Consistent with these results, a significant retention of the Na,K‐ATPase was observed in cultured epithelial cells exposed to hypoxia. The results suggest that the hypoxia‐induced ER retention of the Na,K‐ATPase slows down the maturation and hence the delivery of the newly synthesized pumps to the plasma membrane, resulting in a decrease in ATP consumption as an adaptive response to hypoxia.

Grant Funding Source: Supported by 1R01HL113350‐01A1
Structural investigation of the folding of an immunoglobulin domain on the ribosome using NMR Spectroscopy (LB197)
Authors: Cassaignau Anais , Cabrita Lisa , Waudby Christopher , Wang Xiaolin , Launay Hélène , Camilloni Carlo , Karyadi Maria‐Evangelia , Chan Sammy , Vendruscolo Michele , Dobson Christopher , Christodoulou John
Published: June 2021

Correct protein folding is key to a functional proteome, as globular proteins must acquire their correct three‐dimensional native fold in order to fulfil their biological role. A significant amount of folding occurs co‐translationally on the ribosome and this process is likely to differ from folding of isolated proteins not least through the attachment to the ribosome and the vectorial emergence of the nascent polypeptide from the ribosomal exit tunnel. We are using solution‐state Nuclear Magnetic Resonance (NMR) spectroscopy to study structural aspects of the co‐translational protein folding of stalled ribosome‐bound nascent chain complexes (RNCs) which mimic the progressive emergence of the nascent polypeptide and its acquisition of structure.

The current state of our study on the co‐translational folding of a model immunoglobulin domain will be presented including some of the structural models built from chemical shift data of RNCs. These findings contrast with analogous studies of C‐terminal truncations of this domain, indicating some exciting differences between folding on the ribosome and that in bulk solution.

[1] Waudby CA, Launay H, Cabrita LD & Christodoulou J. Protein folding on the ribosome studied using NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 74, 57‐75 (2013).
Identification of a Sperm acrosome associated protein (SPACA7) splice‐variant in human testis (LB212)
Authors: Rashid Mohammad , Parimi Ramya
Published: June 2021

Transcriptome in a eukaryotic cell can vary based on the alternative splicing (AS) of genes giving a cell unique characteristic. AS can be a mechanism of gene regulation at the transcriptional level or it may be a way of generating diverse proteins from the same gene allowing them to function differently. Alternative splicing may well be a reason to develop male infertility, for example, studies on X‐linked gene glucocorticoid‐induced leucine zipper (GILZ) that is essential for survival and differentiation of spermatogonia shows six genetic variants of GILZ and two of them are involved in male infertility. Recently Sperm Acrosome Associated 7(SPACA7) protein has been identified in mouse. The analysis of SPACA7 using MyHits motif scan indicated the presence of a putative anaphylatoxins‐like domain motif in its N‐terminal domain that may be responsible for such activities like penetration of the zona pellucida. The human counterpart of SPACA7 gene is located on chromosome 13 (13q34) and its product is a195 amino acid protein.

Sperm acrosome structure is formed on the sperm head known to be specifically involved in the process of egg‐sperm fusion, called acrosome reaction, where it is believed to release the enzymes that allow the sperm to fuse with the egg.

The role of SPACA isoforms is still under considerable debate. SPACA7 is also known to be unique compared to other isoforms of SPACA that it is exclusively expressed in testis. SPACA7 in human has never been cloned till date and its specific role is still unknown. We have sub‐cloned a SPACA7 splice variant in our laboratory while screening a human testis cDNA library to study protein‐protein interaction in the regulation of human inducible nitric oxide synthase exploiting yeast and mammalian two hybrid assay systems. We hypothesize that this specific variant of SPACA7 may be the preferred version of the protein that interacts with iNOS to modulate nitric oxide synthesis during acrosome reaction.

Grant Funding Source: NIH/ARRA
Urea and thiourea‐based tipodal ligands induce anti‐angiogenic milieu on human first trimester cytotrophoblast cells (LB213)
Authors: Castor Juan , Horvat Darijana , Khansari Maryam , Sprague David , Hossain Md Alamgir , Beeram Madhava , Kuehl Thomas , Uddin Mohammad
Published: June 2021

Objective: Preeclampsia (preE) is a hypertensive disorder unique to pregnancy with abnormal placentation. We have demonstrated that cardiotonic steroids (CTSs) impair cytotrophoblast (CTB) function. The cell surface receptor for the CTSs has not been identified; we have used highly functional tripodal ligands to explore the receptor/ligand affinity using CTB cells: Para Nitro Tripodal Urea (PNTU), Para Nitro Tripodal Thio‐Urea (PNTTU), Penta Fluoro Tripodal Urea (PFTU), and TRSPHNO2. Design Method: The human extravillous CTB cells (Sw.71) cells were treated with 0.1, 1, 10, and 100 nM PNTU, PNTTU, PFTU, and TRSPHNO2 for 48 hours and the cell function was studied. The levels of angiogenic and anti‐angiogenic factors were measured in culture media, while the cell lysates were utilized to measure the VEGF‐1, AT1 and AT2 receptors expression. Results: Tipodal ligands impaired the CTB function. sFlt‐1 and sEnd secretion were increased while VEGF and PIGF were decreased in the culture media of CTB cells treated with 蠅 1 nM tipodal ligands. The AT2 receptor was upregulated in 蠅 1 nM tipodal ligands‐treated CTB cells compared to basal; however, the AT1 and VEGR‐1 were downregulated. Conclusion: The anti‐angiogenic effects of tipodal ligands on CTB cells are similar with those of CTS. The receptor/ligand affinity of tipodal ligands on CTB cells provides us the clue to design a potent inhibitor to prevent the CTS‐induced impairment of CTB cells.
Selective down‐regulation of IL‐2‐mediated cytokine expression in human T cells by protein phosphatase 1 inhibitors (LB217)
Authors: Kaminuma Osamu , Kitamura Noriko , Mori Akio , Hiroi Takachika
Published: June 2021

Protein phosphatase 1 (PP1) represents a major family of serine/threonine protein phosphatase involved in a variety of cellular events. Activation of PP1 is also regulated by its own phosphorylation/dephosphorylation as well as through interaction with its regulatory subunits. In the process of searching for IL‐2‐mediated signaling molecules in T cells by a proteomic approach, we discovered that PP1 was phosphorylated in an IL‐2 receptor (IL‐2R) β‐dependent manner. IL‐2Rβ signaling was crucial for the production of several Th2 cytokines in T cells, especially of human origin, even if it was evoked by T cell receptor (TCR) stimulation. Therefore, the role of PP1 in IL‐2‐mediated cytokine expression in human T cells was investigated by employing intracellular and low‐molecular weight PP1 inhibitors. Stimulation with IL‐2 directly induced expression of IL‐5 as well as IL‐13 in human peripheral CD4+ T cells. The expression of these cytokines evoked by IL‐2 but not by TCR stimulator was suppressed by treatment with tautomycin, a PP1 inhibitor, but not by ocadaic acid, a PP2A inhibitor. Overexpression of PP1 regulatory subunit 2, an endogenous inhibitor of PP1, also diminished IL‐2‐mediated cytokine expression in T cells. In conclusion, PP1, phosphorylated in IL‐2Rβ signaling, may play an important role in IL‐2‐mediated cytokine synthesis in human T cells.
Functional effects of a pathogenic mutation in Cereblon on the regulation of protein synthesis via the AMPK‐mTOR cascade (LB223)
Authors: Park Chul‐Seung , Lee Kwang Min , Yang Seung‐Joo , Choi Ja‐Hyun
Published: June 2021

Initially identified as a protein implicated in human mental deficit, cereblon (CRBN) was recently recognized as a negative regulator of adenosine monophosphate‐activated protein kinase (AMPK) in vivo and in vitro. Here, we present results showing that CRBN can effectively regulate new protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway, a downstream target of AMPK. Whereas deficiency of Crbn repressed protein translation via activation of the AMPK‐mTOR cascade in Crbn‐knockout mice, ectopic expression of the wild‐type CRBN increased protein synthesis by repressing endogenous AMPK. Unlike the wild‐type CRBN, a mutant CRBN found in human patients, which lacks the last 24 amino acids, failed to rescue mTOR‐dependent repression of protein synthesis in Crbn‐deficient mouse fibroblasts. These results provide the first evidence that Crbn can activate the protein synthesis machinery through the mTOR signaling pathway by inhibiting AMPK. In light of the fact that protein synthesis regulated by mTOR is essential for various forms of synaptic plasticity that underlie the cognitive functions of the brain, the results of this study suggest a plausible mechanism for CRBN involvement in higher brain function in humans, and they may help explain how a specific mutation in CRBN can affect the cognitive ability of patients.

Grant Funding Source: Supported by Ministry for Health and Welfare [HI13C1412] and Ministry of Science, ICT & Future Planning of Korea [2007‐0056157] [2011‐0028665] of Korea.
Hyperglycemia down‐regulates the cgmp‐dependent protein kinase i expression in cytotrophoblasts (LB241)
Authors: Ahsan Gias
Published: June 2021

Objective: The signaling pathway of nitric oxide (NO), cGMP, and cGMP‐dependent protein kinase (PKG) down‐regulates under diabetic conditions. High glucose exposure reduces PKG activity in smooth muscle cells; however, there is no data in cytotrophoblasts (CTBs). The present study evaluates the effect of high glucose on PKG‐I expression in CTBs. Study Design: Human CTBs (Sw. 71) were treated with 100, 150, 200, 300 or 400 mg/dL glucose for 72h. Some cells were pretreated with a p38 inhibitor (10 μM SB203580). After the treatment, the expression of protein kinase G1α (PKG1α), protein kinase G1β (PKG1β), soluble guanylate cyclase 1α (sGC1α), and soluble guanylate cyclase 1 β (sGC1β) were measured in the cell lysates by western blot analysis. The p38 MAPK phosphorylation was evaluated by western blot. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: The expression of PKG1α, PKG1β, sGC1α, and sGC1β were significantly down‐regulated (p<0.05) in CTBs treated with 蠅150 mg/dL glucose compared to basal (100 mg/dL) that were attenuated by SB203580. The p38 MAPK phosphorylation was up‐regulated (p<0.05) in excess glucose‐treated CTBs. Conclusions: Exposure of CTBs to excess glucose down‐regulates the PKG. The attenuation of hyperglycemia‐induced down‐regulation of PKG proteins by SB203580 pretreatment suggests the involvement of stress signaling in this process.

Grant Funding Source: Mohammad Uddin
Identifying functional mimetics of histone deacetylase using isothermal titration calorimetry (LB242)
Authors: Robinson Sophia , Jin Lihua , Grice Kyle , Karver Caitlin
Published: June 2021

Inhibition of histone deacetylase (HDAC) is known to induce tumor cell death, making it a promising approach for the treatment of cancer. To determine the energetic contribution of the active‐site Zn2+ to HDAC interaction with inhibitors as superoylanilide hydroxamic acid (SAHA), we have explored Zn2+ chelators as functional mimetics of HDAC as an alternative to HDAC enzyme. Our goal was to first identify Zn2+ chelators that are capable of forming tight 1:1 (Zn2+:chelator) binary complex and then to determine the energetics of inhibitor binding to the binary complex. One chelator, N‐(2‐(1‐methylimidazolyl)methyL)iminodiacetic acid (DA2Im), was synthesized and three others, Nitrilotriacetic acid (NTA), Tris(2‐pyridylmethyl)amine (TPA) and Tris(2‐aminoethyl)amine (TREN), were purchased from commercial sources. Their Zn2+ binding stoichiometry and thermodynamic parameters were determined using isothermal titration calorimetry (ITC) in either a NEM buffer (pH 6.8) or a methanol:buffer mixture (pH 6.8) with the methanol added to increase the solubility of the less polar chelators. DA2Im and NTA were found to bind with a stoichiometry equivalent to three chelators for every two Zn2+ and an equilibrium constant of (1.2±0.3)×108 and (2.1±0.6)×107 M‐1, respectively, in the buffer without methanol. Conversely, TREN and TPA were found to bind Zn2+ with the desired 1:1 stoichiometry and an equilibrium constant of (1.5±0.1)×106 and >107 M‐1, respectively, in the methanol:buffer mixture. We conclude that TPA and TREN are promising candidates as HDAC functional mimetics for studying Zn2+ inhibitor interactions. ITC experiments are currently underway to use the Zn2+:chelator binary complexes to obtain the energetic contribution of active site Zn2+ to HDAC interaction with inhibitors.
The Circadian Clock Acts Through PIF4 and PIF5 to Regulate Gibberellin Signaling in Arabidopsis (LB251)
Authors: Filo Julie , Wu Austin , Thines Bryan
Published: June 2021

The circadian clock confers 24‐hour rhythmicity to biological events and phases these processes a particular time of day. Plants, being sessile, use the clock to anticipate predictable environmental conditions, such as night and day, and they restrict growth to specific parts of the 24‐hour cycle. The Arabidopsis early flowering 3‐1 (elf3‐1) mutant has an arrested oscillator and shows excessive petiole and hypocotyl growth. Introducing a second gene mutation, phytochrome interacting factor 4 (pif4), to the elf3‐1 background restores a normal growth phenotype to the elf3‐1 mutant. PIF4, and closely related protein PIF5, are bHLH transcription factors that impact plant growth. We hypothesize that the clock acts through PIF4 and PIF5 to influence hormone pathways, such as auxin (IAA) and gibberellin (GA), and this ultimately regulates hypocotyl and petiole elongation. Analysis of transcript levels with quantitative PCR (qPCR) shows genes in both IAA and GA pathways are up‐regulated in the elf3‐1 mutant, but are restored to normal levels in the elf3‐1 pif4‐2 and elf3‐1 pif5‐3 double mutants. Growth of elf3‐1 on paclobutrazol (PAC), a GA biosynthesis inhibitor, also restores wild type like growth. Collectively, these results demonstrate that aberrant growth in elf3‐1 is dependent upon excessive GA signaling, and further implies that GA is responsible for time‐of‐day dependent growth in Arabidopsis.
Inhibition of the Type IB GTP Cyclohydrolase (LB262)
Authors: Alvarez Jonathan , Swairjo Manal
Published: June 2021

The type IB GTP cyclohydrolase (GCYH‐IB) is a tunnel‐fold enzyme that catalyzes the complex conversion of GTP to 7,8‐dihydroneopterin triphosphate in the biosynthesis of folate and the tRNA modified nucleoside queuosine in bacteria. Because it is an essential enzyme in several pathogenic bacteria, and is structurally and functionally distinct from the canonical type IA enzyme (GCYH‐IA) involved in biopterin biosynthesis in humans, GCYH‐IB has been proposed as a potential antibacterial target. Based on lack of sequence homology, different metal dependencies and active site architectures, we propose that the two enzymes utilize the same chemical mechanism but employ different strategies for catalysis. Here we describe the 2.77 Å‐resolution crystal structure of N. gonorrhoeae GCYH‐IB in complex with an 8‐oxoguanine derivative of GTP (8‐oxo‐GTP), a potent competitive inhibitor, and comparative analysis with the ‐IA enzyme in complex with the same inhibitor. The structure reveals a similar mode of binding for the guanine moiety but different modes of binding for the ribose and triphosphate tail. In both enzymes, the O8 atoms of 8‐oxo‐GTP acts as a ligand in the tetrahedral coordination of the catalytic Zn2+ ion. However, three key differences are observed in their interactions with the inhibitor: 1) The water molecule proposed to be the first nucleophile in the GCYH‐IA reaction is absent from the active site of GCYH‐IB. 2) In GCYH‐IB, the ribose hydroxyl groups of 8‐oxo‐GTP are coordinated by two active‐site side chains instead of one. 3) The inhibitor O8 atom is additionally coordinated via a water‐mediated hydrogen bond with a conserved His, an interaction only present in GCYH‐IB. These results explain the different inhibition properties of the two enzymes and confirm that they employ different catalytic strategies.
Purification of Histidine‐tagged and GST tagged proteins using ÄKTA™ start (LB264)
Authors: Haskins Kristi , R Sangeetha , Rao Devavratha , Rao Veena
Published: June 2021

Expression of recombinant proteins using affinity tags is a commonly used purification strategy. Histidine and glutathione‐S transferase (GST) are the most common and versatile of these tags. Histidine tags are small and therefore less disruptive to the protein structures and functions. GST tags enhance solubility of the target protein, hence improving the yield.

Manual purification of tagged proteins often face issues with time to purify, low reproducibility and low yield etc. In addition a researcher who is purifying manually needs to spend time in setting‐up the run, sample loading, collection and measuring the final protein concentration.

We describe here the purification Histidine‐tagged and GST‐tagged protein using ÄKTA start with Frac30 fraction collector and UNICORNTM start system control software.

We purified an N‐terminal Histidine tagged protein expressed in E.coli on a pre‐packed HisTrapTM FF 1mL column. The purification method was completely automated for unattended operation by applying large volume of sample using pump and collecting peak fractions using Frac30 fraction collector. We have consistently obtained high levels of purity (> 95%) of the tagged protein, which is 1.6 fold higher when compared to a conventional manual purification method.

In the second example, we describe the purification of GST tagged protein expressed in E.coli on a pre‐packed GSTrapTM FF 1ml column using ÄKTA start with Frac30 fraction collector. The target protein was purified using pre‐defined method workflow available from the instrument display. The result file that was stored on USB memory stick is imported for viewing and report generation using UNICORN start software.

ÄKTATM start is the newly developed laboratory scale protein purification system that can address the challenges faced during manual purification. Automated purification of tagged proteins has showed higher yields and purity with high consistency.
LipL41, a Hemin Binding Protein from Leptospira santarosai (LB265)
Authors: Sun Yuh‐Ju , Lin Ming‐Hsing
Published: June 2021

Leptospirosis, a widespread zoonotic disease, is caused by pathogen Leptospira. Outer membrane lipoprotein is the potential virulence factor of Leptospira. LipL41 is one of the major lipoprotein and highly conserved in Leptospira spp. Previous study suggests that LipL41 shows hemin binding ability and might have a potential role in iron regulation and storage. The hemin binding ability of LipL41 is determined with a Kd = 0.59 ± 0.14 μM. Two possible heme regulatory motifs (HRMs), C[P/S], are found in LipL41 as 140Cys‐Ser and 220Cys‐Pro. The mutation study indicates that Cys140 and Cys220 might be cooperatively involved in hemin binding. A supramolecular assembly of LipL41 was determined by transmission electron microscopy. At the C‐terminus of LipL41, there are two tetratricopeptide repeats (TPRs), which might involve in the protein‐protein interaction of the supramolecular assembly.
structural insights into ERα methylation by histone methyltransferase SMYD2, a cellular event implicated in estrogen signaling regulation (LB267)
Authors: Jiang Yuanyuan , Trescott Laura , Holcomb Joshua , Brunzelle Joseph , Sirinupong Nualpun , Shi Xiaobing , Yang Zhe
Published: June 2021

Estrogen receptor (ER) signaling plays a pivotal role in many developmental processes and has been implicated in numerous diseases including cancers. We recently showed that direct ERα methylation by the multi‐specificity histone lysine methyltransferase, SET and MYND domain‐containing protein 2 (SMYD2), regulates estrogen signaling through repressing ERα‐dependent transactivation. However, the mechanism controlling the specificity of the SMYD2‐ERα interaction and the structural basis of SMYD2 substrate binding diversity are unknown. Here we present the crystal structure of SMYD2 in complex with a target lysine (Lys266)‐containing ERα peptide. The structure reveals that ERα binds SMYD2 in a U‐shaped conformation with the binding specificity determined mainly by residues C‐terminal to the target lysine. The structure also reveals numerous intrapeptide contacts that ensure shape complementarity between the substrate and the active site of the enzyme, thereby likely serving as an additional structural determinant of substrate specificity. In addition, comparison of the SMYD2‐ERα and SMYD2‐p53 structures provides the first structural insight into the diverse nature of SMYD2 substrate recognition and suggests that the broad specificity of SMYD2 is achieved by multiple molecular mechanisms such as distinct peptide binding modes and the intrinsic dynamics of peptide ligands. Strikingly, a novel potentially SMYD2‐specific polyethylene glycol (PEG) binding site is identified in the C‐terminal domain, implicating possible functions in additional substrate binding or protein‐protein interactions. Our study thus provides the structural basis for the SMYD2‐mediated ERα methylation, and the resulting knowledge of SMYD2 substrate specificity and target‐binding diversity could have important implications in selective drug design against a wide range of ERα‐related diseases.

Grant Funding Source: Supported by Leukemia Research Foundation, Aplastic Anemia & MDS International Foundation and American Heart Association.
Novel condensed workflow for recombinant protein purification from E.coli (LB275)
Authors: Dedeo Anja , George Jesmi , Moskow John
Published: June 2021

Many scientists require purified proteins for their research applications. To meet this need, a multitude of recombinant expression techniques have been developed. While many expression systems are available, E. coli represents the most optimized and widely used vehicles for the expression of 6x Histidine tagged recombinant proteins. Traditionally, the recombinant protein purification workflow consists of five distinct phases: Harvest, Lysis, Clarification, Purification and Analysis. Lysis typically requires multiple time consuming, hands‐on steps, such as freeze/thaw cycling and sonication, which may be harsh and may negatively impact protein quality and contribute to sample variability. Clarification is typically performed in a centrifuge, which makes automation challenging and is primarily needed to reduce clogging in the downstream purification step. Purification is typically performed using columns packed with agarose beads with specificity for the tag on the recombinant protein. While columns provide an easy approach for manipulation of the affinity media, they are greatly affected by lysate consistency and carry‐over of cell debris, which can lead to clogging and poor protein purification and recovery. To guarantee maximum recovery of protein activity and integrity, we have developed a protocol leveraging gentle enzymatic cell lysis and purification with magnetic beads to condense the traditional His‐tag protein purification workflow. A significantly improved workflow was developed by combining Lysis and Purification and eliminating the need for Clarification, resulting in a greater than two hour time savings over the traditional workflow. In addition, this condensed workflow facilitates automation, resulting in increased sample throughput while further reducing actual hands‐on processing time.
Anatomy education in a new distributed MD program ‐ prospects and challenges (LB28)
Authors: Oyedele Olusegun
Published: June 2021

MD undergraduate teaching at the University of British Columbia (UBC), Canada occurs in a distributed setting, with 288 students spread across four locations in the province per year of instruction. Hence, UBC has the largest medical school in Canada. Although experienced anatomy faculty exist on each UBC campus, large class teaching must be performed using videoconferencing technology supplemented by small group interaction in the cadaver labs. The Southern Medical Program at UBC Okanagan received the first set of MD undergraduates at the Kelowna campus in January 2012 and is the newest of UBC’s distributed sites. Teaching anatomy in such a pioneer setting offers unique perspectives on anatomy education as is currently practiced provincially and nationally, along with salient lessons on the prospects and challenges of distance learning in an MD program. This paper reflects on the impacts of location, pedagogic methods and curriculum on the teaching of anatomy in this unique locus and examines wider implications for the anatomy education of future doctors in Canada.
Activation of caspase‐dependent apoptosis by intracellular delivery of cytochrome c‐based nanoparticles (LB290)
Authors: Morales Moraima , Figueroa Cindy , González Tania , Delgado Yamixa , Molina Anna , Griebenow Kai
Published: June 2021

Cytochrome c (Cyt c) is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm. This process normally takes place in response to DNA damage, but in many cancer cells (i.e., cancer stem cells) it is disabled due to various mechanisms. However, it has been demonstrated that the targeted delivery of Cyt c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. Consequently, in this work we designed a nano‐sized targeted Cyt c drug delivery system. First, we precipitated Cyt c with a simple solvent‐displacement method to obtain protein nanoparticles (NPs). Cyt c NPs’ surface was chemically linked to poly(lactic‐co‐glycolic) acid‐ thiol endcap (PLGA‐SH) using succinimidyl 3‐(2‐pyridyldithio)propionate (SPDP) to prevent premature dissolution during delivery. SPDP is a heterobifunctional linker containing a disulfide bond which allows the Cyt c release under intracellular reducing conditions. This construct is further on referred to as PLGA‐S‐S‐Cyt c NPs. The size of the Cyt c NPs obtained was 100‐300 nm in diameter depending on the conditions used, indicating good potential to passively target tumors by the Enhanced Permeability and Retention (EPR) effect. A cell‐free caspase‐3 assay revealed more than 80% of relative caspase activation by Cyt c after the nanoprecipitation and SPDP modification, when compared to native Cyt c. Incubation of HeLa cells with PLGA‐S‐S‐Cyt c NPs showed significant reduction in cell viability after 6 hours. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with DAPI and propidium iodide (PI) after incubation with the Cyt c‐based NPs. Our results demonstrate that the PLGA coating, mediated by SPDP linker, stabilizes Cyt c NPs allowing their delivery to the cytoplasm of the target cells. After smart release of Cyt c into the cytoplasm it was able to induce programmed cell death.

Grant Funding Source: Supported by the NIH‐RISE Program Grant Number 2R25GM061151‐12
Supplementation of Korean black raspberry improves endogenous antioxidant capacity in overweight adults (LB308)
Authors: Kim You Jin , Lee You Jin , Oh Sang A , Huh Iksoo , Park Saejong , Kim Joohee , Kim Suhkmann , Kim Kyu‐Bong , Park Taesung , Kim Ji Yeon , Kwon Oran
Published: June 2021

The natural phytochemicals have been well reported as antioxidants. The fruit of Rubus coreanus Miquel (RCM), referred to as the Korean black raspberry, is a rich source of phytochemicals. We hypothesized that response to RCM supplementation would be more sensitive to detect when applied in combination of clinical assessment and nuclear magnetic resonance‐based metabolomics approach. In 4‐week, randomized, double‐blind, placebo‐controlled study, overweight participants (23蠄body mass index (kg/m2)<30) were randomly allocated to receive either RCM or placebo (n=24 per group). At baseline and at the end of the study, each subject underwent an acute bout of treadmill exercise at 60% VO2max for 30 min immediately after ingestion of a single dose of RCM and blood and urine samples were taken at fasting and post‐exercise state. In comparison with placebo, oxidized glutathione, reduced glutathione/oxidized glutathione ratio and superoxide dismutase activity in erythrocyte significantly improved in the RCM group (P‐value<0.05). Urinary metabolites involved in oxidative phosphorylation, amino acid metabolism, glycerophospholipid metabolism and purine metabolism responded significantly to RCM (P‐value<0.05). Taken together, ingestion of RCM may boost the antioxidative defence mechanism and this can be explained by alteration of endogenous metabolites.

Grant Funding Source: Supported by the RDA “Cooperative Research Program for Agricultural Science & Technology Development (Project No. PJ0084502013)”, the MSIP through the NRF (Bio‐synergy Research Project NRF2012M3A9C4048761), and the MEST (BK21PLUS 22A20130012143)
A randomized clinical trial to determine the efficacy of manufacturers’ recommended doses of omega‐3 fatty acids from different sources in facilitating cardiovascular disease risk reduction (LB329)
Authors: Laidlaw Maggie , Cockerline Carla , Rowe William
Published: June 2021

A randomized clinical trial to determine the efficacy of manufacturers’ recommended doses of omega‐3 fatty acids from different sources in facilitating cardiovascular disease risk reduction Maggie Laidlaw1, Carla A. Cockerline1, William J. Rowe:1 1 Nutrasource Diagnostics Inc.

Omega‐3 fatty acids confer beneficial health effects, but North Americans are lacking in their dietary omega‐3‐rich intake. Omega‐3 supplementation is an alternative to dietary consumption of fish; however, not all omega‐3 products are created equal. The trial objective was to assess changes in cardiovascular risk following supplementation with four omega‐3 supplements.

This was an open‐label, randomized, cross‐over study with thirty‐five healthy subjects. Supplement daily doses (as recommended on product labels) were: Concentrated Triglyceride (rTG) fish oil: EPA 650 mg, DHA 450 mg Ethyl Ester (EE) fish oil: EPA 756 mg, DHA 228 mg Phospholipid (PL) krill oil: EPA 150 mg, DHA 90 mg Triglyceride (TG) salmon oil: EPA 180 mg, DHA 220 mg For the duration of the study, participants were randomly assigned to consume one of the four products, in random order, for a 28‐day period, followed by a 4‐week washout period. This process continued until each subject had consumed each product. Blood samples before and after supplementation were quantified for fatty acid analysis by gas chromatography flame ionization, and statistically analysed using ANOVA for repeated measures.

At the prescribed dosage, rTG was statistically superior to all three comparators in the bioavailability of EPA+DHA. Risk reduction in several elements of cardiovascular disease was achieved to a greater extent by rTG than by any other supplement. PL and TG were relatively unsuccessful in this aspect of the study.

For the general population, the type and dose of omega‐3 supplements may be immaterial. However, the type and dose of omega‐3 supplementation may be important for those interested in reducing their risk of cardiovascular disease.
Effectiveness evaluation of nutrition counseling for adults with risk factors for dyslipidemia (LB342)
Authors: Kim Jung Hee , Nam Tae Young
Published: June 2021

Dyslipidemia is a component of the metabolic syndrome and a risk factor for cardiovascular disease. Nutrition counseling is important to improve dyslipidemia.The purpose of this study was to evaluate the effectiveness of nutrition counseling to adults with risk factors for dyslipidemia diagnosed by national health screening program. The nutrition counseling for adults with risk factors for dyslipidemia was carried out at a public health center in Gyeonggi‐do of South Korea. Thirty four patients out of forty five participants in the program completed individualized nutrition counseling 3 times during 12 weeks after examining participants' dietary habits through questionnaires. Data about serum lipid profiles, body composition, nutrition knowledge, the practice of dietary guideline, and dietary behavior were collected before and after nutrition counseling to evaluate its effectiveness. All data were statistically analyzed by SPSS program(Korea ver.18.0) and significant difference was examined by paired t‐test and χ2‐test. Body weight, body fat and WHR were significantly decreased after nutrition counseling. Total‐cholesterol, TG, and LDL‐cholesterol were also significantly decreased but HDL‐cholesterol did not show significant change. Both scores of nutrition knowledge and the practice of dietary guideline improved significantly(p<0.001). This study shows that nutrition counseling helps to encourage healthy eating practices and to improve the serum lipid profiles of adults with risk factors for dyslipidemia. Overall results indicate that nutrition counseling gives positive change to lower the reliance on medication. Therefore nutrition counseling should be considered for the initial treatment of dyslipidemia.
Dietary prebiotics modulate fecal microbiota and impact cognitive functions in developing rodents (LB376)
Authors: Waworuntu Rosaline , Chichlowski Maciej , Cox Stephen , Berg Brian
Published: June 2021

We previously observed that providing dietary prebiotics polydextrose (PDX) and galactooligosaccharide (GOS) to rodents at weaning positively influences behavior and cognitive functions later in life. Specifically, weanling Long Evans (LE) rats fed PDX‐GOS (7 g/kg each) diet for 35 days had significantly better memory than rats fed a control diet (P<0.05). Here, we compared the microbiota from fecal samples of the rats fed control and prebiotic diets to assess whether alteration in the gut microbiota is associated with the improved cognitive functions. Pyrosequencing of the V3‐V5 region of 16S rRNA genes showed a significant shift of the gut microbiota in prebiotic fed rats relative to control. Firmicutes were dominant in both groups, while Tenericutes and Bacteroidetes were abundant in prebiotic and control fed rats, respectively. There was a decrease in bacterial diversity from postnatal day (pd) 21 to pd 55 in the prebiotic group. At the genus level, the microbiome of the control diet rats was dominated by Clostridium while prebiotic feeding resulted in significantly higher proportion of the short‐chain fatty acid producing bacteria, Allobacculum (p<0.0001), and significantly lower proportion of Clostridium (p<0.05). These findings indicate that LE rats with improved cognitive functions in response to PDX‐GOS diet displayed a considerable shift in fecal microbial communities.
Evaluation of messages to motivate parents to promote intake of calcium‐rich foods in early adolescents (LB377)
Authors: Martinez Yannica Theda , Ballejos M , Cluskey M , Johnston P , Reicks M , Richards R , Wong SS , Bruhn C , Gunther C , Misner S , Banna J
Published: June 2021

Parental practices such as role modeling and making calcium‐rich foods available have an influence on intake of calcium‐rich foods and beverages (CRFB) in early adolescents. This study aimed to test two messages promoting these practices with respect to comprehension, cultural and personal relevance, and ability to motivate parents to encourage CRFB availability and intake. Interviews were conducted with 17 Hispanic and 18 Asian parents from five states to evaluate two messages containing images and taglines tailored to the target audience. Responses were reviewed for common themes. For the message, “Good play starts with calcium,” Hispanics recognized the connection between calcium and strong bones. Both groups understood the importance of providing CRFB for adequate calcium intake. For the message, “Strong families start with good nutrition,” both groups had difficulty understanding that the foods pictured contained calcium and should be provided to ensure adequate intake. For both messages, respondents did not indicate motivation to provide CRFB specifically. Both messages were considered culturally acceptable. Given the issues identified regarding comprehension and ability to motivate parents to encourage CRFB intake, modifications are needed to emphasize the connection between messages and calcium intake for a future intervention to improve CRFB practices among parents of early adolescents.
A counterfactual analysis of changing economic context and its impact on dietary intakes in Chinese children (LB384)
Authors: Xue Hong , Wu Yang , Wang Youfa
Published: June 2021

Background: Little is known about how food prices affected children’s nutrient intakes after China’s entry to the World Trade Organization, which has substantial policy implications.

Objective: To assess the differential effects of food price change on the distributions of macronutrient intakes in children (6‐18yrs) in Urban China and to simulate potential policy effect.

Methods: The China Health and Nutrition Survey (CHNS) data (2000‐2006) with about 1000 children. Quantile regression (QR) and counterfactual analysis were used.

Results: Every one Chinese Yuan increase of oil price was associated with about 3 kcal/day (p<0.01) reduction in energy intake from fat for those whose fat intake was below 70 percentile of the fat intake distribution in 2000. In 2006, oil price effect became insignificant. One Chinese Yuan increase in rice price was associated with about 85 kcal/day (p<0.1) energy intake decrease from carbohydrates among those above 90 percentile of the carbohydrate distribution in 2000 with insignificant effect at lower portion of the distribution, which was in contrast with 2006, when rice price effect had a peak impact of 30 kcal/day (p<0.01) at 60 percentile. Protein intake was insensitive to price changes. Counterfactual analysis suggested a 10% increase in rice price would lead to the most effective energy intake reduction ranging from 7‐14 kcal/day.

Conclusions: Food prices have differential effects on dietary intakes. Reducing carbohydrate intake could potentially be most effective through pricing instruments.

Grant Funding Source: NIH/NICHD (U54 HD070725‐01)
Proteominer facilitates high‐throughput proteomic analysis of low abundance proteins in the cartilage secretome (LB39)
Authors: Williams Adam , Smith Julia , Liddell Susan , Mobasheri Ali
Published: June 2021

Proteomics is increasingly used for biomarker identification. We have developed high‐throughput mass spectrometric (MS) methods to identify proteins in the secretome of articular cartilage explants, which we are utilizing as an in vitro model of joint inflammation. However, the identification of lower abundance proteins of potentially higher biological significance in this model is hampered by the presence of highly abundant glycoproteins in the secretome. Development of protein depletion techniques that allow the identification of low abundance proteins is important for novel biomarker discovery, especially in the context of identifying pathophysiologically relevant biomarkers of joint inflammation and cartilage degradation. Proteominer protein enrichment kits decrease the dynamic range of complex biological samples, allowing improved analytical coverage of the proteome. The objective of this study was to apply high‐throughput MS techniques following proteominer enrichment to expand the range of proteins that can be identified in the secretome of cartilage exposed to proinflammatory cytokines such as IL‐1β. Explant cultures were established in DMEM + 2% Pen/Strep, using metacarpophalangeal joints of horses. Explants were either untreated controls or treated with 10 ng/ml IL‐1β. After incubating for 6 days at 37⁰C in a humidified environment (95% air, 5% CO2) proteins in culture supernatants were concentrated ~24 fold using Centriplus YM‐3 and Centricon‐3 (3000 MWCO) centrifugal filter units. Concentrated samples were processed with a Small Capacity Proteominer™ Protein Enrichment Kit (Bio‐Rad). After elution, samples were reduced, alkylated, acetone precipitated and trypsin digested overnight, before digested peptides were applied to C18 spin columns, followed by nanoLC‐MS/MS analysis on an amaZon speed ETD. Extracellular matrix proteins identified in untreated control secretome included cartilage oligomeric matrix protein (COMP), fibromodulin and fibronectin. Macrophage migration inhibitory factor (MIF), serum amyloid A protein (SAA) and Heat shock 70 kDa protein 1‐like (HSP70) were identified in explants exposed to IL‐1β. Identification of new low abundance proteins in this model will facilitate a greater understanding of inflammatory processes occurring within cartilage.

Grant Funding Source: Supported by Biotechnology and Biological Sciences Research Council (BBSRC) (Contract grant number: BB/G018030/1) and the European Commission Framework 7 program (EU FP7; HEALTH.2012.2.4.5–2, project number 305815).
SDE‐GC‐MS analysis of the Volatile Components in the Wild Grape (Vitis amurensis Rupr) Skin Originated from Zhangjiakou (LB395)
Authors: Zhang Zhisheng , Shu Ying , Li Guijie , Chen Feng , Zhao Congzhi , Tang Xia
Published: June 2021

In this study, the Wild grape (Vitis amurensis Rupr) which was supplied by a local wine factory of Zhangjiakou City, Hebei Province was analyzed for the volatile compounds in the skin. The volatile flavor compounds were extracted by using the method of simultaneous distillation extraction (SDE) and then analyzed by gas chromatography‐mass spectrometry (GC‐MS) with phenylacetate as the internal standard. Generally the volatile compounds of the wild grape are quite complicated. The analytical results showed that 65 kinds of volatile compounds mostly consisting of 12 esters compounds (2.94 mg/kg), 11 aldehydes compounds (39.96 mg/kg), 14 terpenes compound (4.97 mg/kg), 4 ketone compounds (0.44 mg/kg), 4 phenols compounds (1.33 mg/kg) 7 eneyne compounds (7.69 mg/kg) , 1 alcohol (2.37 mg/kg), 5 heterocycle compounds(1.11 mg/kg) ,5 acid compounds(10.94 mg/kg) and 2 amide compounds (3.47 mg/kg) were detected. The total content of these bioactives is 75.22 mg/kg, in which aldehyde compounds account for the most proportion. Fourteen terpenes were found and the kinds were the most among all of the bioactive compounds; also, they were the main aroma compounds in the wild grape skin.

Grant Funding Source: Supported byResearch Center of Primary Product Processing and Engineering of Hebei Province, Baoding