bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural, Functional, and Mechanistic Diversity in Protein Ubiquitination
Authors: Klevit Rachel
Published: June 2021

Protein ubiquitination is a regulatory process with far‐reaching impacts on all aspects of eukaryotic biology. It is involved in every known biological process and is implicated in a growing range of diseases including, but not limited to, cancers, neurodegenerative diseases, and muscle wasting. The language of ubiquitin signaling is incredibly rich. Cellular proteins may be modified with one or more single ubiquitin (Ub) moieties or with poly‐Ub chains. The most common configuration of poly‐Ub is the so‐called Lys48‐linked chain, long known to target proteins for degradation by the proteasome. But it is now clear that any of seven possible lysine linkages as well as linear linkages (N‐terminus‐to‐C‐terminus) and mixed chains are all generated in cells. This results in a remarkable array of outcomes, both at the level of a modified protein and at a cellular level. Three basic enzymatic activities (i.e., E1, E2, and E3) work in series to carry out protein ubiquitination and are ultimately responsible for the variety observed. Decades of research have provided insights regarding shared structural and mechanistic features in each class of enzyme. More recently, novel and unexpected features of individual members of the E2 and E3 families have also led to important conceptual advances in our understanding of Ub transfer and signaling. I will present the general structural and mechanistic features of protein ubiquitination and several recent examples of how highly related enzymes have evolved to perform in specialized ways. Not only do such discoveries contribute to our understanding of the source of diversity in protein ubiquitination, they also provide new avenues with which to understand the rich and varied functions of this essential regulatory system.
Following Lipid Signaling in Intact Cells
Authors: Schultz Carsten
Published: June 2021

Following lipids in cells is usually achieved by expressing fluorescently tagged lipid binding domains or proteins. A location change of these fluorescent constructs is indicative of changes in lipid concentrations. One application is based on the extremely fast switch‐off of PIP3 biosynthesis and the quantitative determination of its metabolism in intact cells. Alternatively, we look at lipid‐protein interactions by using photo‐crosslinkable lipid derivatives equipped with a clickable alkyne group. After illumination, click chemistry permits visualizing the lipids in fixed cells directly by fluorescent tagging. Alternatively, we click biotin on the crosslinked lipid‐protein conjugates and purify the latter for mass spectrometry analysis. Recently, we employed caged lipids that are crosslinkable and clickable to prevent the rapid lipid metabolism and to produce cleaner proteomic results.

Caged lipids are exquisite tools for studying the effects of changes in lipid concentrations in a live cell context. Here we introduce caged sphingosine and caged lysophosphatidic acid and demonstrate some unexpected signaling functions of these lipid.

Haberkant et al., Angew. Chem. Int. Ed. 52, 4033‐4038 (2013)

Feng et al., Angew. Chem. Int. Ed. 53, 6720‐6723 (2014).
How Gut Bacteroides Eat Your Veggies
Authors: Koropatkin Nicole
Published: June 2021

The human gut Bacteroidetes display a greatly expanded capacity for glycan degradation via the expression of multiple outermembrane protein complexes, termed Sus‐like systems. These systems are comprised of proteins that sequentially bind, degrade and import specific carbohydrates. Two classes of carbohydrate‐binding proteins, the SusD‐like proteins and the carbohydrate‐binding lipoproteins (CBLPs) respectively, are functionally conserved within the Bacteroidetes Sus‐like systems and found in all systems. We have recently determined the crystal structures two such cell surface liopoproteins, XgusD, a SusD‐like protein, and XgusE, a CBLP, from Bacteroides ovatus. These proteins are involved in the recognition of xyloglucan, a plant cell wall polysaccharide abundant in many “salad” vegetables including lettuce, tomato and peppers. Xyloglucan is comprised of a beta‐glucan backbone appended with xylose and galactose side chains, and we demonstrate that recognition of this polymer by the XgusD and XgusE proteins is driven by different aspects of the carbohydrate structure. More over, the XgusE protein displays a novel xyloglucan‐binding domain, and likely reflects a new class of carbohydrate‐binding modules. While XgusD is essential for all xyloglucan utilization, mutants of XgusE in B. ovatus are impaired in the uptake of some xyloglucan oligosaccharides. Taken together our data support our previous work demonstrating that the SusD‐like proteins and CBLPs have evolved to play non‐redundant roles in carbohydrate uptake.
ECM Remodeling Events during EC‐Pericyte Tube Co‐Assembly in 3D Matrices
Authors: Davis George
Published: June 2021

We have been investigating the molecular events controlling capillary tube formation in 3D matrices, whereby human endothelial cells (ECs) and pericytes co‐assemble to form tubes with associated pericytes that are polarized on the EC abluminal surface. Dynamic EC‐pericyte interactions lead to vascular basement membrane matrix assembly in either 3D collagen or fibrin matrices, a process that requires the presence of both cell types and which occurs between the cells. Normally, pericyte recruitment leads to more narrow EC tube diameters that occur as a result of basement membrane formation. A major emphasis of our laboratory over the past several years has been to define the growth factor requirements for EC tubulogenesis and EC‐pericyte tube co‐assembly. We have developed models of these processes under serum‐free defined conditions, which require the addition of SCF, IL‐3, SDF‐1alpha, FGF‐2 and insulin (Factors). Addition of these Factors allows for both EC tubulogenesis and EC‐pericyte tube co‐assembly. In contrast, the addition of VEGF or the combination of VEGF and FGF‐2 fails to support these events. Of great interest is that we have identified four additional EC‐derived growth factors that are necessary for EC‐pericyte tube co‐assembly and they are: PDGF‐BB, HB‐EGF, TGFbeta1, and ActivinB. Blockade of these growth factors (individually or combinations) or their receptors leads to marked decreases in pericyte recruitment, pericyte proliferation and vascular basement membrane formation in vitro and in vivo during quail development. In addition, EC tube widths increased when these growth factors are disrupted. Overall, these data indicate that 9 or more growth factors are necessary for human capillaries to assemble in vitro; a novel concept regarding vascular morphogenesis.
Quaternary Structure and Activity Modulation in Human Ribonucleotide Reductase
Authors: Asturias Francisco , Brignole Edward , Tsai Kuang‐Lei , Drennan Cahterine , Stubbe JoAnne
Published: June 2021

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to 2´‐deoxynucleotides, providing the monomeric precursors necessary for DNA replication and repair. Precise regulation of RNR activity is essential for fidelity of DNA replication, as RNR is responsible for maintaining both the correct absolute cellular concentration of deoxynucleotides, as well as the appropriate relative concentration ratios. The class Ia RNRs found in many aerobic bacteria and all eukaryotes consist of 2 protein subunits. The larger subunit α houses the active site where nucleoside diphosphate substrates (CDP, ADP, GDP, UDP) are reduced, and two additional sites for allosteric effector (ATP, dGTP, TTP, and dATP) binding. The specificity site dictates which substrate is reduced, while the activity site binds ATP/dATP and regulates the overall reduction rate. Nucleotide reduction depends on precise interaction of α with a dimeric smaller subunit β2 that contains a diferric‐tyrosyl radical cofactor essential for catalysis. We are using single particle electron microscopy and advanced image analysis methods to understand the way in which nucleotide‐induced structural rearrangements and shifts in complex subunit equilibria combine to modulate the activity and specificity of human RNR.
Biogenesis and Mechanism of Action of Enhancer RNAs
Authors: Shiekhattar Ramin
Published: June 2021

Enhancers are distal regulatory elements that mediate tissue‐ and temporal‐specific gene expression during development. Recent evidence has revealed that active enhancers are transcribed and that such enhancer RNAs (eRNAs) play an important role in transcriptional activation. We show that the metazoan‐specific RNA polymerase II (RNAPII)‐associated complex, Integrator, is recruited to enhancers and super‐enhancers in a stimulus‐dependent manner. Functional depletion of Integrator subunits diminished the signal‐dependent induction of eRNAs at enhancers and super‐enhancers. Global nuclear run‐on and RNAPII profiling demonstrated that Integrator regulates the maturation (3′‐end processing) of eRNAs. We show that the production of mature eRNAs require the catalytic activity of Integrator complex. We propose a role for Integrator in biogenesis of eRNAs in metazoans.
To Grow or Die:Regulation of Plant Sphingolipid Metabolism
Authors: Cahoon Edgar , Kimberlin Athen , Han Gongshe , Dunn Teresa
Published: June 2021

Maintenance of sphingolipid homeostasis is critical for eukaryotic cell growth and regulation of programmed cell death (PCD). Serine palmitoyltransfersase (SPT), a heteromeric enzyme composed of LCB1 and LCB2 subunits, catalyzes the first step in sphingolipid biosynthesis. Our research is directed at understanding the regulation of sphingolipid synthesis via post‐transcriptional mediation of SPT activity in plant cells. We identified genes encoding two 56‐amino acid polypeptides AtssSPTa and AtssSPTb by homology to mammalian small subunits of SPT (ssSPT). Co‐expression of either polypeptide in a yeast lcb1Dlcb2D mutant stimulated SPT activity from the Arabidopsis LCB1/LCB2 heterodimer by >100‐fold through physical interaction with LCB1/LCB2. Though both genes are constitutively expressed, AtssSPTa transcripts were more enriched in all vegetative organs examined, and >400‐fold more abundant in pollen than those for AtssSPTb. Homozygous AtssssSPTa T‐DNA mutants were not recoverable due to loss of pollen viability. Overexpression of AtssSPTa in Arabidopsis stimulated SPT activity and enhanced sensitivity to the sphingosine‐analog mycotoxin fumonisin B1 (FB1), while RNAi suppression reduced SPT activity and fumonisin B1 sensitivity. In converse to results with ssSPT, an orosomucoid‐like protein AtORM1 recently identified in Arabidopsis reduced FB1 sensitivity when overexpressed in Arabidopsis, and RNAi suppression of the AtORM1 gene increased FB1 sensitivity. These findings indicate that ssSPT polypeptides are stimulators of SPT activity and essential in Arabidopsis, whereas AtORM1 functions to suppress SPT activity. Our results also show that ssSPT and ORM genes are viable targets for modulating sphingosine‐analog mycotoxin sensitivity in plants.
A Chemical‐Proteomic Platform to Investigate Cysteine S‐Nitrosation
Authors: Weerapana Eranthie
Published: June 2021

Cysteine residues on proteins play numerous functional roles as catalytic nucleophiles, redox‐active disulfides, or metal‐binding residues. Outside of their direct role in catalysis, cysteines can also regulate protein activity via posttranslational modification by endogenous electrophiles and reactive oxygen species. These modified cysteine residues do not conform to a conserved sequence or structural motif, rendering the global identification of regulatory cysteines a considerable challenge. We hypothesize that cysteines that are hypersensitive to different oxidants or nitrosating agents are likely to be enriched in functional residues that serve to modulate protein activity. To explore this hypothesis, we focused our initial efforts on investigating cysteine susceptibility to nitrosation. S‐nitrosation is a unique posttranslational modification that serves to temporally and spatially control protein activity and localization. In order to identify cysteine residues hypersensitive to nitrosation, we developed a chemical‐proteomic platform that ranks cysteines in the human proteome by sensitivity to a variety of nitric oxide donors. Our proteomic studies identified several known sites of nitrosation, as well as previously unannotated cysteines. We then proceeded to functionally characterize several of the unannotated cysteines using a variety of cell and molecular biology approaches. We show that many of these cysteines are located distal from the active site, and yet mediate essential protein functions such as substrate binding and localization. Overall, our studies aim to unearth novel functional cysteine residues in the proteome, with the long‐term goal of identifying new modes of protein regulation through reactive cysteines.
Obesity is Associated with Changes in Plasma Oxylipids
Authors: Pickens Charles , Sordillo Lorraine , Comstock Sarah , Fenton Jenifer
Published: June 2021

Oxylipids, oxygenated fatty acids, are implicated in inflammation, immune cell signaling, and tumor survival. The link between obesity, inflammation, and cancer is well‐accepted, but it is unknown if obesity is associated with specific oxylipids. The objective of this study was to identify associations between non‐esterified plasma oxylipids and body mass index (BMI). 126 Caucasian males (48‐65 years) were categorized: lean (BMI<25), overweight (25£BMI<30) or obese (BMI³30). Plasma non‐esterified oxylipids were analyzed using solid phase extraction and quantified using a HPLC tandem mass spectrometric analysis. Obese individuals were 1.2 (p<0.05) times more likely to have elevated plasma arachidonate derived 8,9‐epoxy eicosatrienoate (8,9‐EET) compared to lean individuals. Plasma auto‐oxidative arachidonate products, 9‐hydroxy eicosatetraenoic acid (9‐HETE) (P<0.01) and 11‐HETE (p<0.005), were elevated in obese men. Also, lipoxygenase products 15‐HETE (<0.005), 15‐keto ETE (p<0.01), and cytochrome p450 product 20‐HETE (p<0.05) were increased in obese men compared to lean. 16, 17‐epoxy docosapentaenoate was 0.6 (p<0.01) and 0.7 (p<0.05) times less abundant in plasma from overweight and obese men respectively, compared to that of lean. Overall arachidonate enzymatic and non‐enzymatic oxygenation is increased in obesity, including “proinflammatory‐like” oxylipids. Future studies should examine the role of these oxylipids in the initiation of obesity‐associated diseases such as cancer. Research Support: NIH R03CA142000 and MSU CTSI
Relationship between Transferrin Receptor and Two Acute Phase Proteins in Women of Reproductive Age
Authors: Addo O.Yaw , Ashour Fayrouz , Namaste Sorrel , Sullivan Kevin , Suchdev Parminder , Mei Zuguo , Rohner Fabian , Northrop‐Clewes Christine , Flores‐Ayala Rafael , Raiten Daniel
Published: June 2021

Transferrin receptor (TfR) is a biomarker for iron reported to be less affected by inflammation compared to ferritin. We examined the associations between two acute phase proteins (APP), α‐1‐acid glycoprotein (AGP) and C‐reactive protein (CRP), with TfR. A multi‐national database of 8,410 non‐pregnant women aged 15‐49 y from 6 countries (USA, Laos, Ivory Coast, Cameroon and Papua New Guinea) as part of the Biomarkers Reflecting Inflammation and Nutrition Determinants of Anemia (BRINDA) project was analyzed. Elevated TfR was defined as >8.3 mg/L, elevated CRP >5 mg/L, and elevated AGP >1 g/L.

Prevalence of elevated TfR was 20.0%, ranging from 5.7% in Laos to 33.8% in Cameroon. Correlations between APP and TfR ranged from 0.01 – 0.27. Based on preliminary analyses, women with elevated AGP had a higher prevalence of elevated TfR [overall prevalence odds ratio (POR) 2.0 (95%CI: 1.6–2.5), with the strongest association in Laos where the POR was 3.1(1.5–6.7). Elevated CRP was not associated with elevated TfR in any country, except USA. When modeling both AGP and CRP as predictors, the POR for elevated TfR was higher for AGP but it remained non‐significant for CRP. Inflammation, as measured by elevated AGP but not CRP, is associated with TfR in women of reproductive age and thus may overestimate the prevalence of elevated TfR. In this presentation various approaches to account for inflammation in their relationship with TfR will be presented. Funding:Gates Foundation, CDC, GAIN, NICHD.
Ellagic Acid Supplementation Attenuates Sucrose‐Induced Obesity and Metabolic Complication in C57BL/6 mice
Authors: Kang Inhae , Carr Timothy , Chung Soonkyu
Published: June 2021

High‐sucrose diet causes hepatic steatosis and exacerbates obesity and metabolic complications. Previously, we have demonstrated that ellagic acid (EA), a polyphenol abundantly found in berries, grapes and nuts, inhibits hepatic lipid accumulation in human hepatoma huh7 cells and hyperplastic‐ and hypertrophic‐adipose expansion in human adipocytes. In this study, we hypothesized that EA supplementation inhibits sucrose‐mediated hepatic steatosis and its metabolic consequences. To test this hypothesis, obesity‐prone C57BL/6 male mice were randomly assigned into three isocaloric high‐fat (HF) diets (41% calories from fat) of 1) no‐sucrose (HF), 2) high‐sucrose (37% calories from sucrose, HFHS), or 3) high‐sucrose plus raspberry seed flour equivalent to 0.15% of EA (HFHS‐R). HFHS diet for 8 weeks resulted in a significantly increase in body weight, liver, and adipose tissue mass compared to HF alone, which was normalized by HFHS‐R without altering food intake. Consistently, increases in plasma cholesterol and TG levels in HFHS‐fed mice were markedly dampened by EA supplementation in HFHS‐R. HFHS‐induced hepatic TG accumulation, lipogenic gene expression, and VLDL secretion were also significantly decreased in HFHS‐R comparable to HF alone. Furthermore, systemic levels of glucose and insulin tolerance, and hepatic insulin sensitivity were improved in HFHS‐R group compared to HFHS. Our results implicate that EA supplementation compromises sucrose‐triggered metabolic insults by inhibiting hepatic lipid accumulation. Taken together, our work provides an insight into EA‐containing fruits and vegetables could be developed as promising dietary strategies to prevent sucrose‐triggered obesity and metabolic disorder.
Chronic Activation of Pattern Recognition Receptors Suppresses Brown Adipogenesis of Multipotent Mesodermal Stem Cells and Brown Preadipocytes
Authors: Zhao Ling , Bae Jiyoung , Chen Jiangang
Published: June 2021

Brown adipose tissue (BAT) holds promise to combat obesity through energy‐spending, non‐shivering thermogenesis. Understanding of the regulation of BAT development can lead to novel strategies to increase BAT mass and function for obesity treatment and prevention. Here, we report the effects of chronic activation of pattern recognition receptors on brown adipogenesis of multipotent mesodermal stem C3H10T1/2 cells and immortalized brown preadipocytes derived from classical interscapular BAT of mice. Activation of NOD1, TLR4, or TLR2 by their respective synthetic ligand suppressed brown marker gene expression and lipid accumulation during differentiation of brown‐like adipocytes of C3H10T1/2. Surprisingly, activation of the PRR only during the commitment was sufficient to suppress the differentiation. PRR activation suppressed PGC‐1alpha mRNA, but induced PRDM16 mRNA at the commitment. Consistently, PRR activation suppressed the differentiation of immortalized brown preadipocytes. Activation of PRR induced NF‐κB activation in both cell types, which correlated with their abilities to suppress PPARgamma transactivation, a critical event for brown adipogenesis. Taken together, our results demonstrate that chronic PRR activation suppressed brown adipogenesis of multipotent mesodermal stem cells and brown preadipocytes, possibly through suppression of PPARgamma transactivation. The results suggest that anti‐ inflammatory therapies targeting PRRs may be beneficial for the BAT development.
Combination Treatment with Histone Deacetylase 3 and 6 Selective Inhibitors Attenuates Lipopolysaccharide‐Induced Acute Lung Injury (ALI) in Mice.
Authors: Birmpas Charalambos , Joshi Atul , Barabutis Nektarios , Thangjam Gagan , Gregory Betsy , Catravas John
Published: June 2021

Endothelial barrier dysfunction plays a pivotal role in ALI. We have previously shown that inhibitors of the 90KDa heat shock protein (Hsp90) reduce the magnitude of LPS‐induced ALI in mice. Inhibition of histone deacetylases (HDAC) 3 or 6 suppresses the Hsp90 chaperone function. We now show that selective inhibition of HDAC 3 or 6 has a protective role in LPS‐mediated ALI. In vitro experiments utilized human lung microvascular endothelial cells (HLMVEC). In vivo experiments were performed in C57BL/6 mice. Selective inhibitors of HDAC3 (5µM RGFP966) or HDAC6 (5µM Tubastatin A), alone or combined, decreased total HDAC activity in HLMVEC; furthermore, RGFP966 and Tubastatin attenuated LPS‐induced phosphorylation of the Hsp90 client protein, Akt. Additionally, either RGFP966 or Tubastatin A protected against the LPS‐induced decrease in HLMVEC permeability. In subsequent experiments, mice were injected intraperitoneally with either vehicle or a combination of RGFP966 and Tubastatin (at 10mg/kg, each). 24h later, vehicle or LPS were given intratracheally. 24h after LPS, bronchoalveolar lavage (BAL) was performed. LPS produced a profound inflammatory response, characterized by alveolar cellular infiltration, as reflected in an 8‐fold increase in BAL fluid cellular content. Additionally, LPS increased lung capillary permeability, as indicated by a 6‐fold increase in BAL fluid protein content. Pretreatment with the HDAC inhibitors significantly reduced both the inflammatory and hyper‐permeability response to LPS. These data suggest HDAC3 and HDAC6 as potential therapeutic targets in ALI/ARDS. (Supported by HL101902).
Matrix metalloproteinase‐12 (MMP12) inhibits myofibroblast differentiation and lung fibrosis
Authors: Hu Biao , Wu Zhe , Bai David , Tang Ragan , Phan Sem
Published: June 2021

Matrix metalloproteinase 12 (MMP12) is implicated in pathogenesis of emphysema; however its role in lung fibrosis is controversial. C/EBPβ represses MMP12 expression in lung fibroblasts, while C/EBPβ deficiency attenuates bleomycin (BLM) induced lung fibrosis and myofibroblast (MF) differentiation accompanied with de‐repression of MMP12 expression. In this study, the hypothesis that MMP12 inhibits MF differentiation and accompanying fibrosis was evaluated. Promoter analysis, gel shift assay and real time PCR analysis confirmed that C/EBPβ was a direct repressor of MMP12 gene expression. C/EBPβ deficiency induced MMP12 expression in fibroblasts, which was accompanied by diminished α‐smooth muscle actin (α‐SMA) expression. Moreover ectopic over‐expression of MMP12 suppressed α‐SMA expression. In contrast, MMP12 deficiency or treatment with an MMP12 inhibitor enhanced α‐SMA expression, thus confirming its suppressor role in MF differentiation. A potential mechanism was suggested by the ability of MMP12 to cleave N‐cadherin, which was inhibited by an MMP12 inhibitor; while over‐expression of N‐cadherin stimulated α‐SMA gene expression. Furthermore MMP12 deficient mice exhibited significantly enhanced BLM‐induced pulmonary fibrosis relative to that in wild type mice as evaluated by analysis of collagen deposition, MF differentiation and histopathology. These findings suggest that fibroblast MMP12 is an endogenous repressor of MF differentiation, perhaps by digestion of N‐cadherin, thus playing an important homeostatic role to maintain fibroblasts in an undifferentiated state and keeping fibrosis in check. This work was supported by National Institute of Health grants (HL052285, HL091775, HL112880 and DK020572).
Using Protein Chips to Develop a Highly Specific HER2 Antibody for HER2 Amplification Testing
Authors: Zhou Lixin , Yuan Kehu , Qi Lili , Li Zhongwu , Wu Guiyin , Huang Xiaozheng , Shen Yi , Zhao Min , Fu Wei , Liu Huibo , Chu Boyang , Wang Guangli , Shu Youmin , Ma Donghui , He Weiwu , Chen Jian
Published: June 2021

Human epidermal growth factor receptor 2 (HER2) is an orphan receptor tyrosine kinase member of the EGFR families and is found to be a key tumor driver gene. In breast cancer and gastric cancer, HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, the HER2 immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted to another family member HER4. IHC staining also indicated that it has strong non‐specific cytoplasmic and nucleus staining in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have successfully identified and confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non‐specific binding accounts for the unexpected cytoplasmic and unclear staining of 4B5 on normal gastric epithelium. Finally, we have developed a novel HER2 mouse monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. UMAB36 could provide an alternative high specific IHC reagent for HER2 amplification testing in gastric cancer population.
The Role of Talin‐1 and Kindlin‐3 in Neutrophilic Mac‐1 Activation
Authors: Watts Lauren , Lefort Craig
Published: June 2021

Neutrophils rely on ß2 integrins, including Mac‐1, to mediate the cell to cell adhesions underlying trafficking events. Integrin mediated adhesiveness depends on ligand binding affinity, which is controlled through intracellular signaling pathways that induce conformational changes in the integrin extracellular domain in a process known as “inside‐out” signaling. In related integrins, binding of Talin‐1 and Kindlin‐3 to cytoplasmic integrin tails is required for these structural rearrangements. As Mac‐1 holds unique functions apart from other integrins, particularly in the etiology of autoimmune disease, the further characterization of Mac‐1 activation is key to gaining an understanding of how integrin affinity is regulated and ultimately how affinity changes promote autoimmune disease states. To investigate the role of Talin‐1 and Kindlin‐3, we employ functional assays and flow cytometry based reporter antibody assays to probe Mac‐1 conformation in human cell lines and transgenic mice. Preliminary studies suggest that in response to G‐protein coupled receptor signaling, both Talin‐1 and Kindlin‐3 are required for complete integrin activation. This contrasts with previous data that indicates Kindlin‐3 is dispensable for LFA‐1 ectodomain extension following P‐selectin glycoprotein ligand‐1 ligation, suggesting that the mechanisms of ß2 integrin activation may differ for divergent signaling pathways. Overall, determining a direct role for Talin‐1 and Kindlin‐3 in Mac‐1 activation may provide a clearer picture of the etiology of chronic inflammation while highlighting the specific structural confirmations that can be therapeutically targeted in order to stunt the severity of autoimmune states.
The absence of MuRF1 protects against Calpain1‐induced systolic dysfunction in vivo
Authors: Brown David , Robbins* Jeffrey , Willis Monte
Published: June 2021

Protein degradation to maintain the cardiac contractile machinery is essential to the ongoing performance required of the heart. Multiple enzyme systems have been implicated in this process, including the ubiquitin‐proteasome system and Calpain(Capn) enzymes. In the heart, Capn1 is the primary calcium‐activated cysteine protease that physiologically degrades protein aggregates and sarcomere proteins upon activation. Activation of Capn1 results from pathological injury or stress, such as ischemia reperfusion injury and cardiac hypertrophy/heart failure. Recent evidence supports Capn1 involvement in regulating ubiquitination and subsequent degradation of sarcomeric proteins by an unidentified ubiquitin ligase. Muscle RING‐finger protein‐1(MuRF1) is an ubiquitin ligase expressed in striated muscle with many of the same muscle‐specific substrates as Capn1 (eg. troponin I). We hypothesized both Capn1 and MuRF1 are required for the pathological phenotypes associated with increased Capn1 activity. To test this, we crossed α‐MHC Capn1 transgenic mice (Capn1 Tg+) with MuRF1‐/‐ mice and followed their cardiac phenotype by conscious echocardiography. At 6 months, the Capn1 Tg+//MuRF1+/+ mice exhibited a significant reduction in fractional shortening associated with systolic dysfunction; in contrast, sibling Capn1 Tg+//MuRF1‐/‐ mice were largely protected. Capn1Tg+//MuRF1‐/‐ mice also did not exhibit the increased βMHC and ANF mRNA observed in the Capn1Tg+//MuRF1+/+ mice, which supports the absence of MuRF1 protects against pathological hypertrophy. Taken together, these data support a mechanism by which Capn protease and Murf1 ubiquitin‐proteasome pathways work in concert to degrade cardiac proteins in Capn1‐induced heart failure.
Bag3+ P209L Transgene Provides a Cardiac‐Specific Murine Model of Protein Misfolding and Aggregation
Authors: Quintana Megan , Yates Cecelia , Takayama Shinichi , Willis Monte
Published: June 2021

Bag3 is a heat shock protein expressed in skeletal muscle and cardiomyocytes that regulates proteasomal and lysosomal protein elimination pathways. Bag3, in association with Hsp70 and HspB8, assists in chaperone‐assisted selective autophagy. Several mutations in Bag3, including the P209L mutation, results in myofibrillar myopathy, a childhood‐onset disease with protein aggregation. The P209L mutation has been suggested to result in a toxic gain of function and Bag3 insufficiency. To test the role of P209L in the heart, we created a transgenic mouse with cardiac specific (α‐MHC) Bag3P209L. Longitudinal conscious echocardiography was performed over 12 months on mice, with histological analysis of apoptosis, protein aggregation, and fibroblast analysis performed upon harvest. In 8 months, Bag3 P209L Tg mice had significantly depressed fractional shortening compared to sibling wildtype control mice (FS% 38.9±1.0% vs. 48.1±0.95%, p=0.0005). However, no differences in apoptosis nor fibroblast number was identified at 12 months. No differences in amyloid protein aggregation were identified at 12 months (26.8±0.9 AU vs. 30.0±6.6 AU, p=0.76). However, analysis of cardiac tissue for pre‐amyloid oligomers will allow the more clinically relevant detection of proteotoxic intermediates that can lead to cytotoxicity, as is found in neurodegenerative diseases and other causes of heart failure. Since Bag3 is required for autophagosome formation during CASA, future studies will focus on pathological changes in cardiac autophagic flux and its related MAPK signaling. This novel Bag3 transgenic mouse model provides a useful model to characterize cardiac‐specific dysfunction we will use to identify therapeutic targets that protect against the development of cardiomyopathy.
Analyzing Macromolecular Complexes in Situ Using Cellular Cryo‐Electron Microscopy
Authors: Nicastro Daniela
Published: June 2021

Cryo‐electron tomography (cryo‐ET) is a powerful technique for imaging biological structures in their native state and in an unperturbed cellular environment. We integrate high resolution imaging by either cryo‐ET and sub‐tomogram averaging or TYGRESS (Tomography‐Guided 3D Reconstruction of Subcellular Structures), with comparative genetics, biochemical methods and EM‐visible labeling to deconstruct the in situ 3D structure and functional organization of macromolecular complexes. We use cilia and flagella as model systems to advance techniques and approaches for high‐resolution imaging of complex cellular structures. Cilia and flagella are ubiquitous organelles with important biological roles in motility and sensation; defects in their assembly or function cause severe human diseases. The normal beating of cilia requires the precise coordination of thousands of dynein motors, but much remains to be learned about dynein's motility and regulation. We are in the process of generating a molecular blueprint of major ciliary complexes to gain a better understanding of the inner workings of these intriguing nano‐machines.
Recessive Forms of Osteogenesis Imperfecta (OI) and Implications for Mechanisms of Disease
Authors: Byers Peter
Published: June 2021

More than 90% of people with OI are heterozygous for mutations in one of the type I collagen genes (COL1A1 and COL1A2). The mutations affect the production and structure of type I procollagen. In the last decade, studies of animal models, analysis of candidate genes, and whole exome analysis identified 11 genes in which biallelic mutations result in recessively inherited forms of OI. The genes affect 1) collagen folding and processing (CRTAP, LEPRE1, PPIB, SERPINH1, PLOD2, FKBP10 and BMP1), 2) calcium homeostasis in the RER (TMEM38B), 3) recruitment of cells to the osteoblastic pathway (SP7, WNT1), and 4) response to accumulation of abnormal proteins (CREB3L1). The clinical outcome of these mutations ranges from lethal in the perinatal period to a moderate severity and clinical signs are often difficult to distinguish from those encountered in individuals with mutations in type I collagen genes. While it is clear that the list of genes that harbor causative mutations that result in OI is incomplete, it has been thought that the diverse nature of the newly recognized recessive OI genes would provide insights into common pathways from genotype to phenotype. The most recent to emerge is the concept of disturbed TGFβ‐signaling as a consequence of accumulation of abnormal molecules in the matrix, stress in the secretory pathway, or both (see Grafe et al, Nat Med 20:670, 2014). Thus the possibility of a common, potentially treatable, aberrant pathway is an, at last hopeful, sign.
Intestinal epithelial ecto‐5′‐nucleotidase CD73 regulates the homeostasis of Salmonella typhimurium and commensal bacteria
Authors: Kao Daniel , Saeedi Bejan , Schwisow Kayla , Colgan Sean , Kominsky Douglas
Published: June 2021

Purine metabolism is central to the virulence of some enteric pathogens. CD73 is an ecto‐5′‐nucleotidase expressed abundantly on the apical surface of intestinal epithelial cells (IEC) and is rate limiting in the generation of extracellular adenosine. Previous work has shown that epithelial expression of CD73 facilitates transepithelial bacterial translocation and dissemination of Salmonella typhimurium. Based on these results, we hypothesized that apical expression of CD73 by IECs induces an invasive phenotype of Salmonella and the absence of CD73 expression results in a proliferative phenotype. Using mice with IEC‐specific deletion of CD73, we show that in a Salmonella colitis model, the abundance of Salmonella increases nearly 100‐fold within the contents of the cecum and colon of knockout animals in comparison to control animals. Furthermore, we show that adenosine inhibits growth of Salmonella and alters expression of some Salmonella pathogenicity islands in vitro. These data suggest that adenosine may directly impact Salmonella virulence and may occur through induction of an invasive rather than proliferative phenotype. The influence of CD73 on gut microbes does not appear to be specific to Salmonella. The microbiota of animals with IEC‐specific knockout of CD73 was analyzed by 16S rRNA pyrosequencing and differs dramatically from control animals. In conclusion, these findings show that expression of CD73 promotes virulence of the enteric pathogen Salmonella typhimurium through the production of luminal adenosine and is an important regulator of homeostasis of commensal bacteria.
Extracellular RNA Induces Complement Factor B in Macrophages via MyD88
Authors: Zou Lin , Xu Ganqiong , Feng Yan , Chao Wei
Published: June 2021

Objective

We have recently found that complement factor B (cfB) contributes to cardiac dysfunction and mortality in a mouse model of polymicrobial sepsis. However, the upstream signaling that leads to cfB production is still poorly known. During sepsis, RNAs are released into extracellular space from host necrotic cells and from pathogens. However, the biological function of the extracellular RNAs (exRNAs) during sepsis remains largely unknown. We hypothesize that exRNAs induce cfB production through Toll‐like receptors (TLRs) signaling pathway.

Methods

Polymicrobial sepsis was created by cecum ligation and puncture (CLP). Three mg RNase was injected i.p. 1 h before and 12 h after surgery. Complement mRNA was determined by qRT‐PCR. RNA was extracted using Trizol. Bone marrow‐derived macrophages (MΦ) from WT or MyD88‐/‐ mice were stimulated with 10 μg/ml of RNA in the presence of lipofectamine for 24 h. Medium cfB was analyzed by Western blot.

Results

In vivo, 24 h after surgery, CLP led to a significant cfB up‐regulation in the heart but had no impact on other complements tested such as C3, C4, C5. RNase administration led to a 46% reduction, suggesting that RNA may play a role in cardiac cfB production during sepsis. In vitro, there was a significant increase in cfB protein in the medium collected from WT MΦ cultures that had been treated with 10 μg/ml of spleen‐derived RNA for 24 h. Deficiency in MyD88, the adaptor for TLR7/8 (the sensor for single‐stranded RNA), almost completely blocked the cfB increase induced by exRNA.

Conclusion

We demonstrate that polymicrobial infection leads to specific cardiac cfB gene expression and RNase partially reduces the increase. In vitro, exRNA induces cfB expression in MΦ through a MyD88‐dependent mechanism.
Mitochondrial‐Targeted Antioxidant Treatment in a Mouse Model of Non‐Alcoholic Fatty Liver Disease
Authors: Thompson Michael , Cismowski Mary , Bid Hemant , Lucchesi Pamela
Published: June 2021

As obesity has become a more common problem in childhood, the prevalence of non‐alcoholic fatty liver disease (NAFLD) among children has increased. Approximately 70‐80% of obese children have some degree of NAFLD, a precursor for liver failure and/or hepatocellular carcinoma as well as an independent risk factor for cardiovascular disease. While the molecular mechanisms of this disease are beginning to be elucidated, there is no standard pharmacologic therapy. Oxidative stress is a significant factor in disease progression, thus antioxidant therapy has potential utility. In the current study, 10 week old normal (Db/db) and obese diabetic mice with NAFLD (db/db) were treated for six weeks with vehicle or Mitotempol, a mitochondrial‐targeted antioxidant. The effect on oxidative stress, inflammation and metabolism in liver was evaluated through measurement of gene (RT‐PCR) and protein expression (immunoblots), along with hepatic triglyceride levels. Enzymes involved in oxidative stress were not affected by treatment. However, lipocalin‐2, a marker of oxidative stress and inflammation, was significantly decreased following treatment. We also observed a decrease in levels of NFκB and caspase‐3. While there was no change in hepatic triglyceride levels or expression of seven genes involved in fatty acid metabolism, an increase in expression of 4 genes involved in glucose metabolism/transport occurred following Mitotempol treatment. The current study shows that treatment with Mitotempol does not affect hepatic lipid metabolism, but does decrease markers of oxidative stress and inflammation. Mitotempol may be effective in targeting oxidative stress in fatty liver and warrants further evaluation as a potential therapy.
Integrated Computational Model of Intracellular Signaling and microRNA Regulation Predicts the Molecular Components and Timing Critical to Hepatic Stellate Cell Activation
Authors: Parrish Austin , Kuttippurathu Lakshmi , Vadigepalli Rajanikanth
Published: June 2021

Activated hepatic stellate cells (HSCs) play a major role in the development of liver fibrosis. It has been shown that autoregulatory feedback loops between pro‐fibrogenic pathways and microRNAs influence the fate of HSCs. However, the relative contribution of these pathways and regulatory microRNAs to the activation process is unknown. We pursued a computational modeling approach using a Petri net formalism to explore the timing and regulatory balances that are critical to HSC activation. We developed an integrated model incorporating three signaling pathways (NF‐κB, STAT3 and TGF‐β) and two microRNAs (miR‐146a, miR‐21) that are differentially regulated by these pathways. Simulations demonstrated that TGF‐β‐mediated regulation of microRNAs is critical to drive the HSC phenotypic switch from quiescence to activation, whereas NF‐κB and STAT3 alone were unable to induce this switch. Our results show that the relative timing between cytokine stimuli in HSCs alters initial microRNA expression dynamics. Earlier NF‐κB activation resulted in an initial transient increase of both microRNA levels, whereas later activation of NF‐κB versus STAT3 resulted in an initial monotonic decrease in miR‐146a while miR‐21 gradually increased to steady state levels. We observed re‐quiescence from the activated HSC state upon termination of cytokine stimuli. Our model provides a new computational platform for studying HSC activation and the mechanisms where microRNAs act as fine‐tuning markers modulating the system behavior, thereby informing potential therapeutic approaches. Funding: NIAAA R01 AA018873, R21 AA022417, T32 AA007463.
Different Pattern of ATP‐mediated Increases in [Ca2+]cyt Contributes to ATP‐induced Increase in Bcl‐2/Bax Ratio in Lung Cancer Cells But Not in Normal Control Cells
Authors: Song Shanshan , Jacobson Krista , McDermott Kimberly , Makino Ayako , Yuan Jason
Published: June 2021

ATP (extracellular messenger) is in much higher concentration in tumor microenvironment. It regulates cell functions through acting on purinergic receptors to activate a series of intracellular signal transduction pathways, including Ca2+ signaling pathway. Here we report that ATP caused a long‐duration Ca2+ influx in lung cancer cells compared to normal control cells. Ionotropic (P2X3, P2X4 and P2X5) and metabotropic (P2Y2, P2Y4 and P2Y6) receptors were expressed in much higher lever in lung cancer cells than in normal control cells. UTP (P2Y receptor agonist) induced a transient increase in [Ca2+]cyt without obvious Ca2+ influx in lung cancer cells. Activation of P2X receptor with α,β‐meATP and D‐β,γ‐meATP caused Ca2+ influx in lung cancer cells. PLC inhibitor U73122 inhibited Ca2+ transient, while P2X receptor inhibitor TNP‐ATP significantly attenuated Ca2+ influx. Store‐operated Ca2+ entry was not significantly enhanced in lung cancer cells. ATP also increased Bcl‐2/Bax ratio in lung cancer cells, but not in normal control cells. BAPTA‐AM abolished and significantly inhibited ATP‐mediated increase in [Ca2+]cyt and Bcl‐2/Bax ratio in lung cancer cells. The ATP‐mediated increase in Bcl‐2/Bax ratio was also significantly attenuated by removing extracellular Ca2+. These data indicate that upregulated expression of P2X3, P2X4 and P2X5 receptors in lung cancer cells leads to a long‐duration Ca2+ influx induced by ATP that subsequently causes a sustained increase in Bcl‐2/Bax ratio and eventually a significant increase in cell viability or cell proliferation.
Implementing Clinical Scenarios in an Undergraduate Physiology Course to Enhance Critical Thinking Across Multiple Body Systems
Authors: Shaw Jennifer
Published: June 2021

Many medical schools have adopted a clinical problem‐based learning approach for 1st year students. It is essential that undergraduate institutions prepare pre‐health students for this shifting trend in medical education. Undergraduates at OSU are rarely required to apply their knowledge of physiology in the context of a patient exhibiting symptoms involving interplay between multiple body systems. Therefore, the objective of this project was to measure student performance in a senior level physiology course on critical thinking questions covering clinical scenarios involving concepts across multiple body systems taught in lecture. The first assignment provided a baseline measurement of reiterative versus critical thinking skills using a closed note assignment on a clinical scenario covering knowledge acquired in a pre‐requisite Human Physiology course. Half of the questions in the scenario required a reiteration of the lecture notes. The other half of the questions required students to critically ascertain and apply the physiological concepts at work within the scenario. Thereafter, students (n = 54 in 2013; n = 41 in 2014) were tested on six clinical scenarios during the semester. We hypothesized that by the end of the semester students would improve in applying physiological concepts to clinical scenarios. Overall, students performed better on reiterative questions compared to critical thinking questions, although by the end of each semester students increased their performance from an average of 52.9% (+/‐3.6%) to 71.1% (+/‐3%) correct answers on critical thinking questions. We conclude that this approach increases students' critical thinking abilities when faced with clinical scenarios thereby improving student preparation for medical school curricula.
Transformation Of The Introductory Biology Experience For All Students: Progress Of The Northwest Biosciences Consortium
Authors: Kruchten Anne , Baumgartner Erin , Brown Jeffrey , Duncan Jason , Gaiser Chris , Kayes Lori , Kiser Stacey , Shriner Walter , Stavrianeas Stasinos
Published: June 2021

The Northwest Biosciences Consortium is a group of two‐year and four‐year college faculty at public and private institutions in Oregon. Funded by an NSF RCN‐UBE grant, this group is working towards creating modern, student‐centered, integrated, and investigative introductory biology experiences for all students aligned with Vision and Change: A Call to Action. In our second year of the grant we report on the activities of this group including 1) data about the diversity of enrollments and delivery methods in courses at our institutions; 2) insights arising from observations of each others' courses and discussions with faculty; 3) observations of how our courses align with Vision and Change and how they facilitate student transition towards upper division biology courses; 4) obstacles we have encountered especially in the area of smoothing the transition for transfer students taking an introductory sequence at a two year college followed by advanced coursework in biology at a four year college; and 5) plans for professional development activities for faculty to encourage the adoption of student‐centered pedagogies. As a long‐term goal, we plan to help faculty develop scaleable student‐centered pedagogies and assessment documents that will encourage consistency in introductory biology outcomes across our diverse two‐year and four‐year institutions. Supported by the NSF RCN‐UBE grant (DBI‐1248121).