bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Hepatic PLIN2 and PLIN4 in The Development of Western Type Diet Induced Hepatosteatosis
Authors: Griffin John D , Salter Deanna M , Bowman Thomas , Greenberg Andrew 
Published: November 2023

Nonalcoholic fatty liver disease (NAFLD) is characterized by the excessive accumulation of triglyceride (TAG) in the liver. In hepatocytes TAG is stored intracellularly within cytoplasmic lipid droplets (LD). Intracellular LD are coated with various LD associated proteins in the perilipin (PLIN) and FSP27 family. These LD associated proteins are thought to promote LD formation by regulating the hydrolysis of stored TAG. Hepatocyte‐specific knockout of the LD‐associated protein perilipin 2 (PLIN2) protects mice from developing NAFLD and associated inflammation when fed a methionine‐choline deficient diet for 15 days, indicating an essential role for PLIN2 in the development of fatty liver disease. However, the protective effects of hepatocyte‐specific knockout of PLIN2 on hepatic steatosis in mice fed a high fat, western diet (WTD) are unknown. To determine the function of hepatic PLIN2 in the development of WTD‐induced liver steatosis, 16 week old hepatocyte‐specific PLIN2 knockout mice (PLIN2Alb‐Cre) and floxed controls (PLIN2fl/fl) were fed either a WTD previously shown to induce significant hepatosteatosis, or a low fat control diet (LFD). 20 weeks of WTD (44% fat, 39% carbohydrate, 34% sucrose by weight) feeding significantly increased weight gain (14.9g WTD vs. 1.6g LFD, p<0.001), reduced insulin sensitivity as measured by intraperitoneal (IP) insulin tolerance test (AUC increased 56% vs LFD, p<0.05), impaired glucose tolerance as measured by IP glucose tolerance test (AUC increased 59% vs LFD p<0.05) and increased accumulation of hepatic TAG (10.6ug/mg WTD vs 67.3ug/mg LFD, p<0.01). Surprisingly, the absence of hepatocyte PLIN2 expression did not prevent liver steatosis in mice fed a WTD. Interestingly, hepatocyte‐specific knockout of PLIN2 reduced the accumulation of liver triglycerides following LFD feeding (3.3ug/mg PLIN2Alb‐Cre vs. 8.1ug/mg PLIN2fl/fl). To determine whether other LD‐associated proteins may compensate for the absence of PLIN2 we measured the expression of PLIN1, PLIN3, PLIN4, and FSP27. PLIN4 was the only LD‐associated protein affected by either diet or genotype, significantly upregulated by WTD feeding in both lines of mice (increased 4.1 fold vs. LFD). At the present time there are no studies on the role of PLIN4 in the development of hepatic steatosis. We hypothesize that during LFD feeding PLIN2 is necessary for hepatic TAG storage. However, during WTD feeding we hypothesize that PLIN4 is a critical determinant of liver TAG accumulation. Future experiments using newly developed anti‐sense oligonucleotides directed against PLIN4 are underway to determine the relative contribution of PLIN4 to hepatic TAG storage during WTD feeding.Support or Funding InformationWork supported by National Institutes of Health Grant T32DK062032
Chikungunya Virus Infection Alters New Bone Formation Associated with Pre‐Existing Post‐Traumatic Osteoarthritis
Authors: McNulty Margaret A. , Goupil Brad A. , Moses Amber , Mores Christopher N. 
Published: November 2023

Chikungunya virus (CHIKV) is a mosquito‐borne alphavirus found throughout tropical and subtropical regions and has recently experienced an extensive outbreak throughout Central and South America, resulting in almost 2 million reported suspected cases. CHIKV infection results in severe polyarthralgia 1–3 weeks following infection, which may persist months to years following initial clinical symptoms. Previous work by the authors has identified novel skeletal and joint lesions associated with CHIKV infection. Reports have suggested that people with pre‐existing joint disease, such as osteoarthritis (OA), may be prone to developing more severe and prolonged arthralgia associated with CHIKV infection, though this has not been experimentally examined. Therefore, the goal of this study was to determine if CHIKV infection resulted in more rapid progression of pre‐existing OA in an experimental mouse model. Eleven‐week‐old female C57/Bl6J mice underwent destabilization of the medial meniscus (DMM), including a group that did not undergo surgery (CTRL). One week following surgery, mice were intra‐articularly inoculated with CHIKV or SHAM. Intact stifle joints were evaluated with micro‐computed tomography (μCT) for boney changes and histopathology to evaluate OA severity. ANOVAs were performed on μCT and histomorphometric data, Kruskal‐Wallis tests were performed on semiquantitative data. No significant differences were identified in articular cartilage evaluations secondary to CHIKV, however the DMM surgery did significantly increase cartilage damage when compared to CTRL joints. A significant increase in subchondral bone volume in the DMM+SHAM compared to both the DMM+CHIKV and CTRL+CHIKV (p=0.011 and p<0.001, respectively) occurred. DMM+CHIKV and CTRL+CHIKV had significantly smaller medial periarticular osteophytes when compared to DMM+SHAM (p=0.043 and p<0.001, respectively), and CTRL+CHIKV had significantly smaller medial periarticular osteophytes than DMM+CHIKV (p<0.001). Concurrent CHIKV infection with DMM surgery did not significantly impact articular cartilage degradation. However, CHIKV significantly reduced the size of the periarticular osteophytes that normally develop secondary to the DMM surgery. This suggests that CHIKV may have an inhibitory effect on osteophyte bone formation. In addition, CHIKV infection diminishes the amount of subchondral bone sclerosis that normally develops secondary to the DMM surgery. Therefore, these results indicate that CHIKV infection may have a suppressive effect on bone formation that is associated with compensatory changes during the progression of OA.Support or Funding InformationNIH Health Centers of Biomedical Research Excellence Phase III Award 1P30GM110760‐01, NIH T32 Training Grant 5T32oD011124‐09
Dlk1 Regulates Type II to Type I Cell Transition During Alveolar Repair Through Inhibition of Notch Signaling
Authors: Finn Johanna , Pajcini Kostandin , Liu Yuru 
Published: November 2023

Lung alveoli are lined with two types of epithelial cells: Type I cells and Type II cells. Type I cells are thin squamous cells covering ~95% of the surface area which are responsible for blood‐gas exchange. Type II cells have multiple functions including secretion of surfactant and are also able to act as progenitor cells, proliferating and subsequently converting to Type I cells after lung injury to restore the alveolar epithelial barrier. However, the signaling events responsible for inducing and regulation the Type II cell mediated repair remain poorly understood. Our data demonstrated that the non‐canonical Notch ligand Dlk1 (delta‐like 1 homolog) regulates Type II to Type I cell transition through regulation of Notch signaling. Through in vitro culture as well as by utilizing a mouse model of acute lung injury induced by Pseudomonas aeruginosa (PA) infection, we found a dynamic change in Dlk1 expression and Notch activity correlated with Type II to Type I cell transition. In a mouse model in which Dlk1 was specifically disrupted in Type II cells, we found that the mutant cells were unable to differentiate into Type I cells; instead, there is an accumulation of Type II cells expressing low levels of both Type II and Type I cell markers (Sp‐ClowT1αlow) representing an intermediate cell type in between the Type II to Type I cell transition. Concurrently, Notch activity appeared to be abnormally elevated in these intermediate cells appearing during repair. Thus, we conclude that Dlk1 is required for the Type II to Type I cell transition during alveolar repair, and Dlk1 functions by inhibiting Notch signaling in a population of intermediate cells in between the transition of Type II to Type I cells to allow their full conversion into Type I cells. Further understanding of the involvement of Notch signaling and its regulation in the conversion process after Acute Lung Injury could help lead to novel clinical interventions in the treatment of Acute Lung Injury and Acute Respiratory Distress Syndrome.Support or Funding InformationNIH National Heart, Lung, and Blood Institute Training Grant in Lung Biology and Pathobiology (5T32HL007829) and R01HL105947
Determination of the Residues Necessary for the Self‐Interaction of Atg11, a Central Organizer of the Selective Autophagy Initiation Complex
Authors: Spearin Zach , Cawthon Hayley , Smith Jesse , Backues Steven K 
Published: November 2023

Selective autophagy is a process used by eukaryotes to rid the cell of potentially harmful intracellular content such as damaged mitochondria and toxic protein aggregates. The cell forms a double membrane known as an autophagosome around these targets and transports them to the vacuole/lysosome to be degraded. This process helps to maintain the health of the cell and protects against diseases including Parkinson's and Alzheimer's. Our research uses budding yeast to investigate the basic mechanisms by which autophagy related (Atg) proteins control this process. Atg11, a protein essential for selective autophagy, interacts with other proteins using its second and third coiled‐coil (CC2–3) regions and organizes them into an autophagy initiation complex. These interacting proteins include Atg9, Atg20, Atg29, Atg1, Ypt1, and itself. However, it is not known how these partners are organized ‐ for example, are all of these proteins binding at the same time, or do they compete for binding to the same location on Atg11? Moreover, the role of Atg11's self‐interaction in this process is not known. The goal of this research is to determine which amino acid residues of Atg11 are required for its self‐interaction using a random mutagenesis/yeast two hybrid screen. This will help us better understand the significance of Atg11's self‐interaction in the initiation of selective autophagy.Support or Funding InformationNSF RUI grant #1613653 ““Scaffold or Assembly Line: How Does Atg11 Organize its Binding Partners for the Initiation of Selective Autophagy?”, S. K. Backues (PI)
Influenza Hemagglutinin Fusion Domain by Advanced NMR using Novel Orthagonal Refinement, BICS Curvature Measurements and Native Lipid Environments
Authors: Smrt Sean Thomas 
Published: November 2023

The influenza fusion peptide domain (HAfp23) is comprised of the N‐terminal 23 residues of the hemagglutinin glycoprotein which are highly conserved and have been shown to be essential for membrane fusion and the infection process of the Influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We reveal that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk.Our investigation of the HAfp23 system led to our development of novel NMR techniques including Bicelle Induced Curvature and Sorting (BICS) and application of a new positively charged alignment media (Pinacyanol Acetate) for structure refinement. These methods were applied to the study of HAfp23 in combination with well‐established NMR techniques, differential scanning calorimetry (DSC) and fluorescent lipid mixing assays. Furthermore, we have developed an NMR based approach to study HAfp23 in a more native lipid environment which we anticipate to be highly useful in the study of membrane proteins. The elucidations made on HAfp23 as well as the application of novel techniques to study membrane proteins are of high interest to the biophysical communities.Support or Funding InformationThis work was supported by the Department of Chemistry at the University of Illinois at Chicago (UIC) and a UIC Campus Research Board grant. Portions of this work were carried out in the Center for Structural Biology via the Research Resources Center at UIC.
THE STRUCTURAL BASIS FOR POLYPEPTIDE TRANSLOCATION BY THE HSP104 DISAGGREGASE
Authors: Southworth Daniel R , Yokom Adam , Gates Stephanie , Jackerel Meredith , Shorter James 
Published: November 2023

Heat shock protein (Hsp) 104, found in yeast, is a member of the Hsp100 class of molecular chaperones that contain highly conserved AAA+ (ATPases Associated with diverse cellular Activities) domains and serve critical roles in thermotolerance and protein quality control. Hsp104, and the bacterial homolog ClpB, form large hexameric‐ring structures that cooperate with the Hsp70 system to unfold and rescue aggregated protein states by active translocation of polypeptide substrates through a central channel. Hsp104 controls prion inheritance in yeast by recognizing and remodeling cross‐β structures of amyloid fibrils, such as those found in Sup35 prions. This function has led to studies identifying potentiated Hsp104 variants that reduce the toxicity of proteins linked to neurodegenerative diseases. Despite fundamental roles in protein quality control and promising therapeutic activity in rescuing amyloidogenic states, how Hsp104 and its family members function as powerful molecular motors to solubilize proteins is not fully understood. Furthermore, obtaining high‐resolution views of active complexes has been a significant challenge, thus a definitive structural mechanism for disaggregation has remained elusive. Here we have determined the cryo‐EM structure of wild type Hsp104 in the ATP state to 5.6 Å resolution. This work reveals an unprecedented two‐turn spiral architecture for the hexamer, identifying how the twelve AAA+ domains are coordinated for substrate translocation. The N‐terminal, C‐terminal and middle domains are observed in distinct conformations that explain functions in substrate engagement and ATP hydrolysis control. Remarkably, density for the substrate‐binding pore loops is identified to line the channel in a continuous arrangement that appears optimized for substrate transfer across the AAA+ domains, establishing a directional path for polypeptide translocation.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) grant R01GM099836 (to J.S.). A.L.Y. is supported by an American Heart Association Predoctoral fellowship; M.E.J. is supported by a Target ALS Springboard Fellowship. J.S. is supported by a Muscular Dystrophy Association Research Award (MDA277268), the Life Extension Foundation, the Packard Center for ALS Research at Johns Hopkins University, and Target ALS. D.R.S. is supported by NIH grants R01GM109896, R01GM077430 and R01GM110001A.
Inorganic Polyphosphate: A Mediator of Protein Folding in Osteoblasts via Interaction with Cyclophilin B
Authors: Khong Mei Li , Li Lina , Lang Charlie Yen‐Po , Tanner Julian Alexander 
Published: November 2023

Inorganic polyphosphate (polyP), a structurally simple polymer of phosphates linked by high‐energy bonds found in ATP, is fundamental in all cells. Far from being molecules with a simple function, evidence is emerging that polyP critically influences a diverse set of key processes in mammalian cells. However, many unanswered questions remain regarding polyP's mechanism of action in higher eukaryotes and progress in this study is hampered by the lack of tools to analyse polyP function. Using biochemical techniques, we aimed to identify significant polyP‐protein interactions to understand polyP's function in osteoblasts. An affinity chromatography approach, developed by cross‐linking polyP to biotin and immobilising polyP via biotin‐streptavidin interactions, identified polyP‐specific binding proteins in SaOS‐2 osteoblast‐like cells. We report evidence that cyclophilin B (CypB), a chaperone involved in protein folding and secretion of folded proteins to the extracellular matrix, specifically interacts with polyP at high affinity. In one line of evidence, we used a highly‐selective benzimidazolinium dye for long‐chain polyP and observed co‐localisation of polyP and CypB in the endoplasmic reticulum and secretory vesicles of SaOS‐2 cells via confocal microscopy. In another line of evidence, independent binding experiments showed that polyP binds tightly to purified, recombinant human CypB. Biochemical assays using purified CypB further revealed that long‐chain polyP strongly inhibits the peptidyl‐prolyl cis‐trans isomerase activity of CypB. This study provides mechanistic insight into how polyP affects protein folding mediated by CypB function in osteoblasts.Support or Funding InformationThis research was funded by RGC GRF HKU 778312M.
Understanding the Allosteric Control of Kinase Activation by Phosphorylation‐Regulated Protein Dynamics
Authors: Iverson Dylan Bush , Ahn Natalie 
Published: November 2023

The MAP kinase, extracellular signal‐regulated kinase‐2 (ERK2), is activated dual phosphorylation at pThr and pTyr residues located at its activation loop. NMR (13C,1H) multiple‐quantum Carr‐Purcell‐Meiboom‐Gill (CPMG) relaxation dispersion performed on our lab have shown that ERK2 activation is accompanied by a change in conformational dynamics to enable global motions which can be described as an equilibrium between two conformers. In its inactive, unphosphorylated state (0P), ERK2 remains locked in a single conformer (named “L”) whose methyl groups show fast, uncorrelated local motions. Upon activation by dual phosphorylation (2P), ERK2 undergoes a slow (300 s−1) dynamic equilibrium between two states, including “L” and a new conformer named “R” (released). Ligand binding can shift the equilibrium between L‐R conformers, so that high‐affinity small molecule inhibitors of ERK1/2 differentially stabilize the R and L states in the active kinase. Small molecules may be able to exploit the dynamics of 2P‐ERK2, by selecting for a conformer that confers higher affinity. The goal of this study is to understand the structural basis for the differences between conformers. Comparison of X‐ray structures of 2P‐ERK2 against 0P‐ERK2 shows that the only region undergoing obvious structural rearrangements (>2Å) upon phosphorylation is at the activation loop. Thus, one interpretation of the NMR relaxation results is that the activation loop conformation in 0P‐ERK2 represents L, while the conformation in 2P‐ERK2 represents R. In order to test this, ongoing efforts are to identify the methyls associated with activation loop residues, and examine their dynamics by NMR CPMG relaxation dispersion. If the two‐state populations (pA, pB) and exchange rate constants (kex) match those of the global L‐R exchange, then the loop movement might explain the global motion. But if these parameters differ, then the conformational difference between R and L is elsewhere, and are not obvious in the X‐ray structures. In fact it seems unlikely that activation loop movement accounts for L‐R, given that the ion pair interactions formed with pThr and pTyr residues would need to undergo significant destabilization in order to allow conformational exchange. Instead, the difference between R and L more likely lies within the ATP binding site. 2D TROSY NMR experiments show differences in chemical shift perturbations upon binding AMP‐PNP to 2P‐ERK2 (corresponding mostly to R), compared to 0P‐ERK2 (L). The largest differences occur at Ile72 and Leu143, both located nearby the conserved DFG motif, which coordinates Mg2+‐ATP. The chemical shift perturbations only appear in 2P‐ERK2, suggesting a nucleotide binding mode which forms only in the activated and ligand‐bound state. Therefore, it is likely that very small changes in conformation may account for the change in conformational dynamics observed upon activation.Support or Funding InformationR01 supported project
Inorganic Arsenic Bioaccessibility/Bioavailability from Cooked Rice Using In Vitro Digestion/Caco‐2 Cell Model
Authors: Lee Kwang‐Won , Lee Seul Gi 
Published: November 2023

Rice is one of the major sources for exposing inorganic arsenic (As) in food. Using in vitro digestion/Caco‐2 cell model, the bioaccessibility and bioavailability of As from cooked rice were studied. Inorganic arsenic, especially AsIII, was predominant and bioaccessible species in cooked rice. The percentage of bioaccessibility of total As in white rice (75%) was slightly higher (p=0.061) than brown rice (66%). However, there was no different of bioaccessibility of iAs between white rice (95%) and brown rice (96%). In Caco‐2 cell monolayer, As uptake considering sum of retention and transport was determined to be 16 to 38 %. As bioavailabilities in cooked white and brown rice were 31 % and 21 %, respectively indicating that the bioavailability of As from cooked rice can be affected by matrix environment. The in vitro digestion/Caco‐2 cell model may provide an important information on risk assessment of As in rice.Support or Funding InformationThis research was supported by School of Life Sciences & Biotechnology of Korea University for BK21PLUS.
Simple Model for Screening Anti‐Cell‐Clumping Reagents: Acids
Authors: Zem Gregory , Pastrano K. , Strelnicova A. , Cabrera M.L. , Cuevas J. , Espinoza L. , Voskanian C. , Keshishian R. , Sorayazadeh S. , Alvarado B. , Pizana S. , Weisman‐McCarley A. , DeGuzman K. , Pourkhayat A. , Ellikkal K. , Darmali A. , oppenheimer steven 
Published: November 2023

Clumped cells can be more dangerous than unclumped cells in thrombocytic events, cancer cell survival and infectivity. In this study 3 acids (acetic, benzoic and citric) were tested for their effects on cell unclumping using a fixed yeast assay. Fixed yeast display similar cell surface properties as live cells (Acta Histochemica 104, 2002, 99–106). Matched approximately 0.25 ml drops of Prefer (Anatech Ltd, Battle Creek, MI) fixed yeast (Saccharomyces cerevisae) in deionized water (95% ethanol for benzoic acid and its controls) with and without reagent were microscopically examined on glass microscope slides with stirring over a 60 min time course. Samples were assessed every 20 min for percent single cells and graphically and statistically evaluated. In about 2600 total trials acetic acid unclumped cells in a majority of the trials while citric and benzoic acid unclumped cells in about 20% of the trials. In a sample set of 30 acetic acid trials, counting several thousand single cells and clumps, the percent increase in single cells over controls at 60 min was concentration dependent with 17% increase with 1% acetic acid, 24% with 2% acetic acid and 33% with 3% acetic acid. Standard errors for experimentals ranged from +/− 2–5%. P values comparing controls and experimentals were: for 1% acetic acid, 20 min 0.08, 40 min 0.04, 60 min 0.07. For 2% acetic acid, 20 min 0.01, 40 min 0.00015, 60 min 0.002. For 3% acetic acid, 20 min 0.002, 40 min 0.004, 60 min 0.00006. The validity of this kinetic assay as potentially useful for human applications was suggested by comparison of fixed yeast results with a known human cell unclumping reagent (FASEB J 28, 833.1, 2014).Support or Funding InformationSupport: university funds from grants and donations: including NSF U.S. presidential award 0731633, NIH grant awards and Sidney Stern Memorial Trust (to S. Oppenheimer).