bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and Validation of a Culturally Appropriate Heart Disease Prevention Fotonovela among Spanish Speaking Low‐income Latinos
Authors: Segura‐Pérez Sofia , Damio Grace , Galdámez Gilma , Pérez‐Escamilla Rafael
Published: November 2023

Fotonovelas, booklet versions of soap operas that use pictures to tell dramatic stories, have been a very popular mass entertainment medium in Latin America. Fotonovelas have recently been used as an educational tool among Latinos. The main objective of this project is to describe the development and evaluation of a culturally appropriate heart disease prevention fotonovela based on the American Heart Association “The Simple Seven Steps to Prevent Heart Disease” targeting Spanish speaking Latinos. These steps promote controlling blood pressure, blood cholesterol, blood sugar, and weight as well as getting active, eating better, and stopping smoking. Mixed‐methods community‐based participatory research was used for development and evaluating the fotonovela. To be included, participant's had to be ≥18 years old, Latinos, residents of Hartford and; able to read and write Spanish. The formative qualitative research consisted of three phases. First, two focus groups were conducted with 22 participants to assess the face validity, and to obtain community input about the best way to design the fotonovela plot and format to deliver the target messages. Data from these two focus group was used to develop a first draft of the fotonovela. The second phase involved testing the face validity of the draft fotonovela through a third focus group with 4 participants who were part of the two initial focus group. In the third phase of the study the face validity of the fotonovela was confirmed with a new group of 14 participants recruited to participate in a fourth focus group. Focus groups participants were mostly female (81%) were on average 37±17 years old, 31% had completed high school or more, 50% were unemployed and 75% were receiving SNAP benefits. The knowledge, attitude, and planned behavior evaluation was conducted with 100 individuals answering a survey before and after reading the fotonovela. Knowledge was assessed with an 8‐item questionnaire designed for this study. Of these participants, 84% were female who were on average 42.5±15.1 years old, 78% primarily spoke Spanish, 28% had less than high school education and 61% were SNAP participants. After reading the fotonovela, participants increased their knowledge on the seven steps for heart disease prevention significantly (63.7±16.3 pre‐test vs 78.8±13.1 at post‐test, p=0.000), and almost all were highly satisfied with it (96%). After reading the fotonovela, when participants were asked to mention at least three steps that they would commit to follow, 93% wanted to be more active (93%), 98% wanted to eat better, 76% to avoid smoking, 28% to control blood sugar, and 24% to control their cholesterol levels. In conclusion, this culturally and literacy appropriate increased knowledge had strong face validity and may help motivate Latinos to take steps to prevent heart disease.

Support or Funding Information

Funded by the American Heart Association
Networks of exchanging antibiotic resistance between environmental, commensal, and pathogenic microbes
Authors: Dantas Gautam
Published: November 2023

While the most acute effects of increasing antibiotic resistance in pathogens are observed in clinical settings, it is becoming increasingly clear that the evolution and transmission dynamics of resistance gene dissemination is an ecological problem. Indeed, steady use and abuse of antibiotics over the past century in food animals, humans, and the environment has provided substantial selective pressure for enrichment of resistance genotypes in each of their associated microbiomes. An over‐reliance on culture‐based methods, the standard in the study of clinical resistance, has vastly underestimated these reservoirs of resistance genes (or ‘resistomes’). To address this issue, we have recently developed high‐throughput metagenomic functional selections, aided by next‐generation sequencing, to characterize resistomes encoded by the microbiota of healthy human adults and children as well as diverse soils. By combining these analyses with 16S amplicon sequencing and deep shotgun sequencing, we model the impact of various anthropogenic perturbations on the transmission and evolutionary dynamics of microbial communities and their resistomes across time and habitats. Hundreds of resistance genes we identify from specific taxa in these different microbial communities are identical to resistance genes found in major human pathogens, indicating recent genetic exchange between these microbes. We also find thousands of functionally validated resistance genes which are genetically novel, but flanked by genes involved in horizontal gene transfer, including transposases and integrases. Together, these findings highlight the substantial antibiotic resistome encoded by microbes from diverse environments, which is available for exchange with pathogens, with the potential to severely exacerbate the problems with clinical resistance.
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 Information

Work supported by National Institutes of Health Grant T32DK062032
Introducing Informal Communication of Science within an Undergraduate Anatomy and Physiology Course
Authors: Petzold Andrew M , Dunbar Robert
Published: November 2023

Dissemination of scientific knowledge is a skill that is necessary for any undergraduate student within the sciences to acquire. Traditionally, if addressed, this is accomplished through the instruction of scientific presentation or writing. Though important, this type of education tends to encourage students to focus on peer‐to‐peer communication of scientific knowledge, subsequently abandoning the dissemination of knowledge to a non‐scientific (lay) audience. This has presented itself within the traditional media, popular culture and education as a apprehension in regards to science scientific knowledge general populace, an aloof attitude within scientists and an inability to discuss scientific issues. We embedded the teaching of the use of approachable language in describing complex ideas derived from an Anatomy and Physiology curriculum. Students were assessed on their word usage on associated writing activities and examined on associated course content design. By fulfilling the requirements for this assignment, students were encouraged to examine their ability to communicate the knowledge they had gained throughout their Anatomy and Physiology course to a non‐scientific audience ‐ gaining a skill that is sought in a variety of science related fields.
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 Information

NIH Health Centers of Biomedical Research Excellence Phase III Award 1P30GM110760‐01, NIH T32 Training Grant 5T32oD011124‐09
Visualization of Differential Myelinated Cardiac Plexus Fiber Innervation in Heart Failure Using the Sihler's Stain Technique
Authors: Hilton Alistair , Jorgensen Adam , Gardiner Brett , Robison Scott , Lambert H Wayne , Morton David , Wisco Jonathan
Published: November 2023

A whole organ staining of myelinated cardiac plexus nerve fibers was conducted to visualize the cardiac innervation pathways in relation to heart failure. This visualization could better inform treatment for heart failure and contributory conditions, including arrhythmia, valve disease, and coronary artery disease. Two pathology‐free human cadaveric heart specimens and one acute heart failure human cadaveric heart specimen were acquired as opportunity samples from the West Virginia University School of Medicine (WVUSOM) Human Gift Registry. One pathology‐free specimen was sectioned into three transverse slabs prior to staining, while the remaining pathology free specimen and the acute heart failure specimen were stained whole. Sihler's stain rendered the myocardium semi‐translucent red‐pink to purple, while the myelinated cardiac plexus fibers were stained a deep violet. Definitive nerve density was found in three major locations: along the coronary vessels, at the mitral valve, and in the ascending aorta, including the aortic valve. Due to the Sihler's nonspecific staining of myelin, the fibers exact identity is unknown. The most probable identity is pararsympathetic afferents or efferents, though sympathetic afferents are also possible. The pathology‐free and diseased specimens were compared to determine what affect heart failure had on innervation patterns. Notable differences were found in the left descending coronary artery, the AV node, the left ventricle, the mitral valve, and the pulmonary trunk. The diseased heart showed attenuated nerve staining in both the AV node and mitral valve, supporting the relationship between parasympathetic withdrawal and heart failure.
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 Information

NIH 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 Information

NSF 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 Information

This 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 Information

This 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 Information

This 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 Information

R01 supported project
Development of Novel Small Molecule Antibiotics against a Conserved RNA Element in Gram‐positive Bacteria
Authors: Väre Ville Y.P. , Frohlich Kyla M. , Todd Gabrielle , Bell Janeen , Agris Paul F.
Published: November 2023

tRNA‐dependent control of gene expression, T‐boxes, are RNA regulatory elements located at the 5′‐untranslated region (5′‐UTR) in numerous gram‐positive bacteria. T‐boxes control the expression of several genes involved in tRNA aminoacylation, as well as amino acid metabolism and transport. T‐boxes function by recognizing the aminoacylation status of their cognate tRNA ligand: uncharged tRNA can stabilize an antiterminator complex through an acceptor stem – antiterminator interaction, thus allowing transcription of the downstream operon. Aminoacylated tRNA cannot form this interaction, therefore the thermodynamically more stable terminator hairpin is formed, causing transcription to halt. Given that T‐boxes are found exclusively in gram‐positive bacteria and share highly conserved regions necessary for tRNA binding, this RNA regulatory element represents a prime target for antibacterial drug development. Docking studies using atomic‐resolution T‐box structures identified several potential candidates. 43 putative antibacterial compounds representing distinct chemical families were selected for further characterization. Antibacterial microdilution studies resulted in a moderately active hit compound (PKZ18) that has been selected for further antibacterial drug development. Characterization studies demonstrate that PKZ18 is highly refractory to resistance, displays low cytotoxicity, and possesses activity against a wide range of gram‐positive bacteria, including clinical isolates of Staphylococcus aureus, Streptococcus pyogenes, and Clostridium difficile. Mechanism of action studies confirm binding of PKZ18 to both glycine and tyrosine T‐boxes, as well as inhibition of threonine tRNA synthetase, a protein under the control of a threonine T‐box in S. aureus. Structure‐activity relationship (SAR) studies of PKZ18 have identified the essential chemical moieties for both specificity as well as activity. Hit‐to‐lead optimization of PKZ18 by medicinal chemistry and further SAR studies is on‐going.
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 Information

This 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 Information

Support: university funds from grants and donations: including NSF U.S. presidential award 0731633, NIH grant awards and Sidney Stern Memorial Trust (to S. Oppenheimer).
A cell‐based inhibitor screening platform for S‐adenosylmethionine synthetase
Authors: Parungao Gwenn , Dziobak Miranda , Blumenthal Robert , Viola Ronald
Published: November 2023

Infectious diseases are one of the leading causes of death worldwide. The remarkable social and economic burden they cause in society demands new tools, including novel drug approaches, to deal with these diseases. Our present study is based on a growth‐based assay that we have developed in E. coli for screening of inhibitors against S‐adenosylmethionine (AdoMet) synthetase (MAT) from different bacterial, fungal and protozoan species. MAT is the only known enzyme to catalyze the biosynthesis of AdoMet, an essential metabolite for all living organisms. The multitude of metabolic roles of AdoMet makes it an attractive target for development of inhibitors. The sequence and structure of this enzyme is highly conserved particularly in the active site, from bacterial to eukaryotic species. This conservation contributes to the difficulty of finding selective inhibitors. Our in vivo platform for screening inhibitors employs an E. coli metK‐deletion mutant (ΔmetK) that makes its growth dependent upon extracellular AdoMet. Plasmids carrying one of several bacterial, fungal, protozoan or mammalian MAT genes were found to rescue the ΔmetK deficiency without AdoMet supplementation. The growth rates are similar in each complemented strains except for the significant delay in lag phase with the human MAT. An initial proof‐of‐principle experiment with cycloleucine, a known weak inhibitor of MAT, showed different degrees of inhibition in the complemented MATs. Optimization of the inhibitor properties for species selectivity is being guided by determination of the structures of the enzyme from different infectious organisms. Our results highlight the applicability of this screening platform to identify selective inhibitors of bacterial, fungal or protozoan species, with a counter screening to avoid compounds that inhibit human MAT.
Targeting mER and GLP1R for the Treatment of Atherosclerosis and Type II Diabetes
Authors: Du Jasper , Issa John Paul , Kumar Krishna
Published: November 2023

Cardiovascular disease (CVD) and type II diabetes (T2D) currently affect hundreds of millions lives worldwide and are at the forefront of public health concerns. The two diseases are interrelated: Type II diabetic patients are at a significantly higher risk of CVD and ultimately succumb to CVD related complications. Estradiol (E2) provides vascular benefits, including vasodilation, reducing the risk of cardiovascular disease. E2 mediates these atheroprotective effects by signaling at the estrogen receptor in the cytosol, endoplasmic reticulum and plasma membrane. In addition, E2 has been seen to have a prominent effect on the hypothalamus, increasing insulin sensitivity and lowering blood glucose levels. Insulin insensitivity is a hallmark of T2D, suggesting that E2 can be a viable molecule to treat T2D. However, binding to cytosolic estrogen receptor (ER) eventually leads to upregulation of proliferative genes that can result in breast or uterine cancers.

Glucagon‐like peptide 1 (GLP‐1), a gut‐derived peptidic hormone, resolves high blood sugar levels, body weight, and other T2D complications. Interestingly, GLP‐1 has been observed to have cardiovascular protective effects, reducing risk of CVD. However, GLP‐1 is quickly inactivated in minutes by the endogenous protease dipeptidyl peptidase 4 (DPP‐4) in the bloodstream.

These observations point to a link between T2D and CVD, and a link between E2 and GLP‐1. By combining the two hormones together, tangible synergistic benefits can be realized and more importantly, respective obstacles can be avoided. I have developed an in‐solution coupling to chemically link E2 to GLP‐1 at the Lys26 residue. This conjugate would prevent the ability of E2 to initiate breast or uterine cancer but still signal at ER and GLP‐1R. Further, I have identified several methods that prevent proper formation of the conjugate, namely through a Wagner‐Meerwein rearrangement of E2. To preclude inactivation of GLP‐1 by DPP‐4, I have covalently attached a trifluroethyl group at the N‐terminal His of GLP‐1 that retains potency. Additionally, I have conjugated to E2, a small molecule with high affinity to the serum protein transthyretin, in order to extend half‐life and prevent entrance of E2 into the cytosol.

Support or Funding Information

I am funded by an ASBMB Travel Award that was presented to me at the Northeastern Regional ASBMB Conference.
Pentabromophenol, a brominated flame retardant derivate, suppresses TGF‐β signaling by sequester TGF‐β receptor from cell surface and further degradation through a caveolae‐mediated endocytosis
Authors: Yang Pei‐hua , Chen Chun‐Lin
Published: November 2023

Polybormodiphenyl ethers (PBDEs) have been widely used as flame retardants in a variety of consumer products, the possible adverse health effects of PBDEs have focused on their potential endocrine‐disrupting effects and developmental neurotoxicity. In this study, we found that the major biological metabolite of PBDEs, pentabromophenol (PBP) suppressed TGF‐β responsiveness by accelerating the turnover rate of TGF‐β receptor. PBP suppressed TGF‐β1‐mediated cell migration, PAI‐1 reporter gene activation and Smad2/3 activation in various type of cells, abolished TGF‐β1‐mediated repression of E‐cadherin expression, as well as induction of vimentin expression along with Snail and Slug upregulation; thus, blocking the TGF‐β1‐mediated epithelial‐to‐mesenchymal transition (EMT) in A549 calls. TGF‐β superfamily is a key player in the regulation of a wide variety of biological processes from development to pathogenesis including cell proliferation, migration, and the process of cancer development and progression. The in vitro results in this study provide a basis for studies of more detailed relationships between PBP and the modulation of TGF‐β signaling. Given their similarities to PBDEs, additional laboratory and mechanistic study should be considered to assess PBDEs as a potential risk factor for tumorigenesis and other TGF‐β‐related diseases.

Support or Funding Information

Ministry of Science and Technology in Taiwan
Unraveling White Lupin's Signal Transduction in Response to Phosphorus Deficiency Using iTRAQ Labeling, Phosphopeptide Enrichment, and Tandem Mass Spectrometry
Authors: Amadi Michael , Cole Jason , Li Kathy , Chalkley Robert J. , Burlingame Al , Uhde‐Stone Claudia
Published: November 2023

Phosphorus (P) availability is one of the most limiting factors for crop production worldwide. To breed or engineer crops that do well in poor soils, researchers are investigating plants that are well adapted to P deficiency, such as the legume white lupin (Lupinus albus L.). While great progress has been made in understanding white lupin's strategies to acquire P, not much is known about the signal transduction pathways that link sensing of P deficiency with the appropriate responses. Phosphorylation of proteins plays a central role in signaling processes. Proteins that change phosphorylation status when exposed to P deficiency are potential members of such signal transduction pathways. However, quantifying the dynamics of protein phosphorylation is challenging, because of their transient nature and low abundance. A sensitive method is needed to identify proteins that become phosphorylated after exposure to P deficiency.

To this end, white lupin was grown in hydroponics with sufficient phosphorus (+P). After 3 weeks, half of the lupin plants were transferred into P‐deficient nutrient solution (−P). After 24 hours, total protein was isolated from roots of two +P and two −P24h plants, for a total of two independent biological replications. Proteins were quantified, trypsin‐digested, and subjected to LC‐MS (Liquid chromatography‐mass spectrometry). This successfully identified hundreds of white lupin proteins, indicating good quality of sample preparation. We then used four different iTRAQ (isobaric tags for relative and absolute quantification) tags to differentially label the +P and −P peptides from the two replicates. We are currently in the process of enriching phosphorylated peptides by titanium dioxide chromatography. Next, we plan to perform multiplex UPLC‐MS/MS (Ultra‐Performance Liquid chromatography tandem mass spectrometry) to quantify the iTRAQ‐labeled phosphorylated peptides from +P and −P24h in two biological replicates. Peptides that are not phosphorylated under +P, but become phosphorylated after transfer to −P are likely members of white lupin's signal transduction pathway in response to P deficiency.
ER/K Linked GPCR‐G protein Fusions Systematically Modulate Second Messenger Response in Cells
Authors: Dysthe Matthew , Malik Rabia U , Ritt Michael , Sunahara Roger K , Sivaramakrishnan Sivaraj
Published: November 2023

FRET or BRET approaches are well established for detecting ligand induced GPCR‐G protein interactions in cells. However, current FRET/BRET assays rely on co‐expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR‐G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the beta2‐, alpha1‐, and alpha2‐adrenergic receptors, and adenosine type 1 receptor (A1R), tethered to Gαs‐XL, Gαi1, or Gαq subunits. Agonist stimulation of beta2‐AR and alpha2‐AR increases FRET signal comparable to co‐expressed FRET/BRET sensors. SPASM sensors retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR‐G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist‐induced changes in GPCR‐G protein interactions, and linking these changes to downstream signaling.

Support or Funding Information

The research conducted here was funded by the American Heart Association Scientist Development Grant (13SDG14270009), the NIH (1DP2 CA186752‐01, 1‐R01‐GM‐105646‐01‐A1) to S.S. and by the American Heart Association Pre‐doctoral Fellowship (14PRE18560010) to R.U.M.
The C‐terminal Tail of TCL Localizes the GTPase to the Plasma Membrane of HeLa Cells
Authors: Tader Brooke , Florke Rebecca R , Hamann Michael J
Published: November 2023

TCL/RhoJ belongs to the Rho family of GTPases, and it has been shown to contribute to angiogenesis and tumor growth. To better understand the inherent biochemistry of TCL, our lab has been investigating primary sequence difference between TCL and its closest homologs Cdc42 and TC10. Thus far, amino acids 17–20 in the N terminus have been found to be important for GTP‐loading and localization of TCL to the plasma membrane, and mutation of this sequence leads to TCL localization to vesicles. To better understand how the TCL N‐terminus, C‐terminus, and GTP‐binding regions contribute to membrane and vesicle localization independently of each other, expression constructs were generated that fuse the first 24 and last 21 amino acids of TCL to the fluorescent protein Venus. Interestingly, these constructs indicate that the C‐terminus alone may be mostly responsible for TCL plasma membrane localization. TCL constructs containing the GTPase core without the C‐terminal tail suggest the GTPase region uniquely controls localization to vesicles. Additional mutations in the C‐terminal tail will be generated to test how the C‐terminal CAAX sequence and the polybasic sequence contribute to TCL localization in order to understand how TCL localization regulates its function.