bluuebunny/crossref_metadata_2025 huggingface dataset:
Predicting and Interpreting the Hofmeister Effects of Different Salts with Nucleic Bases and Aromatic Compounds Using Solubility Assay
Authors: Hong Runyu , Cheng Lixue , Yao Yao , Knowles Ben , Zhang Yurun , Kerins Michael , Shkel Irina , Record M. Thomas
Published: November 2023

The interaction between salts and nucleobases or aromatic compounds plays an important role in noncovalent biopolymer assembly processes such as DNA and RNA helix formation and protein‐nucleic acid interactions. The project determines effects of six salts selected from the Hofmeister series (NaF, KBr, KSCN, NaSCN, NH4Cl, NH4Br) on the solubility of 11 model compounds (nucleic bases, their analogs, and naphthalene) to experimentally quantify salt‐model compound preferential interactions (μ23) using UV‐Vis spectroscopy. Values of μ23 are obtained from the derivative of the logarithm of the model compound solubility with respect to the molal concentration of salt. Salt has the ability to either increase or decrease the solubility of the bases, which can be explained by their differences in interacting with various surface types of the model compounds, namely aromatic C, aromatic N, Sp3 C, Sp2 O, and Sp3 N. Results of this study indicates that for all the 11 model compounds investigated in this study, KSCN has the most favorable interactions whereas NaF has the most unfavorable interactions except with 26DAP. The differences in their ability to decrease (unfavorable interaction) or increase (favorable interaction) the solubility of bases corresponds with their rank orders among the Hofmeister series, in which the Na+ and F− are at the stabilizing end but SCN− ion is at the destabilizing end. Through one‐way analysis, the breakdown of nucleobase's surface types allows us to study the specific interactions between salts and biopolymer surfaces. Setting Na+ as a reference point, relative strength of interaction potentials of Hofmeister ions with aromatic and aliphatic C surfaces are obtained, which in general follows the Hofmeister series. The quantities describing accumulation and exclusion of Hofmeister salts, Kp, are also obtained through calculation. Using Na+ or K+ as a reference, a dissection of salts’ Kp values to get ions’ Kp values illustrates that both aromatic and aliphatic C surfaces strongly exclude cations and accumulate SCN−. Further studies are required to make dissections on other surface types possible. The quantified αi values and water accessible area (ASA) data are used to predict μ23 values. These predictions are compared with both our experimental data and published experimental data. The results confirm the effectiveness of our method and indicate potential applications in pharmaceutical, environmental, and biological studies.
Distracting the hungry: mosquito anosmia‐inducing and odor perception‐enhancing compounds of natural origin targeting ORco function for control of transmission of malaria and other mosquito‐borne infectious diseases
Authors: Iatrou Kostas , Tsitoura Panagiota , Konstantopoulou Maria
Published: November 2023

ORco, the ubiquitous co‐receptor of insect olfactory receptors, is an essential constituent of the insect olfactory machinery. Mutations affecting ORco function and RNAi‐mediated reduction in ORco abundance impact negatively on the organisms' capacity to detect the normal odor repertoire. On the other hand, compounds acting as ORco agonists may impact in a positive fashion on the function of olfactory receptors. In the current study, we have employed lepidopteran (Bombyx mori and Trichoplusia ni) cell‐based screening platforms expressing ORco and olfactory receptor variable subunits (ORx) of Anopheles gambiae in conjunction with specific ligand‐dependent functional assays to assess the effects of specific constituents of essential oils on receptor functionality. We show that natural compounds, previously selected through screening work employing A. gambiae odorant binding proteins (OBPs) with female expression bias, and displaying spatial repellent properties against Anopheles and Culex mosquitoes similar to or stronger than those of DEET, are, in fact, ORco antagonists. These antagonists cause chemical ablation of the function of all tested heteromeric olfactory receptor complexes containing ORco, and may induce anosmia‐like disorientation effects on mosquitoes. On the other hand, we show that members of a known class of synthetic ORco agonists induce allosteric changes on receptor heteromers resulting in significant ORx ligand‐specific enhancement in odor perception in vitro. Because these ORco agonists are not volatile and thus cannot be detected by mosquitoes at a distance, we are using the odorant receptor expression platforms to screen essential oils obtained from Mediterranean aromatic plants for the presence of natural volatiles acting as specific ORco agonists with functional properties similar to those of the synthetic agonists. In parallel, we are expanding the screens for the isolation of additional natural ORco antagonists. Due to their volatile nature, the natural anosmia‐inducing compounds would be expected to act at a distance and thus be suitable for use as spatial disorienting agents. The same principle should be applicable to the natural olfactory enhancers, which may be used as additives to human odor blend‐containing mosquito traps for more effective trapping. The combined deployment of anosmia‐inducing and olfactory capacity‐enhancing compounds targeting ORco function may provide a most effective approach for protection against indoor and outdoor transmission of malaria and other mosquito‐borne infectious diseases.

Support or Funding Information

This work has been supported by the ENAROMaTIC consortium project (grant No FP7‐222927 of the EC) and was carried out in facilities of the OPENSCREEN‐GR National (Greece) Research Infrastructure.
Deciphering the Function of NTF2‐like Proteins Associated with Polyketide Biosynthesis in Actinomycetes
Authors: Vuksanovic Nemanja , Zhu Xuechen , Silvaggi Nicholas R. , Melançon Charles E.
Published: November 2023

Bioactive natural products are the active components in many therapeutic drugs, and as antibiotic resistance emerges as a global threat, a quest for novel natural products with antibiotic activity becomes increasingly important. Actinomycetes are known to produce many bioactive secondary metabolites, such as polyketides, some of which are powerful antibacterial agents. The gene cluster involved in production of actinorhodin, a benzoisochromanequinone antibiotic found in Streptomyces coelicolor A3(2), has been extensively studied. However, the role of the ActVI‐ORFA gene has remained controversial. Strains where the gene has been disrupted still produce actinorhodin, only at much‐reduced levels compared to the wild‐type strain. A number of studies have suggested a regulatory role for ActVI‐ORFA. Our in vivo kinetic studies of S. Coelicolor mutants show that strains without functional ActVI‐ORFA accumulate at least one off‐pathway intermediate. We also report the structures of ActVI‐ORFA and a homologue, CA‐Cyc3, from Catenulispora acidiphila. Both proteins exhibit the α‐and‐β barrel fold characteristic of NTF2‐like superfamily proteins and possess a His/Asp diad, a motif found in a variety of enzymes that perform acid/base catalysis. Taken together, our functional and structural data suggest that ActVI‐ORFA may have an enzymatic role, directing the pathway away from the shunt product and toward the synthesis of actinorhodin.
Global substrate specificity of mycobacterial serine hydrolases
Authors: Johnson R. Jeremy , Bassett Braden , Waibel Brent , White Alex , Hansen Heather , Stephens Dominique , Koelper Andrew , Hoops Geoffrey
Published: November 2023

One third of the world's population is estimated to be infected with a dormant TB infection. M. tuberculosis survives within this dormant state by engulfing host cell lipid bodies and then accumulating significant intracellular stores of lipids. Among the proteins required for lipid metabolism in M. tuberculosis are a significant number of uncharacterized enzymes predicted to encode serine hydrolases and specifically lipases. Using a streamlined synthetic method, a library of hydrolase substrates based on acyloxy methyl ether fluorescein were expanded to better represent the natural lipodomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global substrate specificity of mycobacterial serine hydrolases under normal and dormant growth conditions. Conformation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors. The hydrolases responsible for the global substrate specificity of one highly active fluorogenic substrate were then isolated using a combination of native protein gel‐resolved activity measurements and mass spectrometry identification. Substrates that were highly activated in Mycobacterium were also tested in other common bacterial strains to determine the orthogonality of the mycobacterial hydrolase reactions. Using the highly activated fluorogenic substrates, future work will compare the serine hydrolase profiles between different strains of infectious Mycobacterium to aid in diagnostics and drug discovery.

Support or Funding Information

Funded by NIH 1R15GM110641‐01A1
ENOX2‐Selective Aptamer Identification for an Electrochemical‐Aptamer Biosensor in Cancer Diagnostics
Authors: Fetter Lisa C , Jacobs Jena , Bonham Andrew J
Published: November 2023

Human Ecto‐Nicotinamide Dinucleotide Oxidase Disulfide Thiol Exchanger 2 (ENOX2) is a common isoform of a parent protein ubiquitously expressed in humans, ENOX1. The ENOX2 isoform is very highly expressed on the surface of tumors in at least twenty‐six variants of cancer, resulting in ongoing research into ENOX2 as a potential diagnostic biomarker for cancer. Given both that ENOX2 is shed into the sera and that its presence is cancer‐specific, ENOX2 is an attractive target for blood‐based diagnostic screens for cancer. To enable this detection on a rapid and reagentless sensing platform, we have designed an aptamer‐scaffold electrochemical biosensor directed against this cancer‐related protein. This was accomplished by first using systematic evolution of ligands by exponential enrichment (SELEX) procedures and high‐throughput next generation sequencing to identify an ENOX2‐selective aptamer sequence. This novel aptamer may be useful in a variety of applications. In this work, the aptamer was then modified to support a conformation‐changing response to target binding. The result modified oligonucleotide was incorporated into an electrode‐based electrochemical biosensor platform. Electrical current changes in the presence of ENOX2 were measured, creating a dose‐responsive readout. Ultimately, this biosensor against ENOX2 will maintain functionality in whole blood, yielding a novel and clinically‐relevant diagnostic tool for the rapid detection of early stages of cancer.

Support or Funding Information

Support for this work comes from the Metropolitan State University of Denver Provost's and LAS Dean's offices.
Building a Synthetic Library of Fluorogenic Ester Substrates to Analyze Serine Hydrolases
Authors: Koelper Andrew , Johnson R. Jeremy , Hoops Geoffrey
Published: November 2023

Mycobacterium tuberculosis, the organism responsible for the respiratory infection tuberculosis, has evolved multi‐drug resistance strains, introducing the need for novel drug targets. Serine hydrolases with diverse roles in lipid metabolism and dormant growth have been proposed as novel targets for tuberculosis. Although many serine hydrolases from TB have been identified by proteomics and activity based protein profiling, the natural substrates and biological reactions for these serine hydrolases are still unknown. To globally investigate the natural substrates for mycobacterial serine hydrolases, we synthesized a structure activity library of 32 fluorogenic ester substrates to screen against mycobacterial hydrolases. The library contains acyloxy methyl ester derivatives of fluorescein with small variations in ester functionalities to mimic mycobacterial lipids. Synthesis of novel compounds required separation techniques including silica‐gel column chromatography, along with characterization techniques including 1HNMR and 13CNMR and high‐resolution mass spectroscopy. To confirm the utility of the fluorogenic library for characterizing serine hydrolases, two structurally homologous bacterial serine hydrolases, Rv0045c from M. tuberculosis and ybfF from V. cholera, were screened against the library of fluorogenic substrates to determine the catalytic efficiency of these enzymes on each substrate. Rv0045c and ybfF both favor short, 3‐atom, ether chains with the heteroatom in the b‐position. ybfF with the most favored substrates showed catalytic efficiencies of high activity (kcat/kM > 104 M−1s−1), while Rv0045c did not perform as well (kcat/kM 103–104 M−1s−1). In addition, comparative kinetic analysis of hydrolase variants with substitutions in their binding pocket provided insight into the substrate specificity of the enzymes, and thereby the natural substrate. Catalytic efficiency deviations from the WT, could be a result of steric hindrance, hydrogen bond interactions, and localized acid‐base chemistry. Future work would be aimed at making more variants and determining the structural features, which contribute to their substrate specificity.

Support or Funding Information

Funded by NIH 1R15GM110641‐01A1
Development of Ester‐Protected Ethambutol Derivatives for Characterization of Mycobacterial Hydrolase Activity
Authors: Larsen Erik , Stephens Dominique , Johnson R. Jeremy
Published: November 2023

Tuberculosis (TB) is a leading cause of death among infectious diseases worldwide, with nearly 10 million cases reported in 2014. The constant selective pressure of current antibiotic regimens has led to the rise of highly drug‐resistant strains of M. tuberculosis, requiring the development of novel antibiotics and therapeutic targets. The inhibition of serine hydrolases from M. tuberculosis has been shown to impede bacterial growth and reactivation, making them an attractive drug target. Our lab has developed a method to probe the global substrate specificity of mycobacterial serine hydrolases through the use of ester‐protected derivatives of ethambutol, a first‐line antibiotic treatment for TB. These derivatives were synthesized directly from ethambutol in a single step, using a selective o‐acylation to yield products in high yield and purity with minimal workup. A small library of derivatives was screened against M. smegmatis, a non‐infectious mycobacterium model for TB. Early results from this screening has found that mycobacterial serine hydrolases prefer strongly hydrophobic moieties, which mimic their natural lipid substrates. Efforts to expand this library and characterize differential hydrolase activity under various physiological conditions are ongoing.

Support or Funding Information

Funded by NIH 1R15GM110641‐01A1
Global Protease Activity Profiling Provides Differential Diagnosis of Cystic Precursor Lesions of Pancreatic Cancer
Authors: Ivry Sam Lacey , Dominguez Dana , Roy Nilotpal , Hatcher Stacy , Hebrok Matthias , Kim Grace , O'Donoghue Anthony , Kirkwood Kim , Craik Charles
Published: November 2023

Pancreatic cysts are being detected at an increasing rate and are estimated to be present in 2 percent of the adult population. Unfortunately, current preoperative diagnostics do not sufficiently distinguish benign cystic lesions from those that harbor malignant potential. Missregulated pericellular proteolysis is a hallmark of invasive cancer, and therefore, we used a global substrate profiling strategy to discover differentiating protease activities in patient cyst fluid obtained through minimally invasive endoscopic sampling. We determined that the activities of the aspartyl proteases gastricsin and cathepsin E are highly increased in mucinous cysts. Profiling of gastricsin and cathepsin E activity using fluorescent peptide substrates differentiated mucinous from nonmucinous cysts with high sensitivity and specificity in a 110 patient cohort. Of particular note, gastricsin activity analysis demonstrated a specificity of 100% and sensitivity of 93%, significantly outperforming the most widely used biomarker, carcinoembryonic antigen. We propose that activity‐based detection of gastricsin and cathepsin E has the potential to replace current diagnostic tests for differentiating mucinous from nonmucinous cysts, leading to improved clinical management of these challenging and highly prevalent lesions.

Support or Funding Information

This study was supported by the following grants: NIH National Heart, Lung, And Blood Institute grant U54HL 119893 (to C. C.), NIH/NCATS UCSF‐CTSI grant UL1 TR000004 (to C. C.), and NIH grant A119685 (to K. K.).
Toward Cell‐Permeable, Multi‐Fluorophore Protein Labels for Enhanced LRET Imaging.
Authors: Soflaee Mona H. , Vera Claudia Ivette Rivera , Miller Lawrence
Published: November 2023

Genetically encoded biosensors based on Förster Resonance Energy Transfer (FRET) are used to microscopically visualize dynamic changes in protein interactions and activities within living cells. However, the spectral overlap between fluorescent protein (FP) donors and acceptors makes it challenging to accurately quantify changes in FRET signals, and multi‐color imaging of two or more FP FRET pairs in a single cell is extremely difficult. Lanthanide‐based FRET (LRET) biosensors incorporate luminescent Tb(III) complexes with ms‐scale excited state lifetimes and multiple, narrow‐line emission bands as donors and FPs (or other conventional fluorophores) as acceptors. Time‐gated microscopy uses pulsed excitation and delayed detection to eliminate ns‐scale fluorescence background and cleanly detect Tb(III)‐sensitized FP emission. In order to increase LRET signals and achieve faster and more sensitive imaging, we have sought to develop biosensors that combine multiple Tb(III) donors with a single fluorescent acceptor. Due to the mismatch in lifetimes, multiple excited donors can successively transfer energy to one acceptor, thereby generating many photons with a single excitation.

Our lab has previously shown that heterodimers of a protein‐targeting ligand linked to a Tb(III) complex can be efficiently delivered to the cytoplasm of living cells by conjugating the heterodimers to arginine‐rich, cell penetrating peptides (CPPs) such as nonaarginine or Tat. Our results are in line with numerous studies which show that CPPs facilitate direct translocation of small molecules from culture medium into the cytoplasm. Macromolecules linked to CPPs enter cells via endocytosis, and the maximum size of CPP cargo that can bypass endocytosis and directly enter cytoplasm is unknown. In order to resolve this question and inform our design of multi‐Tb(III) probes, we conjugated oligoarginine to polyamidoamine (PAMAM) dendrons of different generations. We used FMOC‐based, solid‐phase peptide synthesis (SPPS) to prepare nonaarginine that was N‐terminally coupled to PAMAM dendrons with 2, 4, and 8 branches (generations 0, 1 and 2, respectively). Each branch terminates in a primary amine, which was coupled to 5,6‐carboxy fluorescein. Fluorescence microscopy showed that 2‐dye and 4‐dye Dendron conjugates entered the cytoplasm of HeLa cells within 30 min when cells were incubated at 4 °C or 37 °C in culture medium containing 5 μM or higher probe concentrations. The 8‐dye Dendron‐CPP conjugate did not enter the cytoplasm under any set of incubation conditions. Based on these results and published values for PAMAM dendrimer sizes, we estimate the molecular (hydrodynamic) diameter cut‐off for CPP‐mediated cytoplasmic delivery to be < 2 nm.

Support or Funding Information

Funding provided by the National Institutes of Health, R01 GM081030.
Measuring Oxytocin Hormone and Oxytocin‐Reactive Autoantibodies to Determine Their Correlation with the Severity of Clinical Depression
Authors: Thompson Sarah Elizabeth , Russo A. J.
Published: November 2023

Autoantibodies are immunoglobulins that are unable to distinguish foreign antigens from the body's own proteins. While the mechanisms behind the generation and persistence of these self‐recognizing antibodies are not clear, hormones, such as oxytocin, may be involved in their production. Oxytocin is important in both reproduction and childbirth. However, it is also a key chemical messenger affecting social behavior, with lower levels of the hormone potentially resulting in depressive disorders.1,2 Clinical depression is a serious mental illness that affects 350 million people worldwide and almost 7% of adults in the United States.3 To date, the main methods used to diagnose depression are the Hamilton Depression Rating Scale and the Montgomery‐Åsberg Depression Rating Scale, two subjective, survey‐style observational tests. Because each individual case of depression is unique, simply observing depressive behaviors may not be sufficient to develop effective treatment plans for patients. Certain biochemical measurements, such as blood plasma levels of oxytocin hormone and autoantibodies to oxytocin in the circulation, may correlate to the severity of depression. In this study, direct and sandwich enzyme‐linked immunosorbent assays (ELISAs) will be used to measure the concentrations of these biomarkers in the blood plasma of patients diagnosed with clinical depression. The quantified levels of oxytocin hormone and oxytocin‐reactive autoantibodies will then be compared to depression rating scale results in order to determine if a correlation between the concentration of these biomarkers and the severity of depression exists. Using oxytocin hormone and oxytocin‐reactive autoantibody levels to “quantify” illness severity may contribute to more effective diagnoses and treatment plans for clinically depressed individuals.

Support or Funding Information

This project is funded, in part, by the Dean M. Graham Foundation.
Biosensor Diagnostic for Congestive Heart Failure via Detection of B‐Type Natriuretic Peptide
Authors: Dubchak Nazar , Bonham Andrew J
Published: November 2023

We developed an electrochemical DNA (E‐DNA) biosensor for point‐of‐care quantitative determination of B‐type natriuretic peptide (BNP) levels in blood. BNP is a commonly used biomarker for the onset of heart failure conditions, as its secreted levels in the blood correlate with the internal pressure of cardiac tissue, and high levels of BNP are typically found in patients with congestive heart failure. Unfortunately, current tests for BNP are largely based on phlebotomic blood draw followed by ELISA, which requires external testing and eliminates the possibility of point‐of‐care diagnosis. To address this unmet need, we have developed a rapid, convenient biosensor for BNP based on a known DNA aptamer specific to BNP that has been modified and incorporated into an electrochemical readout format. E‐DNA biosensors have been shown to function in unprocessed whole blood and yield quantitative results in less than one drop of blood. When an E‐DNA biosensor binds to its target, a conformational change results in a change in electrical current. Using varied concentrations of BNP in PBS buffer we were able to dose‐response behavior from the biosensor. This data is used as a calibration standard to allow quantitative measurement of unknown concentrations of BNP, which would have useful clinical application. Although optimizations remain, ultimately, the biosensor will measurement of BNP levels in whole blood, offering a new bedside or point‐of‐care way to measure and monitor an important biomarker for diagnosis of heart failure.

Support or Funding Information

Support for this work comes from the Metropolitan State University of Denver Provost's and LAS Dean's offices.
Over‐expression and association of serum PPIA, vwf, and FXIIIa with a systemic hypercoagulable state in patients with pulmonary tuberculosis
Authors: Chen Zhongliang , Jiang Tingting , Wei Liliang , Li Xiang , Shi Liying , Li Meng , Liu Changming , Tu Huihui , Yang Su , Chen Jing , Li Jicheng
Published: November 2023

Pulmonary tuberculosis (TB) is associated with a systemic hypercoagulable state, as reflected by the elevated plasma levels of fibrinogen in patients with TB. But the detailed mechanisms remain unknown. In this study, iTRAQ‐coupled 2D LC‐MS/MS technique was used to identify the differentially expressed proteins in serum between TB patients and healthy controls. Bioinformatics analysis showed that Peptidyl‐prolyl cis‐trans isomerase A (PPIA), von Willebrand factor (vwf) and Coagulation factor XIII A chain (FXIIIa), associated with blood coagulation and platelet activation, were over‐expressed (P<0.05) in TB patients compared with healthy controls. The high expressions of PPIA (P<0.01), vwf (P<0.001) and FXIIIa (P<0.001) were further confirmed using ELISA analysis in 64 TB patients and 64 healthy controls. In conclusion, our study showed that PPIA, vwf and FXIIIa were over‐expressed in the serum of TB patients, and may explain the potential mechanisms of coagulation dysfunction in patients with TB.

Support or Funding Information

This work was supported by grants from the National Basic Research Program of China (No.2014CB543002), the National Natural Science Foundation of China (No.81573709, No.81273882).
Assessment of the effects of pollutants in the Great Lakes on the human proteome
Authors: Dupree Emmalyn , Crimmins Bernard , Holsen Thomas , Pagano James , Thompson Brooke , Christensen Krista , Raymond Michelle , Darie Costel
Published: November 2023

The Great Lakes fishery provides a bountiful source of protein to indigenous peoples. However, there are concerns about fish consumption and contaminant exposure to these populations. Sera from fish consumers in the Great Lakes basin were analyzed for legacy chemicals such as polychlorinated biphenyls and organochlorine pesticides to assess the differential accumulation of these contaminants with respect to fish consumption and proteome related to contaminant enrichment. The current study is a double‐blinded study: legacy contaminant residues are being analyzed in random order with those sera exhibiting the lowest levels adopted as controls. In parallel, the sera are also analyzed blindly by proteomics methods. The control/exposed samples will then be segregated based on contaminant levels where samples with the lowest levels of PCBs considered controls and those with elevated levels denoted exposed. Legacy chemical analysis was performed using CDC methods employing isotope dilution mass spectrometry. Proteome analysis was performed by albumin depletion and separation by SDS‐PAGE, followed by the in‐gel trypsin digestion and nanoLC‐MS/MS analysis on a NanoAcquity UPLC coupled to a Xevo G2 Q‐TOF mass spectrometer (both from Waters). In addition, the sera are also digested by trypsin in‐solution followed by nanoLC‐MS/MS analysis. The two methods (in‐gel and in‐solution digestion) complement each other. The initial scout runs for the in‐solution digestion and nanoLC‐MS/MS analysis is completed. Additional more in‐depth analyses are currently underway.

Support or Funding Information

Clarkson University
Quantifying Protein Dephosphoylation as a Function of Mechanical Sample Disruption Techniques Towards an Optimized Sample Prep Protocol for Phosphoproteomics
Authors: Garrett Shari , Atwood James , Easparro Brandon
Published: November 2023

Phosphorylation is one of the most commonly studied post translational modifications. Phosphorylation plays a critical role in many cellular processes and is extensively seen in eukaryotic cells. Quantitative phosphoproteomic studies aim to measure the degree of protein phosphorylation as a function of biological state. These studies are routinely performed using tissues and cells. In all cases the proteins must be first liberated from the target sample. Especially for tougher tissue types, mechanical methods of sample disruption are routinely employed; including bead milling, sonication or rotor‐stator homogenization.

However, with mechanical sample disruption, there is always a concern that excessive processing will generate heat or shear forces that may dephosphorylate the target proteins. Maintaining the native state of phosphoproteins is imperative when detecting the removal or addition of a phosphate moiety through a quantitative phosphoproteomic study.

In this study, we quantify protein dephosphorylation as a function of mechanical disruption methods, processing time, and power to develop an optimized method that both provides adequate sample disruption while maintaining phosphoprotein integrity. Beta‐caesin and protein lysates from eukaryotic cells were processed via manual disruption methods and via bead milling using multiple different parameters. Following homogenization, the effect of desphosphorylation was quantified via western blotting and quantitative LC‐MS/MS.
High resolution mass spectrometry coupled with multivariate data analysis revealing plasma lipidomic alteration in patients with colorectal cancer
Authors: De Oliveira Carvalho Patricia , Messias Marcia Cristina Fernandes , Mecatti Giovana Colozza , Angolini Célio Fernando Figueiredo
Published: November 2023

Colorectal cancer (CRC) is one of the most frequent neoplasias in the adult population worldwide and presents a high rate of morbidity and lethality. In this study, a lipidomic approach based on gas chromatography and liquid chromatography‐electrospray ionization‐qToF‐mass spectrometry (LC‐ESI‐qToF‐MS) combined with multivariate data analysis, including principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS‐DA) was adopted for the investigation of potential diagnostic biomarkers in the plasma of CRC patients. Variable Importance in Projection (VIP) was used to identify the ions that had the greatest discriminatory effect among the groups (cancer patients and healthy volunteers). It was possible to identify 15 differential lipids belonging to the glycerophospholipids class. The most abundant ions identified in the group of cancer patients were the phosphatidylcholines and phosphatidylethanolamines whereas the lysophosphatidylcholine (LPC) metabolite identified as LPC (P‐16:1), which presented a major VIP score through the OPLS‐DA model, was down regulated in patients with CRC. Also, a relative reduction of palmitoleic (C16:1), arachidonic (C20:4) and n‐3 polyunsaturated fatty acids was observed in these patients. This study may provide a new insight into underlying mechanisms for CRC and proves that MS‐based lipidomics is a powerful method for discovering new potential clinical biomarkers

Support or Funding Information

FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)
High‐Throughput, Fluorescent Analysis of Reactive Oxygen Species in C. elegans after Knockdown of mrck‐1
Authors: Brown Callista , Heying Ruth , Russo Matthew , Rigoulot Stephen , Erickson Patti
Published: November 2023

Reactive oxygen species (ROS) produced in living organisms can generate oxidative stress, which contributes to cell damage associated with neurodegenerative diseases, such as Alzheimer's and Parkinson's. Studying responses to oxidative stress in model organisms, like the nematode worm Caenorhabditis elegans, may provide insight into basic underlying cellular mechanisms shared with humans. MRCK‐1 (myotonic dystrophy kinase‐related Cdc42 binding kinase), a highly conserved intracellular kinase, is activated by the G‐protein, Cdc42, which contains a redox sensitive motif. Previous studies have shown that MRCK‐1 regulates cell polarity and cytoskeleton reorganization, particularly during cell division and in response to cellular damage. To determine if MRCK‐1 plays a role in other cellular defense responses, we are testing if RNA interference (RNAi) of mrck‐1 results in the production of more ROS under stressed conditions. The cell permeable fluorescent ROS indicator, carboxymethyl dichlorodihydrofluorescein derivative, CM‐H2DCFDA, shows significantly increased fluorescence in hydrogen peroxide‐treated worms when quantified using the SpectraMax microplate reader. Oxidative stress conditions were induced with hydrogen peroxide (H2O2) in the presence of CM‐H2DCFDA, and fluorescence was measured over a period of 20 hours. Measuring the fluorescence of worms exposed to dsRNA from two independent mrck‐1 RNAi constructs, mrck‐m and mrck‐s, enables the quantification of ROS levels when MRCK‐1 function is decreased. Exploring the levels of ROS in RNAi‐treated C. elegans may give further insight into the cellular role of MRCK‐1 both in worms and in humans.

Support or Funding Information

Special acknowledgments to the Salisbury University Department of Biological Sciences, the Henson Undergraduate Research Award, and the National Science Foundation MRI Acquisition Award DBI‐1337534 for support.
Biochemical Study of A Cancer Driver Fusion Protein, DnaJB1‐PKA
Authors: Lu Tsan‐Wen , Zhang Ping , Cianfrocco Michael , Simon Sanford , Leschziner Andres , Taylor Susan
Published: November 2023

Fibrolamellar Hepatocellular Carcinoma (FL‐HCC), a rare liver cancer disease, usually occurs in young adults without any liver disease history. Until now, surgery is still the main treatment because FLHCC does not respond well to chemotherapy. Given the low survival rate and lack of available treatment options, there is a pressing need for new diagnostic and therapeutic approaches to FLHCC.

Whole genome sequencing and transcriptome sequencing of FLHCC tumors show a single, consistent genetic alteration in the FLHCC tumor cells: a deletion of ~400 kB between the first exon of the heat shock protein, DNAJB1, and the first exon of the catalytic subunit of PKA, PRKACA, on one copy of chromosome 19. This deletion produces a chimeric gene, which leads to a chimeric protein. This chimeric DNAJB1‐PRKACA protein is now recognized as the driver of FLHCC. Inhibition of the tumor driver DNAJB1‐PRKACA chimera offers tremendous potential to cure FLHCC. Full structural and biochemical characterization of DNAJB1‐PRKACA and its comparison to native protein PKA will provide valuable insights toward developing a drug that selectively blocks the FLHCC chimera. Here, we show DnaJB1‐PKA has similar kinase activity as wild type PRKACA. It can also form an R2 DnaJB1‐PRKACA2 holoenzyme with PKA regulatory subunits similar to wild type R2‐PRKACA2 holoenzymes even though they elute at different positions from gel filtration, indicating they may have different cell dimensions. Both cryo‐EM and X‐ray crystallography are being used to elucidate differences between wild type and mutant holoenzymes. Differences in holoenzyme functional properties are also being explored.

Support or Funding Information

Funding for this research was provided by the National Institutes of Health Grants GM034921
The secret life of kinases: insights into non‐catalytic functions from pseudokinases
Authors: Murphy James M , Petrie Emma J , Davies Katherine , Tanzer Maria C , Jacobsen Annette V , Hildebrand Joanne M , Lucet Isabelle S , Silke John , Czabotar Peter E
Published: November 2023

While protein kinases are best understood for their phosphoryl transfer enzymatic activities, recent mechanistic studies have led to recognition of non‐catalytic functions in cell signaling. Studies of pseudokinases ‐ the catalytically‐impaired or dead cousins of conventional protein kinases ‐ have been instrumental in shining light on the vast, varied and ever‐expanding repertoire of mechanisms by which kinase‐like domains can mediate protein interactions to modulate cellular signaling.

Although best characterized as allosteric regulators of conventional, active protein kinases, our work and that of others suggests more broadly that pseudokinase domains are important protein interaction modules that can function as molecular switches, scaffolds and adaptors in cell signaling pathways. Here, I will describe how our work has attributed these functions to the pseudokinase, Mixed lineage kinase domain‐like (MLKL). Overall, these studies suggest how conventional protein kinases could moonlight as protein interaction modules, scaffolds or molecular switches to complement their better‐understood catalytic roles.

Support or Funding Information

National Health and Medical Research Council of Australia (fellowship 1105754, grants 1057905, 1067289, infrastructure 9000220) and Victorian Government Operational Infrastructure Support.
Calcium Signals Alter the Mobility and Localization of CaMKIIδ in Cardiomyocytes
Authors: Wood Brent , Galice Samuel , Bers Donald , Bossuyt Julie
Published: November 2023

Ca2+‐calmodulin dependent protein kinase II δ (CaMKIIδ) is an important regulator of cardiac myocyte ion channels, Ca2+ regulation, contraction and transcriptional control; and its activity is upregulated in pathological states. CaMKIIδ has two main splice variants (δB and δC), which differ only by a nuclear localization sequence, so δB is more concentrated in the nucleus. While activity‐triggered translocation of neuronal CaMKIIα to dendritic spines is established as part of memory formation, very little is known about the spatiotemporal dynamics of CaMKIIδ activation. Prevailing views have been that cardiac CaMKII forms extremely stable dodecamers anchored at target‐rich loci and displays very little mobility (partially due to its size). We tested this using adenovirally‐expressed δC‐GFP and δB‐GFP and fluorescence recovery after photobleaching (FRAP). We found that δC‐GFP is remarkably mobile in the cytoplasm (Tau 11.09±0.27s) in resting rabbit cardiac myocytes. Moreover calcium chelation with BAPTA slowed, but 0.5 Hz pacing increased recovery kinetics (Tau 17.08±1.7 and 8.76±0.42s, respectively). δB‐GFP had similar differences as δC‐GFP in mobility under resting, BAPTA and paced conditions (Tau of 11.41±0.61, 19.17±2.5, and 10.04±0.59s). Autonomous δCT287D‐GFP moved rapidly even without pacing (Tau 9.90±0.41s). Calcium effects on nuclear FRAP followed a similar pattern, but recovery was significantly slower (Tau at rest 70.2±4.5s and 56.68±2.2s for δC‐GFP and δB‐GFP). The recovery plateau in the nucleus was also only 0.394±0.02 and 0.105±0.01 for δC‐GFP and δB‐GFP respectively (vs. 0.865±0.01 and 0.824±0.01 in cytosol), indicating a significant non‐mobile fraction of CaMKIIδ in the nucleus. Immunocytochemistry revealed pacing drives endogenous CaMKIIδ out of the nucleus and away from the Z‐lines. Nuclear localization of endogenous CaMKII was even further decreased in resting heart failure rabbit myocytes compared to age matched controls. These results indicate that CaMKIIδ activation promotes its mobility and suggest that CaMKII translocation may be an essential component of signal spread in cardiomyocytes.

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PO1‐HL80101 to DB and RO1 HL103933 to JB
Structural Elucidation of the Binding of the Vav‐SH2 Domain to the EphA2 Cytoplasmic Region
Authors: Zhang Kaibo , Ma Jia , Post Carol B , Stauffacher Cynthia V
Published: November 2023

The Vav guanine nucleotide exchange factors (GEFs) facilitate the GDP/GTP exchange of the Rac/Rho family of small GTPases to modulate cytoskeletal organization, cell morphology and mobility. In endothelial cells, Vav interacts with the EphA2 receptor tyrosine kinase (RTK) with its C‐terminal SH2 domain and plays a crucial role in EphA2‐mediated angiogenesis in cancers. Two closely‐spaced tyrosines in the juxtamembrane segment (JM) of the EphA2 cytoplasmic region, Y588 and Y594, were the postulated Vav‐SH2 recognition sites. Quantitative mass spectrometry data suggests that both JM tyrosines are phosphorylated with high stoichiometry in mammalian cells. However, they exhibit different rates of dephosphorylation by human cytoplasmic protein tyrosine phosphatases (HCPTPs). To characterize the role of each JM phosphotyrosine in Vav‐SH2 binding, isothermal titration calorimetry (ITC) and 15N‐HSQC titration experiments were performed using bacterially expressed Vav1‐SH2 and synthetic JM peptides with different tyrosine phosphorylation patterns. The ITC data indicates Vav1‐SH2 binds preferentially to the doubly phosphorylated and the Y594‐phosphorylated version of JM peptide (JMpYpY and JMYpY), with a KD 10‐fold smaller than the Y588‐phosphorylated version (JMpYY). The 15N‐HSQC spectra suggests all three versions of synthetic JM phosphopeptides bind Vav‐SH2 in similar fashions. The chemical shift perturbation (CSP) analysis of the titration spectra reveals both the primary and secondary phosphotyrosine binding pockets of Vav1‐SH2 are involved in JM phosphopeptide recognition, suggesting each phosphotyrosine has its designated binding pocket. Subsequent 3D NOESY‐HSQC experiments and crystallization trials are undergoing in order to resolve the peptide configuration in the complex of the Vav‐SH2 domain and the JM phosphopeptides.

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Purdue University Center for Cancer Research
AT THE CROSSROADS BETWEEN TYR AND SER/THR SIGNALING: A NEW PARADIGM IN THE REGULATION OF PP2A BY SRC KINASE
Authors: Sontag Estelle , Sontag Jean‐Marie , Gomez Rey J , Hoffman Alexander , Taleski Goce , Mazalouskas Matthew D , Hanks Steven K , Frohner Ingrid , Ogris Egon , Wadzinski Brian E
Published: November 2023

The major enzyme, Ser/Thr protein phosphatase 2A (PP2A), is a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer disease (AD), PP2A regulation remains relatively poorly understood, precluding the development of effective PP2A‐centric therapies. It has been reported more than two decades ago that the PP2A catalytic subunit (PP2Ac) is inactivated in vitro by phosphorylation at Tyr307 by tyrosine kinases such as v‐Src. Numerous studies relying on the use of commercial “PP2Ac pY307 antibodies” have subsequently linked enhanced phosphorylation of PP2A at Tyr307 with pathological cascades in cancer and AD. In contrast, we present here novel evidence in multiple cell types showing that PP2Ac is not phosphorylated at Tyr307. Rather, we uncover a novel mechanism of regulation of PP2A by Src that affects PP2A substrate specificity. Using specific PP2A mutants, we show that Src‐dependent PP2A deregulation can affect the phosphorylation of tau at Ser/Thr epitopes, a pathological hallmark of AD. It also influences the activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings unveil a novel functional Src kinase/PP2A signalling module that positions these enzymes at the interface between Tyr and Ser/Thr signaling pathways. We propose that dysregulation of this Src‐PP2A signaling node may contribute to cancer and AD.

Support or Funding Information

Supported by NHMRC grant APP1002744 and a grant from the Hunter Medical Research Institute, Australia (ES and JMS), and NIH grants GM051366 and DK070787 (BEW), and T32 GM08554 (MDM).
NADPH Oxidase 4 Expression Is Increased through TRPV4 Channel in D. farinae‐Induced Airway Remodeling and TGF‐β1‐Mediated Fibroblast Differentiation into Myofibroblasts
Authors: Al‐Azzam Nosayba Zakariya , Gombedza Farai , Snyderman Mathew , Thodeti Charles , Paruchuri Sailaja
Published: November 2023

NADPH oxidase 4 (NOX4) is a member of the NOX family of NADPH oxidases, encoded by NOX4 gene and has been implicated in TGF‐β1‐induced human cardiac fibroblast to myofibroblast differentiation. Further, NOX4 has been shown to potentiate lung fibroblast differentiation in vitro and its transcript level is enhanced in fibroblasts isolated from idiopathic pulmonary fibrosis (IPF) patients compared to normal ones. Our lab has previously identified that Transient Receptor Potential Vanilloid 4 (TRPV4) TRPV4, a mechano‐sensitive ion channel is involved in D. farinae‐induced airway remodeling during asthma in vivo and myofibroblast differentiation in vitro. However, the role of NOX4 in asthma, its interaction with TRPV4 and the mechanism of action still remains elusive. In the present study, we show that NOX4 is up‐regulated during lung remodeling in asthma and fibroblast differentiation in vitro in a TRPV4‐dependent pathway.

Using NOX4 antagonist and NOX4 siRNA, we demonstrate that NOX4 is involved in TGFβ‐1 induced α smooth muscle actin (α SMA) and fibronectin (FN) expression in human lung fibroblasts (HLF). Further, TGFβ‐1 treatment up‐regulated NOX4 gene expression as determined by real time PCR.. Further, to investigate the effect of TRPV4 on NOX4, we inhibited TRPV4 channel using a pharmacological antagonist RN 1734, and this led to a reduction in TGFβ‐1‐mediated NOX4 expression and differentiation. Interestingly, NOX4 transcripts were up‐regulated in lung tissues in response to D. farinae in an asthma model. More importantly, TRPV4 KO mice which were protected from D. farinae‐induced asthma also exhibited lower levels of NOX4 gene expression, suggesting that TRPV4 promotes airway remodeling through NOX4. Our results unravel a unique role for NOX4 in TRPV4 mediated fibroblast differentiation and lung remodeling and can be used as a new therapeutic target in asthma treatment.
Up‐regulation of atrial and neuronal Kir3 activity by cholesterol
Authors: Rosenhouse‐Dantsker Avia , Bukiya Anna N
Published: November 2023

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells, and its excess is associated with multiple pathological conditions including cardiovascular and neurodegenerative disease. In recent years, cholesterol has been emerging as a major regulator of ion channel function. Here we focus on G protein‐gated inwardly rectifying K+ (GIRK or Kir3) channels that play a key role in the regulation of membrane excitability in neuronal and cardiac cells.

The most common effect of cholesterol on ion channels is a decrease in channel activity. In contrast, we have recently shown that unexpectedly cholesterol enrichment up‐regulates Kir3 activity in atrial myocytes. Notably, we show here that also in freshly isolated CA1 pyramidal neurons from hippocampus, in‐vitro cholesterol enrichment results in a strong increase in physiological Kir3 currents.

To obtain mechanistic insights into the up‐regulation of cardiac and neuronal Kir3 channels by cholesterol, we employed planar lipid bilayers, mutagenesis studies using the Xenopus oocytes heterologous expression system, and molecular modeling. We first investigated the biophysical properties of the channel. Our data showed that the increase in channel activity following an increase in cholesterol levels is a result of an increase in the open probability of the channel and not in the unitary current amplitude and channel conductance. Next, we investigated why and how cholesterol up‐regulates Kir3 channels. Our studies traced the unique impact of cholesterol on cardiac Kir3 channels (up‐regulation and not down‐regulation) to a transmembrane region that includes residues in both the inner and outer alpha helices. Moreover, we identified putative cholesterol binding sites that overlap with the gating hinge region in the transmembrane domains of both cardiac and neuronal channels. Together, our studies provide novel insights into the molecular mechanism that underlies cholesterol‐driven up‐regulation of Kir3 currents in the heart and in the brain.

Support or Funding Information

Scientist Development Grant 11SDG5190025 from the American Heart Association (to ARD)