bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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.
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 InformationFunded 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 InformationSupport 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 InformationFunded 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 InformationFunded 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 InformationThis 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 InformationFunding 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 InformationThis 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 InformationSupport 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 InformationThis 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 InformationClarkson University