bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimizing a Screen for Incorporating Unnatural Amino Acids into Bacterial Proteins
Authors: Dhar Shilpa , Mammoser Claire , Gunter Amy , Kneusel Alyson , Burkhart Grace , Brown Bayland , Rowe Laura
Published: November 2023

Several unnatural amino acids (UAAs), including some fluorescent variants, were tested in a Green Fluorescent Protein (GFP) –based screening system that assesses the ability of the UAA's to be incorporated into proteins in vivo within E. coli bacteria. The fluorescent UAA variants were tested due to their ability to be utilized as tags or labels. Fluorescent UAAs also provide an alternative to bulkier fluorophores for applications such as more efficient cellular and protein imaging to visually track biomolecules in vivo. A number of research groups have incorporated UAAs, including fluorescent ones, into proteins using the amber suppression in vivo method also applied in this work1. However, in this work, UAA's that had never been incorporated into proteins using this method before were tested. The screening system was miniaturized and optimized so that it can be easily, and quickly used by researchers not expert in the field. Overall, over a dozen UAA's were tested with six different orthogonal tRNA/aaRS pairs2 using the screen, with several UAA's incorporating into the protein using a tRNA/aaRS that was not specific to that UAA. Moreover, the initial screen was miniaturized from 250 mL flasks to 96 well plates, and volumes, incubation times, and UAA solubility parameters were optimized in order to increase the efficiency of the screen.3.
Modification of Bovine Serum Albumin (BSA) with a Maleimido‐functionalized, 8‐arm Polyethylene Glycol Backbone (Mal‐PEG8) as a Drug Delivery System
Authors: Hill Jonathan , Huskic Dzenita , Parker Jacob , Carpino Ross , Olsen Kenneth W.
Published: November 2023

Due to its ability to transport hydrophobic materials through the bloodstream, human serum albumin (HSA) has become an intriguing drug delivery system with several formulations currently available. Key residues in the two main drug binding sites, DS1 and DS2, are conserved in BSA, providing a cheaper, more readily available alternative to HSA. Additionally, BSA may be selectively modified at its lone free sulfhydryl (Cys34), requiring no additional modifications. A multi‐BSA PEG8 complex (PEG8‐BSAx) would reduce the effect on osmotic pressure, avoid immunogenic response, and decrease the risk of drug toxicity while maintaining ligand binding capabilities. Several BSA molecules were successfully conjugated onto Mal‐PEG8 in a one‐pot reaction, requiring only BSA, Mal‐PEG8 and MOPS/NaCl buffer at pH 7.2. High molecular weight species (HMW) of PEG8‐BSAx were separated from the reaction mixture via Size Exclusion Chromatography (SEC). Helicity was maintained following PEGylation, as determined by Circular Dichroism (CD). Using a 4–15% gradient SDS‐PAGE gel, three bands of HMW material were visualized. Two bands corresponding to molecular weights of PEG8 complexed with two and three BSA molecules were seen in the separation zone. A heavy band was maintained in the stacking zone, indicating the presence of at least one other species of PEG8‐BSAx. Thermal stability of PEGylated‐BSA complexes was evaluated using UV‐VIS and CD. Fluorimetric assays were performed with Sulfamethoxazole (SMZ), Phenytoin (PHT) and 8‐Anilino‐1‐naphthalenesulfonic acid (ANS). SMZ has shown preference for DS1 while PHT prefers DS2. ANS is a fluorescent probe commonly used in binding studies. Dissociation constants (Kd) and the number of binding sites were consistent between unmodified BSA and PEG8‐BSAx. These results suggest that PEG8‐BSAx is a viable delivery system for water‐insoluble pharmaceuticals.

Support or Funding Information

Loyola University Chicago
Using Multivalency to Improve the Function of Synthetic Epigenetic Proteins
Authors: Tekel Stefan Josef
Published: November 2023

Epigenetic protein engineering is a valuable new tool for studying cell development and cancer, with the potential for novel medical applications. One major advantage to using synthetic epigenetic proteins with transcriptional effector domains over DNA binding domains is the scale of downstream effects. Typically, CRISPR, TALEs, and zinc finger based fusions influence one target gene, while epigenetic‐based proteins are capable of binding across the genome. However, many natural epigenetic binding proteins have poor binding affinity and multiple ligands, making their use in synthetic systems challenging. In one such case with a fusion of the natural methyl‐histone binding protein CBX8 and the activation domain VP64, reactivation of an artificially silenced gene was modest compared to reactivation from a DNA‐binding domain fused to the same activator. In an attempt to increase effector robustness, we built a library of multivalent constructs inspired by multivalent histone‐binding proteins found in nature (e.g. BPTF). Here, we present an in vitro pipeline for screening synthetic multivalent histone binding proteins for target binding, which is followed by an in cell assay to verify gene reactivation potential. First, the library of fusion proteins, each fused to a C‐terminal mCherry RFP tag, were expressed in an in vitro transcription and translation (IVTT) system. RFP signal indicated protein production efficiency. An ELISA was run to screen the proteins for target ligand (H3K27me3) and off‐target binding and narrow the pool for in‐cell characterization. The candidates with the best relative target binding were then expressed in‐frame with a C‐terminal VP64 activation domain in cells that carried an H3K27me3‐enriched luciferase reporter system and levels of gene reactivation were compared. The novelty of our pipeline lies in the low cost IVTT system coupled with a quantifiable reporter allowing for monitoring of in vitro translation efficiency, as well as in vivo transfection efficiency and nuclear localization of the fusions. Cellular luciferase assays indicated a significant level of gene reactivation with a multivalent fusion that contained a flexible linker (4x glycine‐serine‐serine‐serine) compared to a single domain fusion or a double domain with a rigid, helical linker (4x glutamic acid‐alanine‐alanine‐alanine‐arginine). These results demonstrate the ability to optimize synthetic multivalent proteins to increase effector output, as well as highlights the importance of linkers between functional domains.

Support or Funding Information

S. Tekel is supported by ASU SBHSE
LAFORA DISEASE PREMATURE TERMINATION CODONS (PTCs) ARE LIKELY CANDIDATES FOR SUPPRESSION BY AMINOGLYCOSIDES
Authors: Simmons Zoe R , Sherwood Amanda , Dukhande Vikas , Gentry Matthew
Published: November 2023

Personalized medicine‐based treatments are providing patient specific therapeutics for patients with single nucleotide changes resulting in a premature termination codon (PTC). These PTC mutations often result in truncated protein products. Typically, these truncated proteins provide little to no proper function or activity, and thus disease is imminent. Suppression of PTC mutations with novel compounds, including aminoglycosides, has been an effective therapeutic in Cystic Fibrosis and Duchenne Muscular Dystrophy. Lafora disease (LD) is another disease that can result from PTC mutations. In LD, autosomal recessive PTC mutations within the gene EPM2A cause dysregulation of glycogen metabolism. This glycogen storage disorder results in neurodegeneration, myoclonus, and epilepsy in the patient's second life, followed by death in the third decade. We have developed multiple in vitro models to test LD PTC suppression. Our data demonstrate that PTCs in Lafora disease are likely candidates for suppression with novel compounds. Suppression of PTCs in LD will provide a mutation‐specific genetic‐medicine that will cure patients of the disease as well as corroborate the evidence that PTC‐therapy may be an effective therapeutic across a vast range of diseases.
Discovery and Development of Allosteric Pantothenate Kinase Activators
Authors: Sharma Lalit Kumar , Subramanian Chitra , Yun Mi‐Kyung , White Stephen , Jackowski Suzanne , Lee Richard E. , Rock Charles O.
Published: November 2023

The central position of coenzyme A (CoA) in intermediary metabolism and its role in human neurodegenerative disease, led us to develop chemical probes targeting pantothenate kinase (PANK), the regulatory enzyme that controls CoA biosynthesis. There are four mammalian PANK isoforms that exhibit tissue‐specific expression and differ in their sensitivity to feedback inhibition by CoA and its thioesters. A high‐throughput screen and further medicinal chemistry efforts identified a novel class of orally bioavailable PanK modulators called pantazines. Optimization of the series based on lipophilic ligand efficiency (LipE) culminated in PZ‐2891, a drug‐like molecule that binds to PANK3 with nM affinity. Enzyme kinetics, biophysical studies, and X‐ray crystallography demonstrate that PZ‐2891 binds to the pantothenate substrate site of the PANK3•ATP•Mg2+ complex and extends across the dimer interface to form an induced fit, high‐affinity quaternary complex. PANK3 is a highly cooperative dimer and both protomer active sites simultaneously switch between the closed, active conformation that binds ATP, and the open, inactive conformation that binds acetyl‐CoA. PZ‐2891 inactivates the protomer to which it is bound and locks the opposite protomer in an active conformation that is refractory to feedback regulation by acetyl‐CoA. This allosteric property means that PZ‐2891 is a pantothenate‐dependent activator of CoA biosynthesis in cultured cells, and substantially increases intracellular CoA levels as a function of the media pantothenate supplement. Administration of PZ‐2891 to animals by oral gavage recapitulated the pantothenate‐dependent modulation of CoA levels in liver. PZ‐2891 dose‐response experiments also demonstrated the elevation of brain CoA illustrating the ability of PZ‐2891 to cross the blood brain barrier. PZ‐2891 and its inactive analogs are ready to be deployed as a toolkit to manipulate CoA levels in cultured cells and animals to analyze the role of CoA concentration in the control of intermediary metabolism. These nanomolar PANK allosteric effectors are being further developed as preclinical leads to validate PANK as a target to treat neurologic and metabolic disorders.

Support or Funding Information

NIH, ALSAC/St. Jude
Trehalose‐6‐Phosphate Phosphatase Structure and Inhibitor Design
Authors: Harvey Christine , Liu Chunliang , Globisch Daniel , Janda Kim , Dunaway‐Mariano Debra , Allen Karen
Published: November 2023

Trehalose is a sugar synthesized by many bacteria, fungi, plants and invertebrates. Trehalose is synthesized in a two‐step process involving trehalose‐6‐phosphate (T6P) synthase, which catalyzes the conversion of glucose‐6‐phosphate and uridine‐diphosphate‐glucose into T6P followed by T6P phosphatase (T6PP), which catalyzes the dephosphorylation of the phosphosugar to form trehalose. Without T6PP, the build‐up of T6P and absence of trehalose decreases the viability and virulence of fungi, bacteria and nematodes1,2,3. Therefore, T6PP has become a popular target for therapeutic intervention in diseases such as tuberculosis and lymphatic filariasis caused by infectious agents. Here, we present the 1.89 Å structure of Salmonella typhimurium (St) T6PP. The x‐ray crystallographic structure of apo St T6PP shows features seen in the haloalkanoic acid dehalogenase superfamily (HADSF) type II members4, with a core Rossmann‐like fold (residues 1 – 95, 175 – 246) and an α/β fold cap domain (residues 96 – 174). Overlaying a recently published structure of T6P‐bound fungal T6PP5 with St T6PP shows in addition to the four core motifs that bind the phosphoryl group in HADSF members, the cap residues Glu123, His132, and Glu167 are involved in binding the substrate. Unlike most unliganded HADSF structures, the St T6PP structure without cofactor or bound substrate has a closed conformation of the cap domain with respect to the core domain. Therefore, this crystal form may be amenable to soaking inhibitors into the active site. Inhibitor‐bound structures will help rationally design inhibitors to increase specificity and potency. In our laboratories, inhibitors of T6PP from several organisms have been uncovered by screening of an FDA approved drug library. These include Closantel and Cephalosporin C. Future efforts will explore incorporation of additional binding interactions from the Closantel and Cephalosporin C scaffolds to create a highly specific inhibitor for T6PP.

Support or Funding Information

This work was funded by R21‐AI103484 to KNA and DD‐M.
Tri‐Nucleotide Rolling Circle amplification; a novel method for the detection of RNA and DNA
Authors: Zingg Jean‐Marc , Daunert Sylvia
Published: November 2023

Most natural DNA and RNA are devoid of long Tri‐Nucleotide (TN) stretches of sequences that lack one specific nucleotide (Missing Nucleotide (MN)). Here we developed a novel method that is based on Rolling Circle Amplification (RCA), in which the TN‐information of short TN stretches in DNA or RNA is sequence‐specifically recognized, transferred, extended, amplified and detected by Padlock probes that consist entirely of nucleotides complementary to the nucleotides present in the target sequence (complementary TN‐information). Upon specific head‐to‐tail annealing and legation to the TN‐target sequence, these Padlock probes represent extended complementary TN versions of the target sequence that can be further amplified by Tri‐Nucleotide Rolling Circle Amplification (TN‐RCA) into linear concatemeric long ssDNA. Since during TN‐RCA the MN (as dNTP) is not added to the reaction mixture during polymerization, only the correctly ligated circular Padlock will amplify, and background amplification will not occur with endogenous RNA or DNA (which mostly would require the presence of all fourd NTP). Therefore, in the absence of non‐specific amplification, various labeledd NTP can be added to the TN‐RCA reaction that enables the separation, isolation and detection of the amplified concatemeric linear ssDNA. Here we evaluate the TN‐RCA method with RNA/DNA derived from Zika virus and human papilloma virus(HPV). TN‐RCA is a novel isothermal amplification technique that can be used for rapid and sensitive sequence‐specific detection and diagnosis of natural and synthetic DNA or RNA containing TN stretches (genomic DNA, bacteria, virus, mutations, polymorphisms, DNA methylation) with low background in short time.

Support or Funding Information

Coulter Foundation
Supercritical Fluid Chromatography‐Tandem Mass Spectrometry Method Development for the Detection of the Rac/Cdc42 inhibitor MBQ‐167 in Mouse Plasma
Authors: del Mar Maldonado Maria , Bloom Joseph , Hernandez‐O'Farrill Eliud , Vlaar Cornelis , Rodriguez‐Orengo Jose F , Dharmawardhane Suranganie
Published: November 2023

Metastasis is the principal cause of death for most cancer patients; however, effective treatment options are limited. Targeting pivotal regulators of metastasis the Rho GTPases Rac and Cdc42 is a rational approach for novel metastasis targeted therapy design. Therefore, we developed the small molecule MBQ‐167 as a promising new Rac/Cdc 42 inhibitor, which inhibits mammary tumor growth and metastasis in immunocompromised mice by ~90% and is ten times more potent than other currently available inhibitors. However, further studies are needed to determine the pharmacokinetic (PK) parameters of MBQ‐167 in a mouse model, which are essential to understand the mechanisms of drug distribution and elimination of this compound. Hence, it is necessary to develop a rapid and sensitive method to quantify MBQ‐167 in plasma. Supercritical fluid chromatography (SFC) uses supercritical carbon dioxide (SCO2) as mobile phase and offers advantages such as high sensitivity, increased resolution, and rapid analysis times. Also, it is an environmentally friendly technique, since is requires less organic solvents compared to High Performance Liquid Chromatography (HPLC). The objective of this study was to develop a method for the quantification of MBQ‐167 in mouse plasma using SFC coupled with electrospray ionization tandem mass spectrometry (SFC/MS/MS). Plasma samples were subjected to protein precipitation with acetonitrile. Separation was performed on an ACQUITY UPC2TM BEH (3.0 × 100 mm, 1.7 μm) column at 35 °C with a mobile phase consisting of CO2 and methanol 0.1% formic acid (95:5, v/v) at a flow rate of 1 mL/min. A tandem triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode. The method was sensitive with a lower limit of quantification (LLOQ) of 2.5 ng/mL and a lower limit of detection (LOD) of 1.25 ng/mL. Preliminary linearity was observed over the concentration range 2.5–1000 ng/mL. The data obtained supports the use of SFC/MS/MS as a highly specific method to evaluate the pharmacokinetics of MBQ‐167 in mice.

Support or Funding Information

Susan G Komen for the Cure and Puerto Rico Science, Technology, and Research Trust (PRSTRT) grants (to SD), UPR RCM NIH/NIMHHD grant G12MD007600 (to MM)
Genome‐Wide Screen for Escherichia coli [NiFe]‐Hydrogenase Maturation Factors
Authors: Lacasse Michael Joseph , Côté Jean‐Philippe , Brown Eric D. , Zamble Deborah Beth
Published: November 2023

[NiFe]‐hydrogenases catalyze the reversible oxidation of hydrogen gas at an intricate bimetallic active site to facilitate the anaerobic growth of many bacteria and archaea.(1) The maturation of this enzyme relies on a network of cellular pathways: for instance, the incorporation of nickel and iron cofactors both rely on specific metallochaperones as well as their respective metal homeostasis networks.(2) Therefore, [NiFe]‐hydrogenase activity can serve as a beacon for many related biochemical pathways. Furthermore, the ability to tune [NiFe]‐hydrogenase activity has garnered much interest for potential applications to the hydrogen economy and as a novel antibiotic target.(3,4) However, current methodologies to measure hydrogenase activity are time consuming and require specialized equipment, limiting discoveries of novel hydrogenase‐related applications. In order to answer these issues, we designed, optimized, and validated a [NiFe]‐hydrogenase assay in E. coli that is amenable to high‐throughput screening. We have applied this assay to screen the Keio collection(5) in search of the remaining genes that contribute, directly or indirectly, to [NiFe]‐hydrogenase maturation. Here we report the discovery and characterization of genes that had no previously known function.

Support or Funding Information

This work was supported in part by funding from the Natural Science and Engineering Research Council (Canada) and the Canadian Institutes of Health Research.
Poly(lactic‐co‐glycolic) Nanoparticles for biodegradable Chemotherapy Drug Delivery System
Authors: Liang Barry
Published: November 2023

The use of nanoparticles as chemotherapeutic agents holds tremendous promise to revolutionize pharmacology due to their adjustable target specificity, controlled drug release mechanism, and the ability to increase bioavailability. Recently, biodegradable polymers have been shown to act as effective encapsulating agents for site‐specific drug delivery with the added benefit of natural degradation to non‐toxic byproduct metabolites. Here we have prepared poly (lactic‐co‐glycolic acid) nanoparticles (50 nm – 1 μm) using a wet halogenation synthesis. Particle morphology and size were characterized with scanning electron microscopy, and their composition was analyzed with FTIR. UV/Vis spectroscopy was used to quantify the concentration of the particles and the drug loading within them. Based on our preliminary studies with antimicrobial loading of these polymer particles on the cell death of Escherichia coli bacteria, cancer treatment‐active particles were prepared by mixing camptothecin (DNA enzyme topoisomerase) during the synthesis, and these agents were tested for their treatment efficacy in vitro on the MCF‐7 breast cancer cell line. Here, we studied and compared the efficacy of the bare drug, bare nanoparticles, and washed polymer particles.
Biomimetic Nano‐Patterned Design on the Surface of the Implants to Prevent Bacterial Infections
Authors: Boucher Brant , Wolyniak Michael J , Hargadon Kristian M , Dua Rupak
Published: November 2023

Recent advances in materials and manufacturing processes have allowed fabrication of intricate implant surfaces to facilitate bony attachment and prevent bacterial infections. However, the rise of antibiotic resistant bacteria has become a threat to patients and the healthcare industry. New strategies to combat infection must be developed for use in conjunction with the traditional antibiotic administration. In the case of biomedical implantation, biofilms of these bacteria regularly prevent successful osseointegration with the implant through competition with osteoblasts, ultimately requiring surgical implant removal. Occasionally, these infections become systemic, requiring long‐term hospitalization and resulting in death due to the systemic spread of the infection in some cases. New attempts to promote osseointegration while discouraging biofilm development have focused on physically and chemically altering the surface of titanium, which is commonly used in dental and hip implants because of its natural resistance to corrosion and high strength relative to its weight. Our research, inspired by cicada wing structure, aims to create nano‐pillar structure that will prevent bacterial adhesion while promoting bone cell spreading. An alkaline hydrothermal process was conducted for three different durations of time (3 hours, 8 hours and 12 hours in NAOH 1M solution at 600°C) to modify the surface of titanium with unique nano‐patterns. Surface characterization of the samples for different groups was investigated by Zeiss Evo 50VP Scanning electron microscope. Further antibacterial characteristics for the 3 groups will be studied using fluorescence microscopy of E. coli cells that will be allowed to grow on the titanium samples for 5 days. The SEM images revealed spike‐like structure on the surface of titanium that underwent alkaline treatment for 3 and 8 hours. The structures were more persistent in the 8‐hour group as found in cicada wings but were arranged in a random order. The group that underwent treatment for 12 hours has fewer spike structures but more pits with thick boundaries. In conclusion, based on our preliminary results we think that the 8‐hour group will prevent bacterial adhesion because of the presence of spikes that will pierce the bacterial membrane while the 12‐hour group will favor the growth of bacteria by providing sites for attachment. This kind of surface treatment may seem to be a promising technique to prevent bacterial infection on the implants.
Mass Spectrometry‐Based Salivary Protein Biomarker Discovery in Autism Spectrum Disorder (ASD)
Authors: Wormwood Kelly L , Charette Laci , Ryan Jeanne P , Woods Alisa G , Darie Costel C
Published: November 2023

ASD affects approximately 1/68 children in the United States and is characterized by repetitive behaviors, communication deficits and impairments in social interactions. Males are affected approximately 4.5 times more than females and diagnosis typically occurs when a child is about 3 years old. There is currently no biologically based diagnosis or clear, consistent cause of ASD. There are also no approved medications that treat any of the core symptoms of ASD. Here, saliva samples from people with ASD and matched controls were analyzed using a combination of gel electrophoresis (SDS‐PAGE), in gel digestion or in solution digestion, followed by nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) to investigate differences between the proteomes of people with ASD and matched controls. Several protein differences were identified between ASD and control samples indicating dysregulation in immune response as well as gastrointestinal problems. These protein dysregulations may lead to a potential biomarker signature that could be used for diagnosis, possible therapeutic targets and an altogether better understanding of the disorder.

Support or Funding Information

Walker Foundation and ASPIRE Fellowship through Clarkson University
Proteomics and Functional Genomics of the D. grimshawii Sperm and Associated Male Reproductive Tract Tissues
Authors: Karr Tim , Oses Juan , Burlingame Alma , Oliver Brian
Published: November 2023

High throughput shotgun (bottom up) mass spectrometry has become a staple for the study of male reproduction in Drosophila and other diverse taxa. First applied to the Dmel, a growing list of sperm proteomes in mammals and other arthropods is opening up new areas of comparative and functional genomic studies not previously possible. However, very little is known about the proteomes of the associated reproductive tissues, and here we present a proteomic analysis of the D. grimshawii testis (TE), seminal vesicle (SV), accessory gland (AcG) and sperm (SP). These proteomes represent the first comprehensive study of these major components of the Drosophila male reproductive tract. Overall our study identified 4257 total UniProt IDs (1696 TE; 572 SV; 1084 AcG; 687 SP Flybase genes). From this list we obtained 1989 orthologous Dmel IDs for network, genomic and functional analyses. Comparative molecular evolutionary analysis will also be presented demonstating apparent sub‐functionalization of specific GO protein groups. This work establishes a framework for additional studies designed to provide deeper understanding of the evolution of male sexual reproduction traits.
Dissecting the proteome of Drosophila hybrids
Authors: Bamberger Tom Casimir , Montgomery Miranda , Martínez‐Bartolomé Salvador , Yates John R.
Published: November 2023

A cross between two related species generates hybrid animals that are viable, but sterile. Studying hybrids opens up the possibility to interrogate to what degree protein sequence heterogeneity still supports life of cells and organisms. Specifically, hybrid cells with two different haploid genomes need to cope with the extensive number of highly similar proteins that are present at the same time. Here we use quantitative proteomics to dissect the relative contribution of the two species‐specific, haploid genomes to the proteome in D. melanogaster □ D. simulans hybrid flies. The contribution of each species‐specific proteome to the hybrid proteome is quantified with a new software tool ProteinClusterQuant, which accurately represents all peptide to protein relationships. We find that complexity of the hybrid proteome is high during development and later reduced in adult flies. Although genomes of both species are present in an exact ratio of 1:1 in viable hybrid daughter flies, proteins derived from the maternal genome are predominantly found. We conclude that maternally deposited factors guide the expression of the diploid genome towards stability in adult hybrid flies.
Dissection of Z‐disc myopalladin gene network involved in the development of restrictive cardiomyopathy using system genetics approach
Authors: Lu Lu , Gu Qingqing , Mendsaikhan Uzmee , Khuchua Zaza , Jones Byron , Towbin Jeffrey , Purevjav Enkhsaikhan
Published: November 2023

Background

Genetic differences mediate individual differences in susceptibility to cardiomyopathy and severity of disease symptoms. Myopalladin (Mypn) is one of the genes associated with familial cardiomyopathies including dilated, hypertrophic and restrictive cardiomyopathy (RCM). Mutant knock‐in mice carrying the human Q529X‐MYPN mutation recapitulate the RCM phenotype the same as seen in the patients. The aim of this study was to investigate the regulation of Mypn and identify gene networks involved in RCM development.

Methods

Gene expression values were measured in the heart of a large family of BXD recombinant inbred (RI) mice derived from C57BL/6J and DBA/2J. The proteomic data was collected from Mypn knock‐in and knock‐out mice. Expression quantitative trait locus (eQTL) mapping methods and gene enrichment analysis were used to identify Mypn gene pathway and evaluate co‐expression networks.

Results

We found a wide range of expression variation in Mypn among the large cohort of BXD RI strains. Two upstream genetic loci, modulators of the expression of Mypn, have been identified on chromosomes 1 and 5. Candidate genes within those loci include Ncoa2, Vcpip1, Sgk3, and Lgi2. We also identified 15 sarcomeric genes that interact with Mypn, and constructed the Mypn gene network. Among them, well‐known cardiomyopathy‐associated genes such as Ldb3, Des, Actn2, Fhod3, Tpm2, and Syne1 were found to be members of this network. Several novel genes (Myo18b, Fhod3, Myom1, Bmp10, Myl4, Obscn, and Pdlim5) were also related to the Mypn network. In particular, two members (Syne1 and Myom1) have been confirmed at the protein level as high chance genes working as modifiers to induce cardiomyopathy.

Conclusions

Through a systematic genetic strategy we found several novel genes interacting with Mypn that may play an important role in the development of RCM. This work demonstrates that a systematic genetics approach is a powerful tool for constructing co‐expression networks that define the biological process categories within which similarly regulated genes function.

Support or Funding Information

R01HL128350 (Lu L), R01 HL53392 and R01 HL087000 (Towbin JA)
Prediction of β‐Lactamase Proteins using Random Forest
Authors: White Clarence , Dukka KC
Published: November 2023

β‐Lactamases (BL) are enzymes produced by some bacteria that provide resistance to β‐Lactam antibiotics. Resistance to β‐lactam antibiotics is an especially severe threat because these antibiotics are effective against a broad spectrum of pathogen and have very low toxicity to humans. In this work, we developed an algorithm, termed RF‐PredBL, using Random Forest (RF) to predict whether a given protein sequence is a β‐lactamase enzyme or not and if so, it's corresponding B‐lactamase class. In order to validate our method, we compared our results to a well‐known predictor program called PredLactamase (PL) using various performance metrics. In order to mitigate the problem of inconsistent accuracy detection, we use three distinct methods in this work. We increased the data we use to train our predictor, we then used extreme feature engineering, and finally modified the Feature Extraction from Protein Sequence (FEPS) code. Lastly, we used a powerful ensemble algorithm, known as Random Forest (RF), to train our predictor.

In the testing of our predictor, we have determined that RF‐PredBL performs better or equally as well as the existing algorithms for BL classification. During the 10‐fold cross validation, we increased the accuracy of the classic BL prediction from 90.63% to 98.79%. We also increased the accuracy of Class A (61.82% to 98.07%), Class B (89.09% to 96.79%), Class C (70.91% to 95.94%) and Class D (70.91% to 99.84%) predictions. To further improve our predictor, we plan to add features that include physiochemical properties to our feature set and reevaluate their importance.
Metabolic Mediation of Antifungal Toxicity
Authors: Cabral Damien , Belenky Peter
Published: November 2023

Therapy with the potent antifungal amphotericin B is significantly impacted by dose limiting toxicity to host cells. Thus, to improve the efficacy of this antifungal, we must understand the molecular toxicology for both the target fungi and the mammalian host. Previous work has shown that fungicidal drugs, such as amphotericin B, induce DNA damage in fungal and mammalian cells, leading to considerable cytotoxic effects. Therefore, modulating the susceptibility of cells to DNA damage may provide a mechanism of fine‐tuning antifungal efficacy and reducing side effects. In both fungal and mammalian cells, NAD‐dependent histone deacetylases, or “sirtuins”, play a central role in the induction of apoptosis in response to genotoxic agents. Inducing sirtuin activity by elevating cellular NAD levels leads to reduced cellular death and improved protection from various forms of stress in fungi and mammals. We found that treatment of human embryonic kidney cells, a primary target of amphotericin B toxicity, with NAD precursors provides protection against amphotericin B toxicity. However, treatment of C. albicans with the same precursors did not alter toxicity, suggesting that modulating NAD levels may have a differential impact between fungi and mammals. Furthermore, decreasing intracellular NAD levels using FK866 increases amphotericin B toxicity in HEK293 cells, further suggesting a link between NAD metabolism and susceptibility to antifungals in mammals. Therefore, it is possible that we can identify novel therapeutic methodologies that improve antifungal efficacy while reducing toxic side effects by exploiting the innate differences in fungal and mammalian NAD metabolism.

Support or Funding Information

Research reported in this abstract was supported in full by an Institutional Development Award (IDeA) Network for Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103430
Cloning and Protein Overexpression of Chlamydomonas reinhardtii's Novel Gene Cia7
Authors: Gonzalez‐Cantu Hector , Vazquez Erick , Ynalvez Ruby
Published: November 2023

Heavy metal contamination presents a threat to biological systems. These metals interact with the catalytic‐domain of enzymes, can bind irreversibly to enzymatic residues, and interfere with their normal rate of activity. Biological systems have adapted certain mechanisms to attenuate this; one of them being the expression and synthesis of low‐molecular weight, thiol‐rich proteins, termed as phytochelatins and metallothioneins. One such organism, which may present such metal‐defense mechanism, is Chlamydomonas reinhardtii. C. reinhardtii is an excellent model organism used in studies of heavy metal tolerance, and, with the discovery of a putative, metal‐interacting gene, Cia7 in C. reinhardtii, the objectives of this study are (1) overexpress CIA7 in E. coli, for downstream metal‐binding assays (2) the nuclear transformation of C. reinhardtii, in order to generate insertional mutants, subsequently screened for antibiotic resistance, and heavy metal tolerance, in order to assess CIA7's role in metal homeostasis. The gene Cia7 was cloned into a protein overexpression vector, pMal C5X, in fusion, with a maltose‐binding protein, to be further purified, for downstream metal‐binding assays. From pMal C5X‐Cia7, the gene was subcloned and ligated to psL72 vector, in order to transform it into C. reinhardtii via electroporation. This study will report the cloning and overexpression of the novel gene CIA7, as well as the screening of insertional‐cia7 mutants. This study is of significance within the context of bioremediation and heavy metal contamination, specifically from metal‐contaminated bodies of water. The characterization of CIA7 as a metal‐binding protein would allow for its potential use as a metal sequestering agent. This would correlate to metal‐remediated ecosystems, which would eliminate the potential for metal poisoning in humans, as well as a method to quantify heavy metal contamination, by having C. reinhardtii serve as a biological marker for heavy metal contamination.

Support or Funding Information

Norman Hackerman Advanced Research Program
Design principles of pleiotropic G‐protein signaling through guanine nucleotide exchange modulators (GEMs)
Authors: Rangamani Padmini , Getz Michael , Ghosh Pradipta
Published: November 2023

Heterotrimeric (henceforth trimeric) G‐proteins are molecular switches that control signal transduction. Dysregulation of the G‐protein pathway can lead to aberrant signal transduction and herald oncogenesis. Canonical G‐protein signaling, which is triggered exclusively by G protein‐coupled receptors (GPCRs), is spatially and temporally restricted, i.e. triggered exclusively at the plasma membrane (PM) via a process that completes within a few hundred milliseconds. Recently, a new paradigm has emerged, in which tyrosine‐based signals initiated by growth factor receptor tyrosine kinases (RTKs; e.g., EGFR) are relayed via non‐canonical activation of trimeric G proteins; such signaling has unique spatiotemporal features and is orchestrated by a non‐receptor guanine exchange modulator (GEM), GIV (a.k.a Girdin). GIV‐GEM acts pleiotropically as a guanine exchange factor (GEF) for Gαi and a guanine dissociation inhibitor (GDI) for Gαs; such modulation of two opposing Gα‐proteins integrates downstream cAMP signals depending on the abundance of functional copies in cells, fueling sinister properties such as invasiveness, stemness, survival, chemoresistance and angiogenesis. Here we developed the first network‐based compartmental model for signal transduction between RTKs and G proteins; two pathways of paramount importance in biology. The primary goal of this model was to identify the mechanistic regulation of cAMP, PKA, and CREB in different cellular compartments over time and to predict how network crosstalk affects cAMP dynamics. The model was constrained by experimental data and was robust to changes in kinetic parameters but sensitive to changes in initial concentrations of signaling species. Our model not only captured the unique temporal aspects of RTK→GIV→Gαi/s→cAMP signaling, but also predicted previously unforeseen impacts of EGF signaling [receptor endocytosis, copy number variations, activating mutations, etc.] and PDE activity on GIV‐dependent cAMP modulation. Predictions of the model appear to hold true in various disease states, most prominently in cancers.
Student perspectives regarding the move towards student‐centered learning in the anatomy laboratory
Authors: Thangarasa Tharshika , Leddy John J , Ramnanan Christopher
Published: November 2023

In the compressed (<50 hrs) anatomy component of the University of Ottawa's MD Program, most time is devoted to laboratories that emphasize active learning in small groups with tutors that facilitate student‐driven education. A recent (2014) program evaluation study determined that students perceived the following: (i) the active learning environment was highly variable between groups, and was at times limited by teacher‐centered approaches; and (ii) a lack of direction and feedback was perceived by some to be a consequence of the emphasis on student‐centered learning. In light of these perceptions, the following changes were made in 2015–16: (i) there was increased training for tutors and a reduction in tutor‐to‐student ratio, to promote effective guided inquiry during lab sessions; (ii) itemized checklists were provided to add direction and structure to self‐directed learning; and (iii) online formative quizzes were provided so that students could gain insight and feedback regarding their progress. First and second year (M1 and M2) student feedback regarding the program and the tutors from the 2015–16 academic year was quantitatively and qualitatively analyzed to determine whether these changes effectively responded to student concerns, and assessment data was characterized to determine any impact on learning. Both M1 and M2 students indicated strong value for the formative quizzes, with 96% of students utilizing these resources. All students also noted satisfaction with the itemized checklists, and perceived that these resources facilitated self‐directed study in class time and provided guidance during exam preparation. In particular, M2 students (who experienced their first year labs without these tools) felt less reliant on tutor support and more certain regarding achievement of the learning objectives. While M1 students expressed strong satisfaction with the active learning environment, M2 students expressed some concern with the increased emphasis on student‐centered learning, and perceived their learning was impaired with the reduction in tutors. However, analysis of assessment data indicated that, in all regards (in‐lab quizzes and examinations), performance of M1 and M2 in 2015–16 was not adversely affected, relative to data from previous years (2012–2015). In conclusion, students generally highly rated the shift further along the student‐centered learning spectrum. Despite some concerns of students accustomed to more Faculty support, increased emphasis on student‐centered learning did not impair academic performance.

Support or Funding Information

University of Ottawa, Faculty of Medicine Summer Studentship Program (Innovation in Medical Education)
ERK1/2 Signaling through Scaffold Protein Shoc2 Complexing with H, K, and M‐Ras: A Structure‐Function Analysis
Authors: Norcross Rebecca , Galperin Emilia
Published: November 2023

The extracellularly‐regulated kinase ‐1 and ‐2 (ERK1/2) signaling pathway utilizes a network of proteins and signaling events to control many downstream effectors for cell proliferation, apoptosis, differentiation, and motility. The broad scope of ERK1/2's branched signaling capacity requires integrated fine‐tuning for specific signaling control. To this end, the pathway utilizes scaffolding proteins – essential regulatory proteins to facilitate the activities of enzymes in the pathway. Shoc2, the critical scaffolding protein within the ERK1/2 pathway, serves as a platform to create a temporal and spatial microenvironment for effective protein complex formation and signal transduction. The scaffold facilitates the interaction between Ras and Raf‐1 thereby accelerating ERK1/2 signaling. Our earlier studies show that Shoc2 also transduces ERK1/2 motility signals.

All canonical Ras isoforms (H, K, and M) have been reported to interact with Shoc2. We previously determined that M‐Ras recognizes Shoc2 sequences within the Shoc2's N‐terminal domain (amino residues 1‐82). Yet, the mechanisms responsible for the Shoc2 interaction with other Ras isoforms have not been defined. In this study, we are analyzing the structure‐function relationship of Shoc2 with different Ras isoforms and identifying the underlying mechanisms of these interactions. Biochemical and biophysical methods including immunoprecipitations, fluorescence microscopy and in vitro complex reconstitution are utilized. Moreover, any variation in downstream effects of Shoc2 differential binding to Ras isoforms will be determined. This study will also identify the specificity of Shoc2‐mediated ERK1/2 signals achieved via binding to the Ras isoforms. By deciphering the intricate details of protein interactions within the Shoc2's signaling module, we will expand our knowledge of how specificity of ERk1/2 signaling is achieved and controlled.

Support or Funding Information

Funding sources: National Institute of General Medical Sciences, American Cancer Society
DUOX1 silencing in lung cancer is associated with enhanced nuclear EGFR localization
Authors: Little Andrew , Danyal Karamathullah , Heppner David , Hristova Milena , van der Vliet Albert
Published: November 2023

Non‐small cell lung cancer (NSCLC) remains to be one of the leading causes of cancer‐related mortalities worldwide. The NADPH oxidase homolog, Dual Oxidase 1 (DUOX1), is an H2O2 producing enzyme located in the airway epithelium with key roles in mucosal host defense and wound repair mechanisms. Recent studies indicate that DUOX1 is epigenetically silenced in many forms of NSCLC via hypermethylation of its promoter. We previously demonstrated that DUOX1 silencing in lung cancer cells is closely associated with epithelial‐to‐mesenchymal transition (EMT) and enhanced tumor invasiveness and metastasis. However, the mechanism(s) by which DUOX1 silencing promotes these outcomes are not understood. Previous findings indicate that DUOX1‐dependent epithelial host defense pathways are mediated by redox‐dependent activation of epithelial signaling via the non‐receptor tyrosine kinase, Src, and the receptor tyrosine kinase, EGFR. We therefore hypothesized that loss of DUOX1 in lung cancer may be associated with aberrant regulation of Src and/or EGFR, tyrosine kinases that are frequently overexpressed and activated in lung cancer and strongly contribute to tumor growth and survival. In fact, recent studies have indicated that nuclear localization of EGFR in cancer cells is associated with metastatic cell behavior and poor clinical outcome, and the nuclear EGFR localization depends on Src‐dependent phosphorylation of EGFR at Y1101. We observed that overexpression of DUOX1 in alveolar lung cancer A549 cells, which possess EMT‐like features and in which DUOX1 is normally silenced, results in redistribution Src to the plasma membrane and decreased nuclear accumulation. DUOX1 overexpression in A549 cells also suppressed EGF‐stimulated nuclear translocation of EGFR, which was associated with reduced EGFR phosphorylation at Y1101. Conversely, RNAi‐mediated silencing of DUOX1 in the epithelial cancer cell line H292, which normally expresses DUOX1 expression, was found to promote EGF‐mediated EGFR nuclear translocation and Y1101 phosphorylation. Since nuclear EGFR is thought to enhance the transcription of target genes related to cell cycle progression and proliferation (e.g. CDK1, Myc, others), we evaluated gene expression of these target genes in our cell models. Indeed, in cells lacking DUOX1, EGF stimulation significantly enhanced mRNA levels of CDK1, Myc, and other target genes for nuclear EGFR, whereas no such induction was seen in cells that express DUOX1. Our findings indicate that DUOX1 silencing in lung cancer may be associated with worse prognosis, partly due to altered spatiotemporal regulation of EGFR and Src and increased nuclear targeting. Since both EGFR and Src are subject to redox regulation by cysteine oxidation, we are currently aiming to elucidate the molecular mechanisms by which these mechanism are affected by altered DUOX1 status
Ubiquitin‐like Protein Ubl4A Promotes Actin‐mediated Cell Migration
Authors: Zhang Huaiyuan , Zhao Yu , Affonso Carlos Rafael , Manas Adriana , Bonomo Raiza , Xiang Jialing
Published: November 2023

Ubiquitin‐like protein Ubl4A is a small and multi‐functional protein with no ubiquitination activity. Ubl4A is involved in a broad range of cellular activities from C‐tail protein anchoring to tumor suppression. We have shown that Ubl4A is also critical for translocation of signaling protein and cell migration. Ubl4A directly interacts with Arp2/3 to accelerate actin branching, allowing Akt translocation from cytosol to plasma membrane for activation. Here we show that Ubl4A, F‐actin and Arp2/3 are also co‐localized at the cell leading edges during wound closure. Deficiency of Ubl4A significantly reduces actin‐mediated membrane protrusion, and cell migration for wound healing. In addition, the ability of fibroblasts to migrate out of corneal tissue ex vivo is also impaired in Ubl4A‐deficient mice. Interestingly, cell motility, but not phagocytosis, is significantly decreased in Ubl4A‐deficient macrophages. These results imply an important role for Ubl4A in regulation of cellular signaling and migration‐associated pathophysiological processes.