bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationCoulter 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 InformationSusan 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 InformationThis work was supported in part by funding from the Natural Science and Engineering Research Council (Canada) and the Canadian Institutes of Health Research.
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 InformationWalker Foundation and ASPIRE Fellowship through Clarkson University
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

BackgroundGenetic 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.MethodsGene 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.ResultsWe 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.ConclusionsThrough 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 InformationR01HL128350 (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 InformationResearch 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 InformationNorman Hackerman Advanced Research Program
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 InformationUniversity 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 InformationFunding sources: National Institute of General Medical Sciences, American Cancer Society