bluuebunny/crossref_metadata_2025_split huggingface dataset:
Resolving Protein Interactions in Live Bacterial Cells through 3D Single‐Molecule Localization Microscopy
Authors: Richardson Charles Joseph , Rocha Julian , Gahlmann Andreas 
Published: June 2021

In living cells, proteins form dynamic complexes essential for the functional regulation of cellular activities. While in vitro assays provide key insights into protein binding partners, interactions may be artificially reduced or enhanced once the proteins are removed from their native context. Single‐molecule localization microscopy is ideally suited to measure spatial locations of fluorescently‐labeled molecules in living cells and thus enables single‐particle tracking of the entire distribution of molecular behaviors. Here, we apply high‐throughput 3D single‐molecule localization microscopy to measure the diffusion behaviors of over 100,000 individual intracellular bacterial proteins. Quantitative analysis of the single‐molecule trajectories using Monte Carlo simulations of confined diffusion resolves the co‐existence of multiple diffusive states in the bacterial cytoplasm. Molecular and genetic perturbations show that these diffusive states consist of distinct hetero‐ and homo‐oligomeric complexes. These findings provide new molecular‐level insights into how dynamic molecular complexes function in their native environment.Support or Funding InformationResearch was supported by a Hartwell Postdoctoral Fellowship to CR and startup funds from the University of Virginia to AG.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metal binding specificity in a putative nickel solute binding protein.
Authors: Amato Matthew , Yukl Erik 
Published: June 2021

Researchers are pressured to identify novel antibiotic targets in response to the epidemic of bacterial antibiotic resistance. Transition metal homeostasis is related to pathogenicity in gram‐negative bacteria. In particular, metal transporters of the ATP binding cassette (ABC) transporter family are essential for acquisition of specific metals in highly limited environments such as the human host. These systems depend upon a periplasmic solute binding protein (SBP) to bind the appropriate metal with high affinity and specificity. Transcriptional analysis in our model gram‐negative organism Paracoccus denitrificans showed three operons of note encoding ABC transporters to be upregulated under zinc deprivation. Two of these, aztABCD and znuABC, belong to the cluster 1‐A family, which is predicted to transport zinc. Further, the solute binding proteins (AztC and ZnuA) have been shown to bind extracellular zinc with high affinity and specificity in vitro. The third operon pden1341‐1345 encodes a cluster 5 ABC transporter system predicted to be a Nickel/di‐peptide transporter. Thus, it is perplexing that this operon should be significantly upregulated under conditions of zinc deprivation. To determine the possible metal substrate for the Pden1341‐1345 operon, the SBP Pden1341 was cloned, expressed, purified and binding to various metals was determined. Traditional cloning techniques failed by what is thought to be a complication of its large gene size. Gibson cloning proved an effective alternative to cloning full‐length pden1341. Anion exchange and size exclusion chromatography were used to purify Pden1341, and ICP‐OES analysis suggested Pden1341 purifies without bound metal. Binding to various metals was determined by a competitive fluorescence assay as well as isothermal titration calorimetry. The results of these experiments suggest alternate roles for the cluster 5 ABC transports beside the transport of nickel or dipeptides. Thus, cluster 5 ABC transporters may play unexpected functions in metal homeostasis among gram‐negative organisms that may be relevant to the development of metal transport inhibitors as novel antibiotics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Control of the rate limiting step by active site compactness: Reaction of UDPgalactopyranose mutase with UDP‐arabinopyranose
Authors: Sobrado Pablo , Pierdominici‐Sottile Gustavo , Tanner John J. 
Published: June 2021

Galactose is a common monosaccharide exclusively found in mammals as galactopyranose (Galp), the six‐membered ring form of this sugar. In Aspergillus fumigatus and mycobacteria, galactose in the cell wall is found as galactofuranose (Galf), the five membered ring form of this sugar. In these microorganisms, Galf biosynthesis begins with UDP‐Galp being converted to UDP‐Galf by UDP‐galactopyranose mutase (UGM). UGM is critical for the survival and proliferation of several prokaryotic and eukaryotic pathogens. Kinetic analyses have shown that bacterial UGM can also catalyze the isomerization of UDP‐arabinopyranose (UDP‐Arap). The possibility of using this sugar as a substrate for eukaryotic UGM has not been evaluated. Here, we probe the Galp‐Galf and Arap‐Araf isomerization catalyzed by Aspergillus fumigatus UGM using viscosity effects, solvent kinetic isotope effects, site directed mutagenesis, and computational biology. Kinetic studies show that eukaryotic UGM use UDP‐Arap/f as a substrate but with a much lower catalytic efficiency than UDP‐Galp/f. The data is consistent with a switch mechanism controlling the rate limiting steps, from the chemical step for UDP‐Galp to product release with UDP‐Arap or the Trp315Ala mutant. Computational studies suggest that tightness in the active site is responsible for this distinct behavior when either the substrate is changed or the side chain of Trp315 is deleted. These results provide important insights into understanding general enzyme catalysis and the UGM reaction mechanism, and will facilitate the development of inhibitors.Support or Funding InformationResearch reported in this publication was supported by grants from the National Institute of General Medical Sciences (NIGMS) award R01GM094469, the National Science Foundation award CHE‐1506206, and PICT 2436 préstamo BID. The authors are also very grateful to CONICET for financial support.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutational analysis of the Clu2593c homing endonuclease reveals specific residues required for DNA binding and cleavage.
Authors: Davis Lorraine , Takushi Brittany , Kaiser Brett K. 
Published: June 2021

Homing endonucleases (HEGs) are selfish genetic elements that recognize and cleave long DNA target sequences (18–22 basepairs) within an organism's genome. This cleavage induces homology‐directed repair using the HEG‐encoding gene as a template, facilitating its lateral transmission. The I‐CreI family of HEGs is present within organellar genomes of diverse single‐celled algae species and is incorporated at identical positions within the large ribosomal subunit gene. Homologs of this family, which share 35–70% pairwise amino acid identity, have evolved to recognize nearly identical target sites while tolerating sequence polymorphisms along the DNA target site. Although the I‐CreI and I‐CvuI enzymes have been well characterized, the mechanisms by which other homologs of this family achieve target sequence recognition are not well understood. In order to explore the structure‐function relationship of the I‐CreI family, we carried out a detailed molecular‐level analysis of the I‐CreI homolog Clu2593c (57% sequence identity). Based on a co‐crystal structure of WT Clu2593c bound to DNA, we generated 12 single‐residue mutations at positions in the enzyme‐DNA interface. We then tested the functionality of the bacterially‐expressed and purified mutant proteins using in vitro binding and cleavage assays. Additionally, we solved co‐crystal structures of a subset of the mutants bound to DNA. Our analyses revealed that several of the mutations behaved in a similar manner to analogous mutations in I‐CreI, whereas others indicated divergent mechanisms of DNA recognition. These results highlight that homologs of the I‐CreI family have evolved distinct solutions to recognizing a shared DNA target.Support or Funding InformationMurdock Charitable TrustThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antitumor Activity of Simarouba tulae Extracts in a Panel of Cancer Cell Lines
Authors: Del Moral Isabel Conde , Reyes Jeyshka , Vivas‐Mejia Pablo , Ospina Claudia 
Published: June 2021

Cancer is among the principal causes of death globally. Statistics from the American Cancer Society project an increase of 50% in new cases and 60% in worldwide deaths by 2030. Although several chemotherapeutic agents are currently being used for the treatment of most cancer types, recurrence, drug resistance, and severe side effects demand the need for alternative novel drugs. Even with the evolution of synthetic chemistry, nature remains as a fundamental source of molecular diversity and structural innovation. Of the 140 anti‐cancer agents approved since 1940, over 60% could be traced to a natural product. One of the most popular bioactive molecules found in natural extracts are quassinoids which are highly oxygenated triterpenes found in the Simaroubaceae family. Simalikalactone D is a quassinoid isolated from Simarouba tulae, an endemic tree from Puerto Rico. Our objective was to evaluate the anticancer effect of this Simalikalactone D in a panel of human cancer cell lines. The cytotoxicity was evaluated in vitro through dose‐response assays in A2780CP20 cisplatin‐resistant ovarian cancer and in MDA‐MB‐231 breast cancer cell lines. Our results showed that Simalikalactone D exhibits significant cytotoxicity against both cell lines. Partial organic extracts of Simarouba also were toxic to these cells lines. The IC50 values were around 50–100 nM which indicate that Simalikalactone D is a potential anticancer drug. Ongoing experiments evaluate the cytotoxicity of Simalikalactone D in other cancer cell types including colon and prostate cancer cells, and in normal cells. To assess the mechanism of action of this compound, future experiments will measure the effect of Simalikalactone D on activation of apoptosis, cell cycle progression arrest and in proteins associated – with these biological effects.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103475.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis of Fluorescein‐linked, Zinc‐based Metal Organic Frameworks as Carriers of Targeted Treatments for Retinopathies
Authors: Fralish Zachary , Bromfield‐Lee Deborah , Eubank Jarrod , Shelby Shameka 
Published: June 2021

Metal‐Organic Frameworks (MOFs) are compounds composed of metallic ions linked by organic ligands that form porous units with highly specialized shapes and sizes. Beyond this, MOFs have intrinsic biodegradability, chemical diversity, and a high loading capacity. These characteristics make MOFs optimal candidates as vehicles for drug delivery. More specifically, these programmable, nano‐like particles have the potential to hold specific materials and dissolve in particular environments which makes entrapment and targeted release of various treatments possible. These qualities could be advantageous when treating diseases of the retinal pigment epithelium (RPE). Current treatment involves subretinal injection, which requires a skilled surgeon and often results in retinal detachment and occasional proliferative vitreoretinopathy which leads to acute deterioration of visual facility. MOFs with entrapped treatments can potentially be injected into the vitreous of the eye and targeted to release their payload in the RPE, removing the risk of retinal tearing and the need of a skilled surgeon. To pursue this potential, we modified 4,5‐imidazoledicarboxylic acid (ImDC) ligands with fluorescein via an esterification reaction, while synthesizing MOFs with ImDC. The inclusion of fluorescein will allow for observation of MOF incorporation and processing within the cell. Analysis of the esterified ligand through Gas Chromatography‐Mass Spectroscopy and Nuclear Magnetic Resonance, including homonuclear correlation spectroscopy, indicated that the ligand had been modified. Zinc‐based metal‐organic frameworks with ImDC ligands were synthesized and analyzed by Powder X‐Ray Diffraction to confirm the crystalline structure of the framework. The MOFs were also shown to be partially soluble in potassium chloride and sodium chloride following sonication. Preliminary studies of zinc‐based MOFs in HEK‐293 cells have indicated that the MOFs will likely enter the cells by a phagocytic mechanism. Taken together, these studies will lead to the creation of a system to assess the functionality of MOFs in drug delivery. Future studies will investigate the use of various metals to direct the cellular processing of MOFs.Support or Funding InformationFlorida Southern College Faculty‐Student Collaborative Research GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Fruits on the Cancer Incidence in Cusco Peru
Authors: Jones Janae , Munoz Crystabel , Ledesma Litza , Faustino Vroniqa , Nogaj Luiza A. , Roberts Lia , Deprele Sylvine 
Published: June 2021

Breast cancer remains as one of the most prevalent types of cancer in the world. Although Western industrialized nations rely on a contemporary approach to modern medicine, many developing countries, such as Peru, rely on traditional medicine and alternative natural remedies. Therefore, the focus of this study was to determine the extent to which natural remedies and nutrition, contribute to the prevention and incidence of cancer. Here, we focus on the use of certain fruits, like papaya and the noni fruit, as natural remedies for illness. First, survey data was collected in Cusco, Peru regarding which fruits are consumed as natural remedies for illness. Then, molecular techniques such as MTT assay and LDH Cytotoxicity assay were used to test the effect these fruits have on HeLa cells. The survey results showed, papaya and noni as the most commonly used fruits for illness. Noni powder and papaya fruit were extracted with ethyl acetate and ethanol and used in MTT and cytotoxicity assays. Preliminary results in mammalian cells showed no effect of the ethyl acetate extracts on the viability of HeLa cells. However, MTT assay results for the ethanol extraction of papaya skin showed a decrease in mammalian cell viability at high concentrations. Further experiments will be conducted to test fresh noni and papaya as well as other commonly eaten fruits in Peru for their anticancer properties.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adoption of DNA Aptamer‐Based Biosensors into Nanoparticle‐Based Complexes for Raman Detection
Authors: Clark Derek , Bonham Andrew J. 
Published: June 2021

DNA aptamers have been engineered that bind a variety of targets with high specificity. However, turning that binding into a measurable change to enable detection of the target can be challenging, and has inspired multiple detection approaches. Here, we present a sensitive assay of DNA aptamer‐based detection for biological targets using surface enhanced Raman scattering (SERS) by gold nanoparticles long range plasmonic coupling. Gold nanoparticles have unique electrical and optical properties, including the promotion of SERS, wherein closely spaced nanoparticles dramatically increase the sensitivity of Raman spectroscopy, a molecular fingerprinting technique. We have synthesized 60nm gold nanoparticles (AuNP) that produce increased SERS. These AuNP are directly functionalized with thiolated DNA aptamers that contain a terminally conjugated Raman reporting agent (methylene blue), complementary DNA is conjugated to separate AuNP to allow controlled annealing of functionalized AuNP into multiple‐particle complexes (multiplexes). Controlled introduction of silver nitrate further increases SERS by silver coating AuNP multiplexes, causing roughened surfaces (localized surface plasmon resonance) and reducing multiplex gaps. These silver coated multiplexes provide SERS amplification and minimize signal‐quenching geometries. We demonstrate that the binding of target proteins (including Ricin A chain (RTA) and the Myc/Max complex) to the DNA aptamers cause a change in the conformational dynamics of the aptamer, by taking a signal‐OFF approach for target protein detection. Aptamer conformational changes effectively disassembles previously annealed multiplexes, that emit an intense dye “fingerprint”. Aptamer based conformational changes are favorable in the presence of the target protein. Introduction of target protein effectively breaks hybrid silver coated multiplexed AuNP in a concentration dependent fashion, releasing functionalized silver coated AuNP. Bound target protein effectively extinguishes the dye “fingerprint” as target concentration increases. Released silver coated AuNP reduce long‐range plasmonic coupling and signal‐quenching geometries reform as multiplexes disassemble, creating a reduction in signal. Ultimately, such assemblies should offer a stable optical platform for the creation of highly sensitive biosensors for a range of biological targets.Support or Funding InformationThis work is supported by internal grant funding from the Metropolitan State University of Denver.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolomic Analysis of HER2‐positive Breast Cancer Cells
Authors: Bush Jason A. , Sahni Malika K. , Ravindran Resmi K. , Khan Imran H. , Krishnan Viswanathan V. 
Published: June 2021

PurposeHER2‐positive breast cancer accounts for more than 20% of the diagnosed cases and is characterized by aggressive growth, increased disease recurrence, and poor prognosis. While much of the signaling mechanism(s) have been elucidated, the impact of HER2 positivity on cellular metabolism is not well understood. We examined (1) gene expression profiles of mammalian breast cancer cell lines with increasing HER2 expression levels (MCF10A, MDA‐MB‐468, MDA‐MB‐453, HCC1954, HCC2218) and correlated changes to metabolic expression profiles and (2) the effect of serum starvation and pervanadate treatment on the metabolic profiles of these HER2‐expressing cell lines.MethodsOur study used a combination of 1H‐NMR and 2D‐NMR (TOCSY), multivariate analyses, and protein expression validation in human breast cancer cell lines (untreated, serum‐starved, serum‐starved with pervanadate treatment).ResultsWe found that both gene and metabolic expression profiles of HER2‐positive cell lines correlated with leucine and isoleucine biosynthesis (p‐value < 0.05). Comparative analyses of profiles from untreated, serum‐starved, and serum‐starved with pervanadate‐treated cell samples revealed that lactate concentration (p‐value <0.05) dropped significantly with serum starvation and pervanadate treatment consistent with reduced glycolytic metabolism and potentially mediated through suppression of tyrosine phosphatase activity.ConclusionThis study highlights important metabolites that are potential biomarkers of HER2‐positive breast cancer and suggests alternate/novel pathways to consider for future understanding and treatment.Support or Funding InformationThis work was funded in part through intramural programs at Fresno State to MKS and JAB, and through the NIGMS 1SC3GM122630‐01 to JAB.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Urine Odor Profiling for Diagnosis of Interstitial Cystitis
Authors: Park Rebekah Julie , Shahid Muhammad , Yeon Austin , Kim Jayoung 
Published: June 2021

Interstitial cystitis (IC) is a clinical condition that manifests as a sensory hypersensitivity of unknown cause and is characterized by frequent urination, bladder discomfort, and pelvic pain. Chronic inflammation is a potential systemic risk factor for IC. However, it remains unknown the underlying mechanism through which a healthy bladder protects itself from inflammatory triggers. In this study, we profiled urinary odor, which consists of various volatile organic chemical compounds in urine obtained from IC patients and healthy controls. Using comprehensive solid phase micro extraction‐gas chromatography‐time‐of‐flight mass spectrometry profiling combined with comprehensive bioinformatics analyses, levels of urinary volatile metabolites in healthy individuals were compared to those in IC patients. Among the several metabolites whose levels were significantly altered, menthol showed significantly reduced levels in IC. In an attempt to understand the mechanistic meaning of IC‐associated volatile metabolite, menthol, we performed cytokine profiling and DNA microarray. Our findings suggest that inflammatory events and the activation of signaling networks were suppressed by menthol treatment. Further validation with biochemical experiments and Western blot analysis confirmed that menthol reduced the expression of cytokines and signaling factors that are closely associated with inflammation. These findings will provide potentially new strategies to alleviating both the odor and inflammation associated with IC.Support or Funding InformationThis work was supported by the National Institutes of Health grants (1U01DK103260, 1R01DK100974, U24 DK097154, NIH NCATS UCLA CTSI UL1TR000124), Department of Defense grants (W81XWH‐15‐1‐0415), Centers for Disease Controls and Prevention (1U01DP006079), IMAGINE NO IC Research Grant, the Steven Spielberg Discovery Fund in Prostate Cancer Research Career Development Award, and the U.S. ‐ Egypt Science and Technology (S&T) Joint Fund, funded by the National Academies of Sciences, Engineering, and Medicine and USAID (all to J.K.). Any opinions, findings, conclusions, or recommendations expressed in this article are those of the authors alone, and do not necessarily reflect the views of any of the previously mentioned sponsors. J.K. is former recipient of Interstitial Cystitis Association Pilot Grant, a Fishbein Family IC Research Grant, New York Academy of Medicine, and Boston Children's Hospital Faculty Development.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.