bluuebunny/crossref_metadata_2025 huggingface dataset:
Postmortem Samples from Aviation Accident Victims Maintain Tissue‐specific mRNA Expression Profiles
Authors: Burian Dennis , Hutchings David C , Uyhelji Hilary A , Williams Daniel L , White Vicky L
Published: November 2023

The translation of marker panels for aerospace medical factors from discovery phase to a forensic setting requires that RNA stability and the factors that affect it be assessed in post‐accident samples. Increased understanding of these parameters will allow investigators to optimize assay design and conditions to compensate for expected post‐accident sample degradation.

To assess the utility of samples from accident victims for gene expression analysis, total RNA was purified from accident victim blood and tissue samples. Purified RNA was characterized for integrity, yield, purity and the origin of ribosomal RNA. Data was gathered regarding accident conditions and postmortem interval. Gene expression data was gathered by qRT‐PCR for panels of genes on victim RNA. Live subject data was obtained from microarray data available in public databases. Correlation analyses were performed to determine mRNA expression patterns within victim samples and tissue‐specific expression patterns between live and victim samples. Primers specific for bacterial rRNA were characterized for sensitivity and specificity on purified E. coli RNA, then used on purified accident victim RNAs exhibiting either prokaryotic or eukaryotic rRNA alone, both rRNAs, or no rRNA peaks.

RNA integrity was highly variable between victim samples but there was no correlation between RNA integrity and yield, postmortem interval, trauma level, or accident condition parameters. Across 35 genes in 87 victim samples, mRNA levels were generally very highly correlated suggesting that mRNA degradation is not dependent on accident‐related factors and, for these genes, is mechanistically similar. In a comparison of victim samples to live subject expression levels with 44 genes, blood, brain, and muscle relative expression levels were moderately well correlated. The assay for bacterial rRNA was found to be sensitive for very low levels of purified bacterial RNA and highly specific for bacterial rRNA when challenged with mixtures of bacterial and human RNA but had lower Cts than expected in accident victim samples without prokaryotic rRNA peaks.

In conclusion, aviation accident victims exhibit a wide range of degradation but very few failed to yield qRT‐PCR data of sufficient quality that relative expression values between target genes were not preserved. That victim samples have similar expression patterns to live subject data demonstrates that marker panels developed in human subject studies can be translated to accident investigation and will allow additional information to be incorporated into accident investigation.

Support or Funding Information

FAA Office of Aviation Medicine, FAA Office of Accident Investigation and Prevention
Functional Characterization of a Cysteine‐Less Isoform of the Human GLUT2 Hexose Transporter
Authors: Reyes Alejandro Mauricio , Pérez Alejandra Andrea , Vargas‐Uribe Mauricio , Elgueta Coral , Cuevas Alexei , Ojeda Lorena , Arce Robinson , Salas Monica
Published: November 2023

The GLUT2 carrier is the major glucose transporter isoform expressed in hepatocytes, insulin‐secreting pancreatic beta cells, and absorptive epithelial cells of the intestinal mucosa and kidney. GLUT2 is suggested to play a critical role in maintaining glucose homeostasis because it functions as a low affinity, high‐turnover transport machinery. However, little is known regarding the mechanism of transport at the structural level. Here, we designed a cysteine‐less isoform of GLUT2 (C‐less GLUT2) to serve as platform for functional and structural studies, i.e. cysteine‐scanning mutagenesis in combination with thiol chemical modification. The kinetic properties of the C‐less GLUT2 expressed into Xenopus oocytes were like those of the native GLUT2, except that the overall activity was lower than that of the wild‐type transporter. Like the native protein, the C‐less GLUT2 transported methylglucose and deoxyglucose, and the activity was reduced in the presence of other sugars such as fructose, sorbitol, 3‐O‐methyl‐D‐glucose, galactose and ribose. Similarly, the activity of the C‐less isoform was inhibited by the classical GLUT2 inhibitors cytochalasin B, quercetin and resveratrol. This C‐less protein was refractory to MTSET, a membrane‐impermeable thiol‐alkylating reagent that obliterated the activity of native GLUT2. Our data suggest that the C‐less isoform of GLUT2 retains native‐like function and therefore is a suitable platform to perform structure‐function analysis by cysteine‐scanning mutagenesis.

Support or Funding Information

Supported by grants from FONDECYT 1130386, and FONDEF D11I1131
ALS and Ubiquilin‐2: Effects of ALS Mutations on Ubiquilin‐2 Structure and Function
Authors: Castaneda Carlos , Dao Thuy
Published: November 2023

Amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases are caused by malfunctions in ubiquitin‐mediated protein degradation pathways. Ubiquilin‐2 is a 624‐amino acid multi‐domain adaptor protein critical for maintaining protein homeostasis through the ubiquitin‐proteasome system, the endoplasmic reticulum‐associated protein degradation (ERAD) pathway and autophagy. Mutations in ubiquilin‐2 have recently been shown to cause dominant x‐linked inheritance of ALS and ALS/dementia. Moreover, wild‐type ubiquilin‐2 is present in protein inclusions found in patients diagnosed with either familial or sporadic ALS, further linking ubiquilin‐2 to the pathogenesis of ALS. Interestingly, most ALS‐linked mutations are localized to the proline‐rich repeat (Pxx) region that is unique to ubiquilin‐2 and not present in the other members of the ubiquilin protein family. The consequences of these mutations (specifically to residues P497, P506, P509, P525 and P533) on ubiquilin‐2's structure and function remain unknown. Towards that end, we are using biomolecular NMR spectroscopy and other biophysical techniques to characterize the Pxx region of ubiquilin‐2. 13C‐detect NMR experiments have allowed for the direct and necessary observation of the prolines as well as aided in resonance assignment for a significant portion of this ubiquilin‐2 region. Significantly, even though the Pxx region is intrinisically disordered, we observed sequence‐distant perturbations in several ALS‐linked mutants. Serendipitously, similar to some other ALS‐linked proteins such as TDP‐43 and hnRNPA1, our ubiquilin‐2 constructs exhibit salt and temperature dependent liquid‐like phase‐separation, a behavior that is also affected by ALS mutations. Our data suggest that the C‐terminal region of ubiquilin‐2 is important for modulating phase separation behavior. We are working towards modeling the structure of ubiquilin‐2 containing the Pxx region, as well as identifying protein‐binding surfaces involving the Pxx region.

Support or Funding Information

ALS Association Starter Grant to C.A.C.
Modulatory Role of Anti‐Tau Antibodies on Phosphorylation of Tau‐441 by MARK4
Authors: Ziu Iva , Martic Sanela
Published: November 2023

Tau protein stabilizes microtubules in neurons. The detachment of tau from microtubules, induced by post‐translational modifications, leads to tau aggregation, neuronal cell death, and cytotoxicity. The hyperphosphorylation of tau is one of the main causes of tau pathology in neurodegeneration. The current immunotherapies that target tau protein have shown clearance of tau pathology in animal models. However, it is still unclear how antibody‐based inhibition of tauopathies takes place. We have shown that anti‐tau antibody, targeting R1 epitope of tau, inhibited in vitro aggregation of non‐phosphorylated tau‐441. We also reported that phosphorylation of tau‐441 at S199 may be inhibited by antibodies to phosphorylated tau. Here, we report on the effects of anti‐tau antibodies to non‐phosphorylated tau (epitope at R4 repeat domain) on the phosphorylation outcomes of tau‐441 when MARK4 was used as a protein kinase. Using Dot blots and Western blots we evaluated the phosphorylation at S199, T231 and S262 residues of tau‐441. MARK4‐catalyzed phosphorylation of tau‐441 indicated a successful phosphorylation at S262 and S199, but not at T231. However, in the presence of anti‐tau antibodies (binding to tau R4 epitope) the phosphorylation at T231 was turned on, while phosphorylation at S199 and S262 remained unchanged. These findings suggest that phosphorylation of tau at specific sites may be modulated by using antibodies specific tau epitopes.

Support or Funding Information

NIH/NIGMS R15 GM11905301
Effect of Rad 23 selectivity to polyubiquitin chain linkage on Far‐1 degradation in yeast
Authors: Liu Yixuan , Cooper Eric
Published: November 2023

Ubiquitin is a small, 76‐amino acid polypeptide that can be covalently attached to other proteins to regulate their activities. The role of ubiquitin in tagging proteins for proteasome‐mediated degradation is its most well‐known function, but ubiquitin regulates non‐proteolytic processes as well. Ubiquitin's versatility is due, in part, by its ability to be linked into different types of polymers, depending on which of its seven lysine connect the individual ubiquitin subunits together.

Polymers assembled through ubiquitin's lysine 48 are generally considered to be the primary “flag” targeting substrates to the proteasome for degradation. Although ubiquitin can be recognized by the proteasome directly, there is abundant evidence indicating certain ubiquitinated substrates requiring a “shuttle” protein to deliver them to the proteasome. For example, in yeast, the cyclin‐dependent kinase inhibitor Far1 is only efficiently degraded by the proteasome in cells expressing a wild type version of a shuttle protein called Rad23. We are performing an in vivo experiment to test the hypothesis that Lys48‐linked polyubiquitin is, indeed, the primary degradation in cells. By switching the ubiquitin polymer‐binding selectivity of Rad23 from Lys48‐selective to Lys63‐selective, we will determine whether Far1 is still efficiently degraded in these cells.
Pba3‐Pba4 Plays a Role in Preventing Non‐Productive Interactions Among the α Subunits of the Proteasome
Authors: Panfair Dilrajkaur , Ramamurthy Aishwarya , Hochstrasser Mark , Kusmierczyk Andrew
Published: November 2023

The proteasome is a multisubunit protease complex that plays a major role in the degradation of ubiquitin tagged proteins. Structurally, the 20S proteasome is composed of four coaxially arranged rings. The two outer α rings, and two inner β rings, are each made up of seven homologous but distinct subunits. Proteasome assembly is a complex and highly ordered process that requires the assistance of assembly chaperones. The assembly chaperone Pba3‐Pba4 assists in the formation of the α ring, which is an early event in the proteasome assembly. Due to the rapid nature of these events, the exact order of the α subunit addition to form an α ring, and the precise role of Pba3‐Pba4 in this process, remain unclear. Here, we investigate the early events in proteasome assembly by the recombinant expression of Saccharomyces cerevisiae α subunits in Escherichia coli. We show that the coexpression of several α subunit combinations, such as α7α1, α6α7α1 and α5α6α7α1, results in the formation of high molecular weight complexes (HMWC). Using an engineered disulfide crosslinking strategy, we show that all of these complexes are double α rings. Interestingly, the coexpression of Pba3‐Pba4 with α5α6α7α1 prevents the formation of this HMWC, whereas the Pba3‐Pba4 cannot disrupt a preformed α5α6α7α1 complex. More importantly, the crosslinking strategy suggests that similar HMWCs may form in vivo when Pba3‐Pba4 is absent. Our data suggest that α subunits have a tendency to form non‐canonical ring complexes and one of the roles of Pba3‐Pba4 is to prevent the formation of these likely dead‐end species.
Proteasome Assembly and the Formation of Non‐canonical Complexes in Vivo
Authors: Kusmierczyk Andrew R , Hammack Lindsay J
Published: November 2023

The 26S proteasome is the heart of the ubiquitin‐proteasome system (UPS) in eukaryotes with a central role in the degradation of proteins and the maintenance of protein homeostasis. The 26S proteasome is comprised of a compartmentalized protease, the 20S proteasome or core particle (CP), whose activity is modulated by its interaction with one or more copies of the 19S regulatory particle (RP). The quaternary structure of the CP, found in all domains of life, is absolutely conserved and consists of four heptameric rings stacked coaxially. The rings are made of structurally related α and β subunits. In eukaryotes, assembly factors chaperone the seven different α and seven different β subunits to form a structure with α(1‐7)β(1‐7) β(1‐7) α(1‐7) stoichiometry. Here we demonstrate that CP subunits can assemble into structures other than the canonical CP in vivo. Specifically, the yeast α4 subunit forms high molecular weight complexes (α4 HMWCs) whose abundance increases when proteasome function is compromised. Results from a disulfide crosslinking approach are consistent with these complexes being ring‐shaped. Though several eukaryotic α subunits can form rings when expressed recombinantly in bacteria, this is the first evidence that such non‐canonical complexes exist in vivo. Our results place the α4 subunit at an important crossroads in the assembly of the CP. In addition to being present in the canonical CP, and these newly discovered α4 HMWCs, the α4 subunit also assembles into an alternate CP isoform called an α4–α4 proteasome, whose formation is evolutionarily conserved from yeast to humans. How α4 is distributed among these 3 possible complexes in vivo is not known. We present evidence suggesting that relative levels of the Rpn4 transcription factor might determine the extent to which α4 populates assembly pathways that lead to the formation of α4 HMWCs and α4–α4 proteasomes.

Support or Funding Information

This work was supported in part by a Research Support Funds Grant from Indiana University‐Purdue University Indianapolis (to A.R.K.).
Rolapitant is not a mechanism‐based inactivator or tight‐binding inhibitor of CYP2D6 in vitro
Authors: Glass Sarah , Furge Laura Lowe
Published: November 2023

Rolapitant is a NK1 receptor antagonist that was approved by the FDA in September 2015 as a treatment for chemotherapy‐induced nausea and vomiting. In vivo, rolapitant is primarily metabolized by CYP3A4, a member of the P450 superfamily that is responsible for the metabolism of 45% of pharmaceuticals. In clinical studies, it was shown that rolapitant inhibits the activity of another major drug metabolizing P450, CYP2D6, for at least a week after just one dose. Because the inhibition time is long, we hypothesized that rolapitant might be a mechanism‐based inactivator that irreversibly inhibits CYP2D6. Such inactivation could lead to adverse drug‐drug events. Interactions of rolapitant with both CYP3A4 and CYP2D6 were investigated utilizing AutoDock molecular modeling, spectral binding analysis, mass spectrometry metabolite studies, and in vitro inhibition assays. The results were compared with a known mechanism‐based inactivator, SCH 66712, and a tight‐binding inhibitor, quinidine. From this study, it appears that rolapitant is neither a mechanism‐based inactivator nor a tight‐binding inhibitor. Because of this, the in vivo inhibitory potency could be due to the drug's long mean elimination half‐life (7 days). Understanding the mechanism of inhibition by rolapitant has clinical relevance in preventing drug‐drug interactions caused by CYP2D6 inhibition during polypharmacy often occurring during cancer treatment.

Support or Funding Information

NIH 1R15‐GM086767‐02
Investigation of Possible Inhibitors Against Multidrug‐Resistant New Delhi Metallo‐Beta Lactamase (NDM‐1)
Authors: Hernandez Sonia Lisseth , Shrestha Sabi , Beckhem Josh , Fast Walter L
Published: November 2023

Since penicillin was introduced in the world of medicine, this antibiotic saved millions from infectious diseases. However, in the past decades, antimicrobial resistance has developed into a widespread public health threat causing high morbidity and mortality rates. New Delhi Metallo β‐lactamase (NDM‐1) is associated with many resistant nosocomial infections by organisms such as Klebsiella pneumoniae. This organism has become resistant by producing NDM‐1 which hydrolyzes the beta‐lactam ring found in beta‐lactam antibiotics. It is important to find novel inhibitors against New Delhi Metallo β‐lactamase (NDM‐1). In order to find potential small molecule inhibitors against NDM‐1, a combination of wet lab and virtual screening procedures were performed. Virtual screening was done to identify high scoring compounds using GOLD. Different virtual libraries were screened along with positive and negative controls. In the wet lab, competent BL21(DE3) bacteria cells were transformed with an expression plasmid containing the CDS of NDM‐1 and then expressed using IPTG and ZnSO4 to improve soluble yield. In addition, three methods of purification were performed which included nickel affinity chromatography with 6xHIS tag, ion exchange and size exclusion Fast Protein Liquid Chromatography (FPLC). The protein samples were characterized using SDS‐PAGE gel. Furthermore, protein activity of the enzyme was analyzed in the presence of potential inhibitors using Nitrocefin as a substrate in spectrophotometric assays. Differential Scanning Fluorimetry (DSF) was performed to analyze the binding impact of several potential inhibitors on the melting temperature of NDM‐1. Out of the 12 compounds, only 1 showed modest shifts to the melting curve. Any novel compounds found to inhibit NDM‐1 activity may lead to effective new inhibitors to overcome antibiotic resistance.
High‐Throughput Virtual Screening to Identify Novel Inhibitors of 6‐Phosphogluconate Dehydrogenase in Plasmodium vivax
Authors: Weber Nicole Brooke , Trieu Carolyn , Beckham Joshua
Published: November 2023

The discovery of novel drugs is warranted in the face of rising resistance developed by Plasmodium vivax parasites to a number of malaria medicines. Therefore, the principal aim of this research study is to discover unknown inhibitors for 6‐phosphogluconate dehydrogenase (Pv6PG) in the protozoan parasite P. vivax. First, the full Pv6PG coding DNA sequence was synthesized with overlapping oligo‐primers, amplified via Polymerase Chain Reaction (PCR) methods, and cloned into a pET expression vector. Following transformation into E. coli BL21(DE3), the protein was expressed and then purified with Nickel Affinity Chromatography and size exclusion chromatography. Differential Scanning Fluorimetry (DSF) assays were performed to assess the ligand‐binding interactions through shifts in the denaturation temperature. One best ranking compound from the Maybridge HitFinder library was docked using GOLD and scored 74.19, but did not exhibit a significant shift of the melting curve. To identify potential inhibitors, virtual high‐throughput screening was used. First, a homology model of the target Pv6PG was created using ICM. This docking algorithm made binding mode predictions of a ChemBridge library of over 100,000 ligands. The best ranking compounds will next be tested in wet lab using spectrophotometric assays to determine if they any have significant ability to inhibit the enzymatic functionality of Pv6PG. Successful isolation of compounds that inhibit this protein will play a role in hindering the vital biochemical pathways in the malaria‐causing organism Plasmodium vivax and could lead to greater progress in the pursuit of a novel treatment for this infectious disease.

Support or Funding Information

HHMI
Quantification of the In Vitro Inhibitory Effects of the Arachis Hypogaea Lectin on HIV‐1 Reverse Transcriptase
Authors: Garza Christen Ann , Ynalvez Ruby
Published: November 2023

Lectins are proteins known to bind to specific carbohydrates quite similar to antibody binding to its antigen. Since lectins possess this activity, it is evident that they could interfere during cell‐to‐cell communication and other biological processes. Plant lectins show promising abilities to potentially treat those afflicted with Acquired Immune Deficiency Syndrome or HIV/AIDS. The reverse transcriptase (RT) enzyme performs the role of transcription of RNA to DNA during the replication of HIV‐1. This allows for integration of viral DNA into the host genome and spreading of the virus. However, if this enzyme is inhibited, the replication process of HIV‐1 is directly disrupted. The objective of this study is to investigate the anti‐HIV effects of a lectin found in the peanut plant, Arachis hypogaea, by inhibition of RT. No studies yet have been reported that this lectin shows such activity. By using an Enzyme Linked Immunosorbent Assay (ELISA), it was concluded that the plant lectin from Arachis hypogea, revealed an elevated anti‐HIV RT activity.
Synthesis of Water‐Stable Material‐Organic Frameworks at Room Temperature
Authors: Le Nhat H , Jackson Karl
Published: November 2023

Metal‐Organic Frameworks (MOFs) are crystalline coordination polymers in which metal cations are connected by organic, electron donating “linker” groups. Their rigid struts allow the materials to maintain its porosity when guest solvents are removed. Thus, we can manipulate this class of nanoporous, supermolecular materials to achieve certain characteristics. This is why MOFs are also called “materials with tunable properties.” The developments of MOF in both scientific and industrial fields have been flourishing in the last decade. Indeed, MOF can be used for many different applications such as sensors, semiconductor, gas storage, drug delivery, oil‐spill cleanup, biometric design, and photo‐catalysis. However, MOF typically requires solvothermal conditions to crystalize, which means that it has to be dissolved and heated at a high temperature. Additionally, MOF are usually unstable due to the hydration‐oxidation of metal sites within the lattice. For those reasons, certain environment and economic constraints should be considered when synthesizing MOFs. Here we present a new, low‐cost, widely relevant, direct, fast, and environmentally friendly method to create MOFs with uncommon properties under traditional circumstances. Our MOFs are synthesized in water at room temperature using salts as linker groups.
Landscape of the Regulatory Elements for Lysine 2‐Hydroxyisobutyrylation Pathway
Authors: Huang He , Luo Zhouqing , Qi Shankang , Huang Jing , Dai Lunzhi , Dai Junbiao , Zhao Yingming
Published: November 2023

Short‐chain fatty acids and their corresponding acyl‐CoAs sit at the crossroads of multiple metabolic pathways and play important roles in diverse cellular processes. They are also precursors for protein posttranslational lysine acylation pathways. A noteworthy example is the newly identified lysine 2‐hydroxyisobutyrylation (Khib) that is derived from 2‐hydroxyisobutyrate and 2‐hydroxyisobutyryl‐CoA. Histone Khib has shown association with active gene expression in spermatogenic cells. However, key elements for regulating this physiology‐relevant pathway remain unknown, hindering characterization of mechanisms by which this modification exerts its biological functions. Here we report the first global profiling of Khib proteome in mammalian cells, identifying 6548 Khib sites on 1725 substrate proteins. We showed that Esa1p in budding yeast and its homologue TIP60 in human could add Khib both in vitro and in vivo, respectively. We further confirmed TIP60's in vivo activity for Khib by identifying 497 potential Tip60‐regulated Khib substrate sites. In addition, we identified HDAC3 as the major enzyme to remove Khib. Our study thus discovered both the “writer” and “eraser” for histone Khib marks, and major Khib protein substrates. These results not only illustrate the landscape of this new lysine acylation pathway, but also open new avenues for studying diverse functions of cellular metabolites associated with this pathway.

Support or Funding Information

Our work was supported by National Institutes of Health (NIH) award GM105933, DK107868, and GM115961 (Z.Y.). This work was also supported by Research Fund for the Doctoral Program of Higher Education of China 20120002110022 (J.D.)
The Role of the Na+‐H+ Exchanger Isoform 1 (NHE1) and Calcineurin B Homologous Protein Isoform 2 (CHP2) on Cell Proliferation and Migration in Squamous Cell Carcinoma of the Lung.
Authors: Kooiker Amanda J , Provost Joseph J , Wallert Mark A.
Published: November 2023

Lung cancer is a debilitating disease affecting thousands of people worldwide. Approximately 80% of lung cancers are comprised of non‐small cell lung cancer (NSCLC). Of all NSCLCs, 30% are due to squamous cell carcinoma (SCC), a cancer that begins in squamous epithelium of the lungs before moving deeper into the tissue. The Na+‐H+ Exchanger Isoform 1 (NHE1) acts as a key regulator of cellular proliferation, migration, and metastasis in a variety of solid tumors, including those due to SCC. The calcineurin B homologous proteins (CHP1 and CHP2) appear to act as important cofactors in the regulation of NHE1. While both CHP isoforms have been found to bind to nearly the same amino acid sequence of the cytoplasmic regulatory domain of NHE1, differential expression has been observed. CHP1 is ubiquitously expressed, while CHP2 expression has been shown to be higher in tumor samples from SCC patients and cultured cell lines than in normal healthy tissue. Through proliferation assays, we have shown that the inhibition of NHE1, using cariporide, is able to lead to a decrease in cellular proliferation in SCC cells. In this study, CRISPR‐Cas9 gene editing will be used to characterize the role of NHE1 and CHP2 by preferentially removing them from an SCC cell line (NCI‐H520). We will present data evaluating cell proliferation, migration, and tumor formation in cells lacking NHE1 (H520 NHE1KO) and CHP2 (H520 CHP2KO).
Targeting Integrin‐linked Kinase with Small‐interfering RNA Suppresses Invasion and Metastasis in Cisplatin‐Resistant Ovarian Cancer
Authors: Reyes‐González Jeyshka M. , Báez Perla , Valiyeva Fatma , Vivas‐Mejía Pablo E.
Published: November 2023

Ovarian cancer has the highest mortality rate among gynecological cancers. It continues to be characterized by metastatic bulky disease burden, which presents a major clinical challenge. Advanced disease is often treated with surgery, followed by platinum‐based chemotherapy such as cisplatin. Unfortunately, the majority of ovarian cancer patients become resistant to cisplatin, and relapse. Therefore, the identification of targets that link metastasis and chemoresistance is urgently needed to develop novel and effective strategies for ovarian cancer treatment. Integrin‐linked kinase (ILK), a serine/threonine kinase and adaptor protein, has been shown to play crucial roles in cancer progression, epithelial‐mesenchymal transition (EMT), angiogenesis, and metastasis. Our previous findings indicate that cisplatin‐resistant ovarian cancer cells (A2780CP20 and OV90CIS) express higher levels of phosphorylated ILK (p‐ILK) protein when compared to their parental counterparts (A2780 and OV90, respectively). Targeting ILK with small‐interfering RNAs (siRNAs) significantly reduced the invasive ability of A2780CP20 and OV90CIS cells in vitro (85.5% reduction; p<0.0001 and 61.6% reduction; p<0.0001, respectively). Therefore, to investigate the effects of ILK silencing in vivo, Control‐siRNA or ILK‐siRNA were encapsulated into DOPC‐DSPE‐PEG‐2000‐nanoliposomes. First, to assess the efficacy of the nanoliposomal siRNAs to silence their target in vivo, cisplatin‐resistant ovarian cancer cells (A2780CP20) were intraperitoneally injected into nude mice. Three weeks after tumor implantation, mice were treated twice with either Control‐siRNA or ILK‐siRNA nanoliposomes. ILK protein levels were significantly downregulated by ILK‐siRNA compared to Control‐siRNA, as evident in Western blot analysis (40% reduction; p<0.05). Next, to assess the involvement of ILK in metastasis in vivo, A2780CP20 cells were intraperitoneally injected into nude mice. Seven days after cell inoculation, mice received a weekly dose of either Control‐siRNA or ILK‐siRNA nanoliposomes. At the end of the treatment period, ILK‐siRNA significantly reduced the number of tumor nodules compared to Control‐siRNA (48% reduction; p<0.01). No obvious toxicity during therapy was observed. These results indicate that silencing ILK is a promising therapeutic approach for advanced and chemoresistant ovarian cancer. Further studies are required to investigate the biologic and molecular roles of ILK downstream targets in cisplatin‐resistant ovarian cancer.

Support or Funding Information

This project was supported in part by the RTRN (U54MD008149) grant to PEVM, institutional seed funds from the University of Puerto Rico, Comprehensive Cancer Center (PEVM), and National Institute of Health, Minority Biomedical Research Support (MBRS) RISE Grant Number R25‐GM061838 (JMRG).
Evaluation of the High Mobility Group A1 proteins (HMGA1) as a key mediate in the anticancer activity of ‐EF24.
Authors: Diaz Madelyn Nicole , Travis Lauren , Barber Sommer , Sumter Takita Felder
Published: November 2023

The architectural chromatin binding proteins High Mobility Group A1 (HMGA1) are some of the most overexpressed proteins in malignant cancers and induce neoplastic transformation. The protein is increased as the last step of a prominent colon cancer progression pathway and mediates drug resistance, therefore correlating with a poor patient prognosis. HMGA1‐‐mediated chemoresistance results from a self‐protective process called cellular senescence. Analogs of the antioxidant, curcumin, when used in combination with traditional chemotherapeutic agents, are useful treatment options for drug resistant tumors. This study had two specific aims. The first being to investigate how colon cancer cells HCT116 respond to treatment with EF24. The second aim was to evaluate how hmga1 expression changed as a result of treatment with EF24. Our preliminary findings showed that cell viability decreased after 24‐hour treatment with low‐dose EF24, as indicated by an MTS assay, with notable discrepancies between cells that underwent a pulsed treatment regimen versus those that underwent continuous treatment. Furthermore, we demonstrated that cells exhibited fragmented DNA when treated with low‐dose EF24, which is characteristic of apoptotic cells. At higher, pulsed doses, senescence activity increased indicating the induction of a senescence pathway. Lastly, gene expression studies indicated that hmga1 was significantly down regulated in cells treated with continuous, low‐‐dose EF24. Further investigation of this pathway could lead to decreased toxicity and increased viability of combination cancer therapies.

Support or Funding Information

The research support is provided by the National Center for Research Resources (5 P20 RR16461), the National Institute of General Medical Sciences (8 P20 GM103499) from the National Institutes of Health, and the Winthrop McNair Scholars program.
Individualized proteogenomics in analysis of resistance to BRAF inhibition in malignant melanoma
Authors: Schmitt Marisa , Nalpas Nicolas , Maass Annika , Macek Boris
Published: November 2023

Malignant melanoma is characterized by somatic BRAF and RAS mutations in the mitogen‐activated kinase pathway (MAPK), which strongly correlate with poor prognosis of the disease. The serine‐threonine kinase BRAF is mutated in ~70% of melanoma cases, resulting predominantly in V600E substitution and leading to constitutive activation of downstream MAP kinase signaling pathways. Targeted inhibition of the mutated BRAF with selective inhibitors like vemurafenib or dabrafenib results in the reduction of MAPK signaling and regression of the disease. However, most patients quickly develop resistance to drug treatment. Several mechanisms for the acquired resistance in melanoma have been detected in vivo and in vitro, but key (phospho)proteins involved in resistance as well as somatic mutations that can alter their modification status and can therefore influence development of resistance are still largely elusive. Here we use quantitative proteogenomics based on stable isotope labeling with amino acids in cell culture (SILAC) to investigate the proteome of five drug‐resistant and drug‐sensitive melanoma cell lines. Among 7,808 quantified proteins and 13,452 phosphorylation sites, we detect 53 proteins and 312 phosphorylation sites involved in major biological processes and cancer‐related signaling pathways that were significantly changing in resistant cells and therefore constitute potential therapeutic targets or markers of the resistance to BRAF inhibitors. To validate our data set, we focused on several interesting candidates and validated their levels by western blot across all investigated cell lines. We are currently knocking‐out respective genes using CRISPR/Cas9 and assaying genome‐edited melanoma cell lines for variability, cell migration, cell invasion and cell apoptosis. To study the influence of non‐synonymous somatic mutations on the signal transduction networks, we established a bioinformatics workflow for prediction of non‐synonymous single nucleotide variants (nsSNVs), as well as insertions or deletions (InDels) of nucleotides, and applied it to exome sequencing data from the vemurafenib‐sensitive and resistant melanoma cell line A375. This led to A375‐specific protein database containing 18,786 single amino acid variants (SAVs), of which 1,325 have not been reported previously. Over 8,800 SAVs affected modifiable amino acid residues Ser/Thr/Tyr and Lys, therefore potentially influencing signal transduction networks involved in resistance. To conclude, our (phospho)proteomics data provide important insights into molecular mechanisms underlying resistance to BRAF inhibition and the established proteogenomics workflow can be directly used for a personalized approach to cancer understanding and individualized treatment.
Inulin Acetate Nanoparticles as Vaccine Adjuvants
Authors: Narisetty Susmitha
Published: November 2023

An adjuvant is a substance which is added to a vaccine in order to increase the immune response (humoral and/or cellular) of the body. Alum salts are the most common adjuvants used in currently approved human vaccines in the United States, providing efficacy of vaccines through humoral immunity. For treatment of complex diseases such as cancer, tuberculosis and HIV, both humoral and cellular mediated immune responses are critical. The recent discovery that inulin, derived from dahlia plant tubers, activates cellular immunity has provided researchers with a platform to treat these complex diseases. Inulin acetate (InAc), a novel adjuvant, is not recognized singularly by dendritic cells. However, when presented in an appropriately sized particle, InAc acts as an efficient immune modulator. We explored various approaches to prepare InAc nanoparticles of different sizes using the process of double emulsification and lyophilization for long term storage. Ovalbumin was used as a model antigen/drug and polyvinyl alcohol (PVA) as an emulsifying agent. Nanoparticles in size ranges from 150–350 nm were achieved by altering the concentration of ovalbumin and the volume of PVA. In addition, it was discovered that lyophilization of nanoparticles did not change particle size. The interactions of these two variables, in affecting particle size, are discussed.
AraL Phosphatase From Bacillus subtilis, a Member of the HAD Superfamily
Authors: Martin Cassandra , Armeli Jordan , Madaio Michael , Hill Jacqueline , O'Handley Suzanne
Published: November 2023

The HAD (Haloacid Dehalogenase) superfamily is a diverse superfamily with a majority of the enzymes being phosphatases. One family of the HAD superfamily is the nitrophenyl phosphatase family, so named because the first family members were yeast enzymes found only to hydrolyze p‐nitrophenyl phosphate. Since that time, a number of enzymes with a variety of activities have been identified, including an enzyme from B. subtilis, AraL, originally designated as a sugar phosphatase. The AraL gene has been subcloned to incorporate a HisTag for nickel affinity chromatography, and the enzyme has been expressed, purified and characterized. Interestingly, it was found that although AraL does cleave phosphate from some sugar phosphates (ribose 5‐phosphate, ribulose 5‐phosphate, and arabinose 5‐phosphate), intermediates of glycolysis (glyceraldehyde 3‐phosphate, phosphoenolpyruvate, and dihydroxyacetone phosphate) are much better substrates for the enzyme. Since glycolytic intermediates are better substrates, AraL's role may be to regulate the glycolysis and gluconeogenesis pathways when arabinose is the carbon source. Arabinose is metabolized into intermediates that enter in the middle of the glycolysis pathway. The fact that intermediates after the arabinose point of entry are substrates, and intermediates before the arabinose point of entry are not substrates, suggests that AraL may inhibit glycolysis and activate gluconeogenesis.

Support or Funding Information

NIH AREA GRANT
The Use of Next Generation DNA Sequence to Determine Fungal Diversity on Potato Tubers Treated with 1,4‐dimethylnaphthalene
Authors: Patel Ravi Narendra , Diaz Rene Alfonso , Campbell Michael A
Published: November 2023

Prevention of sprouting in stored potato tubers is a major concern for the agricultural and food industries. The compound 1,4‐dimethylnaphthalene (DMN) has been shown to prevent premature sprouting when fogged onto the surface of potato tubers. Treatment of Saccharomyces cerevisiae cultures with DMN resulted in a temporary suppression of growth. Based on that data we speculate that treatment of stored potato tubers with DMN would result in sprout suppression as well as alteration of fungal communities on the surface of potato tubers. The treatment of potatoes was done with DMN or sterile water (control) for two days. The surface of the potatoes consisted of 4.2 ppm of DMN residue after treatment. Periderm (2 cm) samples were quickly frozen in liquid N2 and stored at −80°C after the treatment. DNA was isolated from frozen tissues using a Power Soil DNA Isolation Kit (mobio.com). Total DNA was measured and prepared for PCR using primers specific to the ITS regions of fungi (F). PCR products were evaluated using gel electrophoresis and sequenced using Illumina technology (Penn State Nucleic Acid Facility). A total of 3.7 million DNA sequences were analyzed using the Mothur (v1.36) software package to determine fungal diversity by alignment to the silva.eukarya.fasta database. Among all samples there were 1124 operational taxonomical units (OTU). It was determined that there is a greater fungal diversity on the control tissue than the DMN treated tubers. This data suggests the DMN alters the fungal diversity on the surface of potato tubers.

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Penn State Behrend, 1,4‐Group
Effects of a junk‐food diet on the rat gut microbiome
Authors: Gutilla Alexis , Campbell Caitlyn , Pikaart Michael , Vollbrecht Peter
Published: November 2023

The human body contains ten times more bacteria than human cells, with hundreds of trillions of bacteria residing in the intestines alone. These microbiota play an important role in the extraction of energy and nutrients from food, and previous research suggests that changes in the gut microbial community may be associated with the development of diet‐induced obesity. The current study aims to observe shifts in the gut microbiomes of rats fed a junk‐food diet. Sprague Dawley rats were fed either a junk‐food diet or regular chow diet (n=12/group) for four weeks. During this period, rats were monitored for changes in the gut microbiota as well as anxiety‐like behaviors and cognitive function. Fecal samples were collected weekly and a portion of the slurried fecal samples were diluted, filtered, and plated on modified mTEC agar plates in order to obtain E. coli counts. Real‐time PCR will be used to analyze the microbial composition of the fecal samples as well as intestinal samples collected later in the study. A better understanding of the interactions between diet, obesity, the brain and the gut microbiome will potentially lead to new methods for the prevention and treatment of diet‐induced obesity.
Sirtuin 3‐ and Diet‐Mediated Regulation of Mitochondrial Function During Aging
Authors: Dhillon Rashpal S. , van Ginkel Paul R. , Fu Vivian , Prolla Tomas A. , Denu John M.
Published: November 2023

Aging is a multifactorial process that results in the gradual damage of a number of cellular systems. Specifically, the decline of mitochondrial function due to hyperacetylation has been implicated in numerous metabolic defects, including senescence. The NAD+‐dependent mitochondrial deacetylase, sirtuin 3 (SIRT3) has a marked impact on mitochondrial acetylation status and may be responsible for attenuating oxidative stress. Furthermore, caloric restriction, which induces SIRT3 expression, has been linked to increased longevity. However, the impact of acetylation on mitochondrial function over the lifespan of an organism remains largely unknown. Here, we used a multi‐tissue approach to establish the role of SIRT3 and acetylation status in governing aging phenotypes by comparing mitochondrial function and structure in aging mice.

C57BL/6 mice in the SIRT3+/+ and −/− condition were fed a control or calorically‐restricted diet, and sampled at 5, 15, and 25 months of age. Gastrocnemius, brain, liver and heart were measured for respiratory capacity, complex enzyme kinetics, mitochondrial morphology, mtDNA content, and the protein expression of key SIRT3 targets. Metabolomic profiling of the liver was used to assess the impact of electron transport impairments on substrate utilization and accumulation. To determine possible mechanisms for the aging phenotypes observed in the present study, we examined expression levels of ATP‐dependent mitochondrial proteases. Generally, all tissues had markedly different respiratory responses to the treatment condition. However, oxygen flux through complex II revealed a significant SIRT3‐CR “rescue” effect at old age in most tissues. These results suggest that the deficiencies in oxidative phosphorylation may be driven by an impaired function at respiratory complexes caused by reduced protein content or complex activity. Mitochondrial structure, as determined by electron microscopy, revealed differences amongst groups, dependent on both genotype and diet. Overall, our findings suggest that SIRT3 in conjunction with caloric restriction may play a pivotal role in the age‐related response of the mitochondrial respiratory chain to aging in a tissue‐specific manner.

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National Institute of Health, National Institute on Aging
Combination Therapy to Enhance the Efficacy of Recombinant Immunotoxins: Evaluating the Role of Mitochondrial Translation in Toxin‐Mediated Cell Death
Authors: Zhu Yuyi , Weldon John
Published: November 2023

Recombinant immunotoxins (RITs) based on Pseudomonas exotoxin A (PE) are targeted cytotoxic proteins currently in clinical trials for the treatment of cancer. PE covalently modifies and inhibits elongation factor 2 (EF2), which halts protein synthesis within the cytosol and ultimately induces apoptotic cell death. Although PE inhibits translation in the cytoplasm, eukaryotes also have a translation system in mitochondria that is similar to the translation system in bacteria. Recent findings have highlighted the importance of mitochondrial stability and function in tumorigenesis, sustaining cancer cell proliferation, and maintaining cancer cell survival. By inhibiting protein synthesis within the mitochondria through the usage of antibiotics, mitochondria may become unable to properly function, resulting in the inability to meet the energy requirements of the cell and the activation of apoptotic cell death. I proposed that combination treatment with both PE‐based RITs and antibiotics that inhibit mitochondrial protein synthesis will reduce cancer cell survival compared to independent treatments of either therapeutic alone. To test this hypothesis, combination treatment of RITs and seven different antibiotics (tetracycline, chloramphenicol, streptomycin, linezolid, fusidic acid, doxycycline, and kanamycin) have been applied to CA46 and HEK293 cell lines followed by the assessment of cell viability through WST‐8 assay. No statistically relevant effects were observed for combination treatments in HEK293 cells, but the addition of a nontoxic level of chloramphenicol to RIT‐treated CA46 cells showed a ~50% enhancement in cell killing compared to RIT treatment alone (p=.05). In response, we are continuing to explore the usage of chloramphenicol in combination therapies with RIT. Though we conclude that targeting mitochondrial translation with antibiotics is not a general strategy for RIT combination therapy, combination therapy may be applicable in specific instances.

Support or Funding Information

This project was supported by funds from Towson University's Office of Undergraduate Research and the Fisher College of Science and Mathematics, as well as an undergraduate research award from the American Society for Biochemistry and Molecular Biology.
Buffer Therapy for Cancer
Authors: Pilot Christian Joseph , Gillies Robert
Published: November 2023

Background

Solid tumors exhibit an increase in glucose metabolism when compared to normal tissue. The increased metabolism generates an acidic tumor environment, allowing for metastasis and local invasion of tumor cells to occur. Previous research has demonstrated that oral buffers can neutralize this acidity and prevent metastasis and local invasion. Buffer therapy utilizes the intrinsic buffering capacity of various GRAS (generally regarded as safe) compounds to raise the pH tumors. While the acidity and alkalinity of foods has been documented, this is not the same as their buffering capacities.

Methods

A Mettler‐Toledo pH titration system was used to measure the buffering capacity of nutritional supplements and foods. The presence of titratable groups within the supplements and food sources led to their selection. Data were expressed as mmoles H+ consumed per 100 g (or per serving) to reach a pH of 4, which is the post‐prandial pH in the duodenum.

Results/Discussion

The buffering capacity of NaHCO3 has yielded promising results in mouse models and has undergone evaluation in phase I/II human trials. Clinically, compliance in buffer therapy remains problematic in patients, as the therapy requires ingestion of 40g‐50g NaHCO3 capsules daily. A plausible solution is supplement NaHCO3 with foods and supplements, expressed on a point‐based system wherein 1 point corresponds to 1g of NaHCO3. Research suggests that the therapeutic properties of buffer therapy occur when ~50 points are ingested daily. A compilation of common items and their corresponding point values has been created as a patient guide.

Support or Funding Information

Moffitt Cancer Center