Faculty-Guided Student Research 2024
Mission
We educate the next generation of leaders to improve the health of our society.
Vision
ACPHS strives to be a global leader in preparing students for transformative health careers to solve problems that change the world.
Greetings!
It is our great pleasure to introduce this collection of faculty-mentored student abstracts from our 2024 Research Symposium. This publication is a testament to the breadth and depth of scholarly activity at Albany College of Pharmacy and Health Sciences, where students are engaged in four main areas of inquiry: biomedical sciences, clinical and health sciences, pharmacy and pharmaceutical sciences, and social and behavioral sciences.
In recognition of our vibrant research culture, ACPHS is included among Research Colleges and Universities classified by the Carnegie Foundation. Students engage in meaningful research guided by our faculty. These experiences not only deepen their understanding of their fields but also position them for future success in graduate studies, clinical practice, industry and more.
We are incredibly proud of our students’ achievements and the impact they will make in advancing science and improving patient care. The commitment of our faculty in this endeavor is essential and invaluable.
We invite you to explore this collection and celebrate the promise and potential of the next generation of leaders who will improve the health of our society.
Sincerely,
Toyin Tofade, MS, PharmD, BCPS, CPCC, FFIP
Toyin Tofade President and Professor
Joseph J. Carreno, PharmD, MPH
Joseph
J. Carreno
Associate Professor and Director of Research and Sponsored Programs
Comparing the BioFire Pneumonia Panel and Conventional Respiratory Culture to Detect the Predominant Bacterial and Viral Isolates Causing Pneumonia
Tofade, C; Parent, M; George, M
Pneumonia is a common infection caused by bacteria, viruses, or fungi resulting in the inflammation of the air sacs in the lungs. The economic burden of pneumonia in the U.S. is estimated annually at over $15 billion.
Intrinsic/mutated resistances to antimicrobial treatment vary by source. Understanding predominating organisms in patients is critical for e cient and accurate treatment. Gold standard procedures for the determination of predominating isolates are currently achieved by respiratory cultures. Reliable culture results performed in the hospital clinical laboratory can range from 2 days to 2 weeks. Molecular testing such as the BioFire assay has proven to be highly sensitive for several isolates and has a short turnaround time (less than 2h). Rapid molecular identification of pneumonia isolates that is comparable to the conventional gold standard culture would allow clinicians e cient access to the predominating organism and treatment options for patients. We designed a project to test the e cacy and reliability of the BioFire Pneumonia Panel assay to determine its feasibility and compare pathogen identification to conventional culture methods. 27 patient sputum samples, were collected and evaluated with both systems. The data demonstrated a 64% correlation between conventional culture and the BioFire molecular identification results, amongst samples (expectorated sputum), across both methods. We believe that the BioFire Assay can be a reliable and e ective system for fast and dependable results to treat patients and reduce delays which can lead to infection exacerbation and increased economic burdens. Due to the limited sample size, the data demonstrates the need for a larger study to adequately assess the reliability of the BioFire assay to improve patient care.
The Stipulations of Gene Therapy and its Ties to Biomanufacturing
Das, J; Allisha, J; Dunnigan, T; Datta, P
As a result of the antibiotic revolution of the 20th century, there has been an unprecedented decline in the incidence of infectious diseases among the populations of developed nations such as the United States of America. While pathogens still pose an imminent health risk, as our population ages, the concerns of the medical community have largely shifted to the rise of cancers and chronic/congenital illnesses. Unlike pathologies of infectious origin, how clinicians can treat and cure these genetic conditions is relatively obscure. A patient cannot simply take a 2-week course of a drug such as penicillin to cure cystic fibrosis. Gene therapy (GT) serves as a unique approach to those who seek to not only treat but also potentially cure diseases of a congenital/genetic origin. GT can thus be defined as the introduction of modified genes via vehicles that are either non-viral or viral to patient cells to correct mutations within the patient genome. While viral vectors utilize classical viral mechanisms of entering host cells and potentially integrating into the genome, non-viral vectors rely on physical/chemical means of host cell entry. Viruses that are utilized as platforms for the development of GT vectors include adenovirus, adeno-associated virus, and lentivirus. Examples of chemical non-viral vectors include cationic lipids, polymers, peptides, and nanoparticles. Examples of physical non-viral vectors include the injection of genetic material and electroporation. While viral vectors display a high transduction e ciency, recent developments in non-viral vectors have made them a promising alternative in the progression of GT technology due to their low immunogenicity, mutagenicity, and cytotoxicity. GT poses as a viable treatment for several di erent diseases including cancers, leukemias, and anemias. In this review, the origins, development, and applications of gene therapy will be discussed before examining its ties and stipulations to biomanufacturing, as well as drawbacks and limitations.
Nuclear tra cking of cell death e ectors during hyperglycemia-induced shift from apoptosis to necroptosis
Dougherty, M; Knapp, A; Singh, M; Deragon, M; LaRocca, T
Extrinsic apoptosis and necroptosis are two regulated programmed cell death pathways that share the same initiation step, which is stimulated by TNF-α or another death-inducing ligand. We have previously established a shift from extrinsic apoptosis to necroptosis under hyperglycemic conditions, which can directly increase cellular metabolism and ROS production, resulting in enhanced necroptotic cell death. In the presence of oxidative stress, Nrf2 is released from its inhibitor (Keap1) in the cytoplasm and translocates to the nucleus, where it interacts with AREs to increase the expression of antioxidants. We have previously observed the disappearance of Nrf2 in the nucleus under hyperglycemic conditions, and this study serves to further investigate the impacts of Nrf2 and hyperglycemic conditions on cell death. All U937 monocytes were treated with TNF-α and CHX to initiate cell death and halt protein translation. Treatment with cisplatin and bypassing necroptosis served to restore Nrf2 nuclear translocation, which was observed via western blot. Treating the cells with sulforaphane, which is an Nrf2 activator, and analyzing them via WST showed improved cell viability. Treatment with Leptomycin-B, a nuclear export inhibitor, and analysis via WST displayed a significant reduction in cell death, while we did not observe a significant improvement in cell viability upon treatment with Ivermectin, a nuclear import inhibitor. Cells treated with sulforaphane were also analyzed with fluorescence microscopy to observe superoxide production. The observed results were unexpected, and we speculate that the positive feedback loop occurring between RIP1 and ROS may be responsible for the retention of Nrf2 in the cytoplasm, limiting antioxidant activity, and promoting necroptosis. Further investigation using nuclear fractionation and co-IP may prove useful to elucidate the potential interactions between RIP1 and Nrf2, and other components of the necrosome and Keap1-Nrf2-ARE pathway.
Identification of
novel regulators of the DNA damage response in Staphylococcus aureus
Dunnigan, T; Jayachandran, P; Ma, Z; Malik, M
With a finite arsenal of antibiotics to choose from, medical professionals are facing an ever-expanding issue on how to treat antibiotic resistant MRSA infections. An estimated 117,247 cases of Staphylococcus aureus blood stream infections occurred in the US in 2017 alone, resulting in 19,832 deaths. The DNA damage response (DDR) is a key contributor to the development of antimicrobial resistance in S. aureus. It has been shown that this resistance can be acquired through DNA mutagenesis induced by the SOS response mediated DNA repair pathway governed by the RecA recombinase and LexA repressor. When activated, these proteins lead to a downstream cascade and expression of SOS response genes. Only a handful of genes have been shown to mediate the SOS response in S. aureus. Given the significance of the DDR in mediating antibiotic resistance, we hypothesized that there are additional genes which mediate the DDR in S. aureus. In Mycobacterium smegmatis, there has been a recent discovery of novel regulatory roles for PafBC in the DDR. This study aims to discover novel molecular regulators and e ectors of the DDR in S. aureus. We screened over 1,000 mutants of the 1,920 transposon mutants of the University of Nebraska S. aureus Transposon Mutant Library (NTML) with the DNA damaging agent Mitomycin-C (MMC). Currently, we are characterizing two candidate genes that could play a role in the DDR. Characterization of the genes involved in the DDR will provide insight into the mechanism by which MRSA gains antibiotic resistance.
Characterization of the Hyperglycemic Shift to Necroptosis in PMA-Di erentiated U937 Cells
Knapp, A; Deragon, M; LaRocca, T
Eukaryotic cells primarily undergo two characterized types of programmed cell death (PCD): apoptosis and necroptosis. Apoptosis is a non-inflammatory pathway that is driven by the interactions of cysteine aspartic proteases or caspases. By contrast, necroptosis is a highly inflammatory pathway that is caspase independent. Necroptosis causes irreparable damage to the body and depends on the formation of the necrosome, a cell death complex driven by the action of RIP1 kinase. Despite producing dramatically di erent consequences on the body, these PCD pathways exist in a delicate balance with one another as they can both be induced by TNF-α. Various stimuli may be responsible for shifting favor to one PCD over the other. We've previously established that hyperglycemic conditions stimulate a TNF-α induced shift from apoptosis to necroptosis. Our previous studies characterized the hyperglycemic shift in a circulating monocyte model: undi erentiated U937 cells. In this study, we aim to compare the mechanism of the hyperglycemic shift to necroptosis in undi erentiated U937 monocytes to PMA-di erentiated U937 macrophages. Results suggest that PMA-di erentiated U937 cells undergo the hyperglycemic shift to necroptosis as exhibited in the undi erentiated cell model. Furthermore, the inhibition of RIP1 kinase and ROS appears to decrease cell death in di erentiated cells grown in hyperglycemic media, reflecting what is seen in hyperglycemic U937 monocytes. Fully characterizing the novel hyperglycemic shift and how it a ects the di erentiated cell model will enable a better understanding of how the PCD shift may exacerbate disease.
The Isolation and Characterization of the Novel Bacteriophage Annalisa
Gutta, N; Louie, E; Vredenburg, I; Meng, B; Jayachandran, P
The goal of the SEA-PHAGES program is to investigate the diversity and evolutionary processes of bacteriophages found in soil samples utilizing aseptic techniques. Annalisa was isolated using an enriched isolation procedure. We used 4 rounds of serial dilution to purify the phage. Amplified phage lysate was used for DNA isolation and using restriction digest the fingerprint of the genome was created and the DNA was sent for sequencing. TEM imaging revealed that it was a siphoviridae phage. DNA sequence results revealed that the genome size was 47,283 base pairs and the phage belonged to the cluster CZ4. Annalisa was auto-annotated in DNA master using Glimmer and Genemark. Using coding potential, RBS score, and comparative tools such as Phamerator, Starterator, and BLAST, the start codons were verified. Analyzing the data provided by these tools we decided to change the start for 14 genes and deleted 3 genes. HHpred, Protein Data Bank, and NCBI BLASTP were used as a functional annotation tool and a predictor of protein function. The annotated Annalisa genome will be archived in the existing phage database and NCBI. Annotated genetic sequences of isolated phages can be used to study the evolutionary processes of phages.
GFP Expression and Purification from Suspension-adapted HEK293 Cells: A Streamlined Workflow for Recombinant Plasmid Purification, Transfection, and GFP Recovery
Sahm, R; Sheppard, S; Swint, H; Datta, P
Expression Vectors (Recombinant plasmids, pDNA) are indispensable components in biomanufacturing. Expression vectors have been routinely used for basic research (e.g., cancer research, drug discovery) and biomanufacturing (e.g., insulin, monoclonal antibodies). Applications also include gene therapy and vaccine production. The current work focuses on establishing a workflow for production and expression of pDNA. A GFP encoded recombinant plasmid (under CMV promoter) was used as the model for establishing the step-by-step approach of plasmid purification and subsequent transient expression in HEK293 cells. The pDNA was purified using a spin column-based method (ZymoPure plasmid kit, Zymo Research). The concentration and purity of pDNA were quantified using UV-Vis Spectrophotometry (NanoDrop™ OneC, Thermofisher). Absorbance at 230 nm, 260 nm and 280 nm are used to evaluate the productivity of pDNA and purity of pDNA. Gel electrophoresis (with and without restriction digestion) was performed to evaluate pDNA size and identity. The purified GFP-encoding pDNA was used for transiently transfecting suspension-adapted HEK293 (Expi293) cells. Next, the GFP the transiently transfected cells were purified using a ion-exchange chromatographic purification method using AKTA FPLC.
Examining the e ectiveness of healthcare influenza vaccination mandates on the burden of nosocomial influenza cases among patients with End-Stage Renal Disease (ESRD)
Saleem, A; Doll, M
Background: Influenza is the seventh leading cause of death in the United States and a major cause of hospitalizations. Nosocomial infections, or infections acquired in a healthcare setting, represent a huge burden in the United States. Persons living with end-stage renal disease (ESRD) may be at higher risk for nosocomial infections due to more frequent and longer hospitalizations. The objective of this study was to examine the burden of nosocomial influenza among persons with ESRD hospitalized with influenza and compare the burden between states with and without healthcare influenza vaccination mandates.
Methods: The dataset utilized in this study was from the United States Renal Data System (USRDS), which provides comprehensive medical record data for persons living with ESRD on Medicare. Nosocomial influenza was identified in medical records as an influenza hospitalization, where influenza was not "present on admission" (POA) using an indicator in Medicare data. The burden of nosocomial influenza was examined by patient demographics. State healthcare worker vaccine law data was ascertained collected for each state between 2013-2016, and nosocomial influenza was compared by law type.
Results and Conclusion: Approximately 3% of all influenza hospitalizations among patients with ESRD were identified as nosocomial infections. Data regarding di erences in nosocomial influenza by demographics and geography are forthcoming.
Bystander cell death during the hyperglycemic shift to necroptosis
Singh, M; Deragon, M; LaRocca, T
The e ects of the programmed cell death pathways of apoptosis and necroptosis have been studied in many diseases, including HI-brain injury. The inflammatory nature of necroptosis causes leakage of cell death stimuli called damage-associated molecular patterns (DAMPs) such as AGEs and ROS to the surrounding environment, where they can encounter neighboring cells. We will test the hypothesis that a cell death shift in U937 monocytes leads to the death of neighboring neurons in the neonatal brain. During a stroke injury, there is an increase in stress-induced hyperglycemia that potentiates this shift from apoptosis to necroptosis.The in-vitro techniques used to execute this study include cell viability assays and western blot analysis with U937 cells and HMC3 microglial cells with or without selected chemical inhibitors to analyze the bystander e ects on these cells. After, interpreting the data from the bystander WST with U937 without the inhibitors, it was observed that there was more death in the bystander U937 cells that had been exposed to high glucose versus normal glucose. This laid down the ground of our previous finding that there is a shift from apoptosis to necroptosis under hyperglycemic conditions. Then we tried a few important inhibitors such as zVAD, Necrostatin (Nec-1) and N-acetylcysteine (NAC). The bystander cell death in 50 mM increases with zVAD (pan-caspase inhibitor), which might indicate necroptosis. To confirm that we needed to perform the triplicate experiment with Nec-1 (RIP-1 inhibitor) that showed significantly less death in 50mM compared to 10mM. Then with NAC inhibitor (whole cell ROS scavenger), We found that there is significantly less death in both 10mM and 50mM. In future experiments, we will perform bystander U937 and HMC3 WST and fluorescence microscopy with JC-1 and Mito Sox red with or without inhibitors to find the type of bystander cell death.
Development of Cell-Based
Assay for the Cytotoxic E ects of Aspirin on HepG2 Human Liver Cancer Cell Line
Swint, H; Sahm, R; Carillo, V; Datta, P
Cell-based assays are highly incorporated into drug discovery and development processes. They can be used to evaluate a drug's e cacy or toxicity. Well-designed and validated cell-based assays synergistically complement preclinical trial studies. The substitution was encouraged in drug clinical trial development through the FDA's Modernization Act of 2021. One of the main reasons for drug failure during clinical trial studies is drug toxicity, commonly found in the form of drug-induced liver injury (DILI). The project focused on developing a well-characterized cell-based cytotoxicity assay using a human liver cancer cell line (HepG2). The cell-based assay was validated with aspirin, an OTC small molecule drug, known to cause DILI in animals and humans. The project is the foundation for developing a high throughput platform to evaluate DILI, drug-drug interaction studies, and extending the technology to 3D high throughput platforms.
RNA Polymerase III May Regulate HIV-1 Replication
Thompson, L; Hong, Z; Dang, C; Katz, D; Bosqu, A; Singh, V
Elimination of latent reservoirs is an extremely challenging task due to the involvement of heterogeneous mechanisms in regulating HIV latency. Thus, we need to identify novel mechanisms to target heterogenous reservoirs uniformly. Our preliminary studies have suggested that inhibition of RNA Polymerase III (RNP III) can lead to strong reactivation (up to 90%) of latently infected cell lines representing T cell as well as monocytic reservoirs. This is highly relevant as HIV genome preferentially integrates near Alu repeats that are often transcribed by RNA Pol III. Furthermore, siRNA mediated knockdown of RNP III also produced consistent results suggesting a direct role of RNP III in regulating HIV latency. In this study we investigated the involvement of RNA Pol III in regulating HIV-1 replication in a variety of cell lines as well as primary cells. We examined the role of RNP III in regulating HIV-1 replication by utilizing two complimentary approaches - i) inhibition of RNP III by its inhibitor ML60218, and ii) genetic knockdown of RNP III using siRNA. These experiments were conducted using HIV-1 pseudotyped (DuoFluo) virus and HIV1-Bal in HEK293T, SupT1, CD4+T cells and macrophages. Results suggested that knockdown or inhibition of RNP III significantly induced viral replication as determined by RT-qPCR and flow cytometry. Results showed a consistent increase in HIV-1 transcripts including transcription initiation, elongation and Tat specific transcripts. However, we did not observe any significant change in total HIV DNA in response to RNP III manipulations. Collectively, this is the first study to investigate the role of RNA Pol III in regulating HIV-1 replication, demonstrating consistent results in cell lines as well as primary cells. Currently, we are performing experiments to evaluate the role of RNP III in the establishment and maintenance of latency using primary CD4+T cell model.
An antimicrobial susceptibility testing pilot study to decrease the time to correct treatment for patients with Gram-negative sepsis
Verma, R; George, M; Parent, M
Antibiotic susceptibility testing on bacterial pathogens found in blood cultures is a critical step in finding viable treatment options for the patients. A study on rapid antibiotic susceptibility testing was conducted from September 2023 to January 2024. A total of thirty-four (n) gram-negative blood cultures were obtained throughout the period of the study. About 44% of the 34 individuals were male (15/34) and 56% were females (19/34). The average age of the patients who were sepsis-positive for gram-negative bacteria was 64 years. Each sepsis-positive blood culture was tested for antibiotic susceptibility using two di erent techniques, Kirby-Bauer disc di usion assay, and an automated MIC system. The data gathered at the end of the study showed 94.11% similarity (32/34) between the results obtained by KB and MIC. The two cases that had di erences in the results are as follows. A blood culture for E. coli obtained on 11/24/2023 had a few discrepancies in the data recorded for KB and MIC. The readings of KB for Ceftazidime showed that the bacteria were susceptible to the drug, however, the MIC results showed that it was resistant. A similar discrepancy was observed for Aztreonam. Blood culture for Enterobacter cloacae obtained on 11/15/2023 tested susceptible to Trimethoprim-sulfamethoxazole (SXT) based on KB readings but showed resistance on MIC results. However, the study shows impressive results that could account for utilizing the traditional Kirby-Bauer technique in place of automated equipment in the future.
Structural and Functional Characterization of Human Cytochrome P450 2C9*2 Genetic Variant in complex with S-warfarin
Belrad, A; Shah, M
The highly polymorphic Cytochrome P450 (CYP) 2C9 enzyme is responsible for the metabolism of up to 15-20% of clinical drugs. The *2 allele represents an amino acid substitution from arginine to cysteine at 144 (Arg144Cys). It is one of the two most common genetic variants of CYP2C9. Individuals homozygous for *2 are known to have decreased capacity to metabolize drugs including s-warfarin, losartan, etc. The aim of the study is to express, purify and crystallize the CYP2C9*2 variant in complex with s-warfarin, and determine the three-dimensional structure. In addition, the aim is to elucidate the binding a nity of s-warfarin to CYP2C9*2 and wild-type using isothermal titration calorimetry (ITC). The CYP2C9*2 was expressed in E.coli Rosetta2 cell line followed by protein purification through a nity and ion-exchange chromatography. The purified protein was crystallized in the presence of s-warfarin. The crystals were screened using X-rays and the data were collected and processed, followed by structure determination using crystallography. The structure of CYP2C9*2-s-warfarin complex indicated the presence of electron density corresponding to s-warfarin near the access channel region of the protein. The Arg144Cys amino acid substitution in the *2 disrupts the hydrogen-bonding interactions with the neighboring residues. The e ect of Arg144Cys is transduced to several important secondary structural elements. Crystallization to achieve higher resolution is in progress that will help understand the binding of s-warfarin. Furthermore, the average KD or dissociation constant for s-warfarin binding to CYP2C9 wild-type was 7 µM ± 0.42 as illustrated by ITC. However, the CYP2C9*2 genetic variant did not show any binding with s-warfarin. Additionally, computational docking studies are ongoing that will aid in predicting interactions of s-warfarin in the active site of CYP2C9*2. Together, the results provide insights into the role of single nucleotide polymorphism and its e ect on the binding of s-warfarin to CYP2C9.
Investigating the influence of human serum albumin concentration on (Z)-4- hydroxytamoxifen treatment of breast cancer
Berghela, M; Musteata, M
Precision medicine in cancer treatment demands accurate dosing and timely interventions for optimal patient outcomes. However, clinicians encounter numerous challenges, including demographic shifts towards an aging population, rising obesity rates, and complex diagnostic dilemmas contributing to elevated mortality rates. A critical aspect within our control is the selection of appropriate treatments and the precise administration of therapeutic doses. Balancing the e cacy and toxicity of anti-cancer drugs is paramount, as overdosing can lead to adverse e ects and financial burden, while suboptimal dosing may result in treatment failure. The therapeutic window for these drugs is narrow yet varies significantly among individuals due to their interactions with proteins and fats in bodily fluids and tissues. Monitoring drug concentrations in the bloodstream provides valuable insights into dosing optimization, yet total drug levels alone do not reflect the active drug fraction within cancerous tissues. To address this limitation, we aimed to establish normalized drug levels by integrating drug quantities with patient-specific body compositions. Our primary objective was to elucidate the optimal tamoxifen levels for breast cancer treatment and investigate the influence of tamoxifen-protein interactions, particularly with albumin, on breast cancer cell growth. Utilizing a fluidic device designed to mimic drug kinetics in murine physiology, we successfully constructed and validated a model mirroring the in vivo drug distribution dynamics. Subsequent analysis revealed normalized tamoxifen levels and demonstrated a significant inhibitory e ect of albumin on tamoxifen's anti-cancer e cacy in breast cancer cell cultures. These findings underscore the pivotal role of albumin in modulating tamoxifen's therapeutic response in breast cancer. Moreover, leveraging patient-specific albumin levels may inform personalized tamoxifen dosing strategies, optimizing therapeutic outcomes while minimizing adverse e ects.
Crosstalk between breast cancer cells with ESR1 mutations (D538G) and fibroblast
Clark, K; Jin, K
Approximately 75% of breast cancers are driven by Estrogen Receptor (ER, ESR1), a transcription factor that is also the target of endocrine therapies. However, drug resistance is a common problem in clinical practice. Although endocrine therapy and CDK4/6 inhibitor therapy have shown promising preclinical and clinical benefits for breast cancer, the development of drug resistance remains a significant challenge. The underlying mechanisms and potential therapeutic targets for metastatic breast cancer with ESR1 point mutations are still unknown. In previous studies, using cytokine array, bioinformatic analysis, RT-qPCR, and ELISA analysis, we found that adipsin (complement factor D) is highly secreted from MCF7 and T47D ER+ breast cancer cells with Y537S and D538G mutations compared to wild type (WT). Adipsin works to promote cell growth through the complement system, which may be exploited as a potential treatment. In preliminary studies, we co-cultured ESR1 mutant cells with normal fibroblasts and screened the secretomes secreted from fibroblasts in the crosstalk of ESR1 mutant cells using cytokine antibody arrays. We identified 10 upregulated secreted factor candidates through bioinformatic analysis. We hypothesize that these factors during crosstalk between the breast cancer cells with ESR1 mutations (D538G) and fibroblasts could serve as key regulators in the resistance of endocrine therapy and the transition of breast cancer cells to metastasis. Currently, I am investigating which secreted factors are upregulated in crosstalk between ESR1 mutant (D538G) cells and fibroblasts. In future studies, I aim to identify a critical secreted factor and its role in ESR1 mutant breast cancer cell growth and migration. This research may lead to the development of new therapeutic strategies to overcome drug resistance in metastatic breast cancer with ESR1 point mutations.
Clinical Significance of CYP2C8 and Its Interactions with Acyl Glucuronides of Gemfibrozil and Clopidogrel
Gladding, L
Human Cytochrome P450 2C8 (CYP2C8) is one of 57 enzymes of the CYP450 family which participates significantly in drug metabolism. It is known to metabolize several drugs, including amodiaquine, cerivastatin, dasabuvir, and enzalutamide. CYP2C8 accounts for approximately 7% of hepatic CYPs, contributing to the metabolism of more than 60 drugs. The acyl glucuronides of gemfibrozil (anti-lipemic) and clopidogrel (anti-platelet) are considered potent inhibitors of CYP2C8 by the FDA. Such inhibition of the enzyme leads to drug-drug interactions resulting from an increased concentration of other CYP2C8 substrates. It is not yet known how the drug glucuronides bind in the active site of CYP2C8. It is currently hypothesized that the dimethyl phenoxy group of gemfibrozil glucuronide and the thiophene group of clopidogrel glucuronide covalently link with the heme in the active site to inactivate the enzyme. The aims of this project are to crystallize CYP2C8 and determine the crystal structure of this enzyme in complex with the drug glucuronides. CYP2C8 was expressed in a recombinant manner in E. coli and purified using a nity chromatography and ion exchange chromatography. The purified protein was then crystallized in the absence and presence of the acyl glucuronides of gemfibrozil and clopidogrel using a variety of commercially available crystal screens. The crystals of CYP2C8 in the absence of glucuronide have been obtained and will be sent to Stanford Synchrotron Radiation Lightsource (SSRL) for X-ray di raction in Stanford California, and graphic data will be collected remotely. Crystallization of CYP2C8 in the presence of the acyl glucuronides is in progress and any resulting crystals will also be sent to SSRL for data collection. The results will help characterize the interactions of these drug glucuronides with CYP2C8, and this in turn may help to develop safer medications and avoid drug-drug interactions in patients.
Investigating the role of RNA Polymerase-III in regulating HIV-1 replication
Thompson, L; Hong, Z; Jamal, I; Dang, C; Sangasani, D; Bosque, A; Singh, V
Elimination of latent reservoirs is an extremely challenging task due to the involvement of heterogeneous mechanisms in regulating HIV latency. In this study we are investigating the role of RNA polymerase-III (RNA Pol III) enzyme in regulating HIV-1 expression. Our preliminary studies demonstrated the enrichment of Pol III transcribed noncoding RNAs in latent cells, namely 7SK, 21A and BC200 that are interspersed among Alu repeats. This is highly relevant to HIV latency because the HIV genome is found to preferentially integrate near Alu repeats. Subsequently, use of an RNA Pol III inhibitor, ML60218, resulted in an unprecedented reactivation (up to 90%) of latent cell lines J89GFP and THP89GFP, in a dose-dependent manner (25 μM-50 μM). Further, we examined the role of RNA Pol III in regulating HIV-1 replication by utilizing two complimentary approaches to diminish RNA Pol III function- i) inhibition of RNA Pol III by its inhibitor ML60218, and ii) genetic knockdown of RNA Pol III using siRNA. These experiments were conducted using HIV1-Bal as well as HIV-1 pseudotyped (DuoFluo) virus in THP-1, HEK293T, SupT cells, and HeLa cells. Results suggested that knockdown or inhibition of RNA Pol III significantly induced the viral replication as determined by increased HIV-1 specific transcripts. Additionally, we performed pNL4.3 HIV-1 plasmid DNA transfection in HEK293T cells and analyzed the e ect of RNA Pol III inhibition/knockdown on HIV transcription. Results showed a consistent increase in HIV-1 transcripts including transcription initiation, elongation and Tat specific transcripts. Collectively, these results unravel a previously unknown but important role of RNA Pol III in HIV-1 replication. Currently, we are performing experiments using human primary CD4+T cell and macrophages to investigate the role of RNA Pol III in regulating HIV expression and latency.
Leveraging Glucose Consumption for the Cell Count of Adalimumab Producing CHO Cells
Li, K; Dean, M; Nelson, E; Datta, P
Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production as a result of their ability to grow in high cell densities, increasing the yield of a desired protein product. This project investigates a potential method in evaluating the growth characterization of an adalimumab producing CHO cell line to be scaled up in high density cell culture systems. Glucose is an important cell culture media component and measurement of glucose consumption is an indirect method for evaluating cell growth. The first part of the project was to evaluate whether glucose consumption correlates with cell growth. A positive correlation was observed between glucose consumption by the CHO cells and their subsequent growth. Our finding suggests that monitoring glucose levels could be a valuable tool for optimizing cell growth within the bioreactors. In parallel, we show that the cells have the capacity to adhere to microcarrier beads and FibraCel disks which are mediums critical to high density cell culture systems, allowing for further optimization of production.
Identification of IL-11 in HOXB7 overexpressing TNBC cells
Lomonaco, D; Jin, K
The HOX gene family is crucial in determining cellular identity during development, with HOXB7 acting as a central regulatory gene for target development and di erentiation molecules. Previous research has shown that HOXB7 amplification is linked to a poor prognosis and that it can drive epithelial cells towards an epithelial-mesenchymal transition (EMT) phenotype. Additionally, HOXB7 has been identified as a key master regulator of tamoxifen resistance through the activation of several receptor tyrosine kinase pathways, including EGFR. HOXB7 overexpression has also been shown to induce angiogenesis and macrophage recruitment via TGF 2 upregulation, indicating a critical role in the crosstalk between endocrine-resistant breast cancer and the tumor microenvironment (TME). To further understand this relationship, MDA-MB-231-HOXB7 overexpressing cell lines were established in this study. The aim is to identify a secreted factor in the crosstalk between HOXB7 overexpressing cells and stromal cells that promotes cancer cell proliferation and migration. Using a human cytokine array, 10 putative candidates of secreted factors were selected, and it was found that IL-11 expression was upregulated in MDA-MB-231-HOXB7 cells. Knockdown of HOXB7 by siRNAs decreased IL-11expression, as confirmed by real-time qRT-PCR and ELISA analysis. Blocking IL-11 protein by anti-IL-11 neutralizing antibodies significantly inhibited HOXB7 cell growth and implied that the autocrine pathway of IL-11 plays a critical role in TNBC cell growth and migration. Overall, this study aims to identify a novel secreted factor as a drug target to inhibit breast cancer cells with HOXB7 expression. Ultimately, the findings will enable the identification of drug regimens with activity against TNBC, which can be used to design and conduct clinical trials.
LC-MS/MS Analysis
of
Tamoxifen
and its Metabolites Using Vials of Di erent Materials and Di erent Sample Solvents
Mattison, D; Jin, K; Musteata, M
The purpose of this research was to determine what type of total recovery vial works best for analysis of n-desmethyltamoxifen(nd-Tx), tamoxifen(Tx), endoxifen(Ex), and 4-hydroxy-n-desmethyltamoxifen(4h-Tx) by LC-MS/MS.
Methods: A solution containing 64 ng/mL of each of the four metabolites in a 50:50 methanol: water solvent and an internal standard mix made of 1 ug/mL of Tamoxifen-13C6 in acetonitrile were vortexed together in a glass vial in a 1:2 ratio. A glass total recovery vial with a glass insert, a glass total recovery vial with a deactivated glass insert, and a plastic total recovery vial were assembled. The mixed solution was carefully pipetted into each vial in small amounts until each was nearly full and the exact time was recorded. The three vials were submitted for LC-MS/MS and set to be injected every 90 minutes for six total injections. The LC-MS/MS software recorded the exact injection times, the areas, and concentrations of tamoxifen and its metabolites nd-Tx, Ex, and 4h-Tx within the identical solutions in the di erent vials. Four trials were conducted, and the injection time, areas, and concentrations were analyzed using Microsoft Excel and n/n-max values were found and averaged between trials. A linear trendline of each curve was generated; those with the least dramatic slopes were most consistent. Results: When using the 83:17 organic: water solution in the media, no significant di erences were observed in any of the metabolite concentrations or areas when di erent vial types were used. The next step is to use less organic solvent which is more relevant for typical analysis to potentially view more di erences between the vial types. Conclusion: These results can be used when doing future pharmacokinetic research involving these tamoxifen metabolites and the use of LC-MS/MS to assist researchers in choosing the best total recovery vials and obtaining the most accurate and consistent results.
Understanding the morphological characteristics and mechanisms of antibiotic collateral sensitivity of synergistic combination through the evolution of antibiotic resistant Klebsiella pneumoniae
Mills, A; Slusarek, D
Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that causes a variety of infections including UTI and pneumonia in immunocompromised/hospitalized patients. K. pneumoniae is a multi-drug resistant (MDR) microorganism frequently resistant to numerous antibiotics. Due to scarcity of new antibiotics, treatment options for MDR infections are limited prompting exploration of e ective combinations of existing antibiotics to identify synergistic combinations. Antibiotic synergy occurs when combinations of drugs are more e ective together than either drug is separately. We are interested in understanding mechanisms of collateral sensitivity that may underly synergistic antibiotic combinations and evolution of antibiotic resistance of K. pneumoniae mutants. Collateral sensitivity occurs when resistance to one drug causes sensitivity to another. Gentamicin-resistant cultures were evolved to levels over 100-fold greater than the MIC of WT K. pneumoniae MKP220 to explore mechanisms of collateral sensitivity, as a way of developing new combination therapies. Purification of gentamicin-resistant K. pneumoniae mutants was achieved by multiple rounds of streak purification and the MIC of gentamicin was determined for each isolate. Sensitivity to additional antibiotics was determined by comparing these strains to WT using disc di usion assays. The growth rates and colony morphologies of the isolated strains' phenotypes were examined for physiological defects under various environmental conditions. Mutants of interest were chosen based on the most phenotypically di erent colonies and most gentamicin resistant then sent for whole genome sequencing to identify specific mechanisms of resistance and/or sensitivity. Multiple mutations were found in the selected strains including genes involved in energy production, cell membrane, stress responses, DNA replication, and other areas. Identified mutations will promote the approach of new combination therapies to combat current challenges of antibiotic resistance with K. pneumoniae.
Time kill analyses of Stenotrophomonas maltophilia when exposed to varying concentrations and combinations of TMP-SMX, minocycline, and levofloxacin
Nahorniak, A; Moolick, K; O'Donnell, J
Stenotrophomonas maltophilia nosocomial infections have been rising nationwide, particularly within immunocompromised populations. Growing resistance to currently available treatment options severely limits appropriate treatment. Currently, there is no standard optimal treatment for S. maltophilia infections, though recent Infectious Diseases Society of America guidance on treatment suggests combination therapy as standard of care. Optimal combinations for use in clinical settings are not well defined. Due to a lack of newly approved antimicrobials with in vitro activity, it is critical to optimize use of currently available agents to improve clinical outcomes and decrease the development of antibiotic resistance. In this experiment, 24-hour static concentration time kill studies will be conducted in triplicate using minocycline, levofloxacin, and trimethoprim-sulfamethoxazole TMP-SMX alone and in combination to determine the most e ective combination regimens against a variety of S. maltophilia isolates. Concentrations simulating humanized exposures (i.e., AUC0-24) of these agents will be used to determine the most e ective combinations. Synergy will be defined as ≥ 2 log10 cfu/mL kill greater than the most active individual agent. Bactericidal activity will be defined as ≥3 log10 cfu/mL kill relative to the starting inoculum. The starting bacterial inoculum for all experiments will be 1x10^6 cfu/mL, with colony counts enumerated at 0, 1, 2, 4, 8, and 24 hours after inoculation. We hypothesize that the combination of TMP-SMX plus minocycline, as well as TMP-SMX plus levofloxacin will exhibit greater bacterial killing relative to each individual agent. Because TMP-SMX has a di erent mechanism of action than minocycline and levofloxacin, this would allow for the drug concoction to a ect bacterial growth via di erent pathways. As one of the top ten most common pathogens in nosocomial pneumonia, optimization of treatment for S. maltophilia infections is essential to improve patient care and prevent development of multi-drug resistance.
Inhibitory e ect of pegcetacoplan, a C3 inhibitor, in breast cancer
Shaik, A
In ER+ breast cancer, the expression of the estrogen receptor allows for the use of targeted endocrine therapies, however, some populations that undergo this treatment become endocrine resistant and can become metastatic as a result. Of this population, the ERα 1 (ESR1) gene was more frequently mutated than in non-resistant population. The most prevalent mutations, Y537S (YS) and D538G (DG), cause constitutive ER activity that is ligand independent. In the previous study, we found that treatment of ESR1 mutants with the C3aR inhibitor SB290157 significantly decreased in all ESR1 mutants cell viability and increased apoptosis compared to wild type cells. ESR1 mutant cells were treated with SB290157 and tamoxifen in combination which revealed an increased sensitivity of these cells to tamoxifen. Our next question was if there is any clinical therapeutic C3 inhibitor. Pegcetacoplan FDA Approved in 2023 was developed for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) which is a X-chromosomal defect which prevents the formation of an anchor on the cell membranes. We hypothesize that pegcetacoplan can be used in breast cancer to inhibit the production of C3 pathway in crosstalk between ESR1 mutant cells and adipose tissues. We have designed the following aims, 1) Screening the inhibitory e ect of pegcetacoplan in endocrine resistant cells and ESR1 mutant cells. 2) Investigate a synergistic e ect of pegcetacoplan with CDK4/6 inhibitors in crosstalk between endocrine resistant cells and adipocyte. We predict that we would successfully find the inhibitory e ect of pegcetacoplan and synergistic e ect with CDK4/6 inhibitors in endocrine resistant breast cancer growth. In this study, we will provide evidence that supports the hypothesis that functional inhibition of the C3 signaling pathway has the potential to circumvent breast cancer growth and metastasis.
Comparison of Ultrafiltration
Devices for the Measurement of the Free and Total Concentration of Tamoxifen and its Metabolites
Tan Lu, W; Mattison, D; Berghela, M; Jin, K; Musteata, M
The purpose of this study was to identify the most useful device for the measurement of the free and total concentrations of tamoxifen and its metabolites (N-desmethyltamoxifen, 4-hydroxytamoxifen, and 4-hydroxy-N-desmethyl-tamoxifen) in serum, cell culture medium, and a physiological circulatory system. Material and methods: Liquid-liquid extraction, microextraction, and ultrafiltration using Amicon Ultra 0.5 mL (molecular weight cuto 10k Daltons) and Centrifree Centrifugal units (molecular weight cuto 30k Daltons) was performed, along with quantitative analysis using Waters Acquity ultra performance liquid chromatography (UPLC) with a triple quadrupole mass spectrometer (TQD). Results: Measuring the concentration of tamoxifen and its metabolites in serum and in cell culture medium has shown that 4-hydroxytamoxifen has a lower binding a nity compared to the other metabolites and tamoxifen has the highest a nity due to its binding to albumin. As of free concentration in serum using Amicon Ultra 0.5 mL, 4-hydroxytmoxifen has shown the highest concentration of 239 ng/mL and in many instances, the results for the binding of the other metabolites resulted in no signal (100% binding, probably due to adsorption onto the centrifugal device). This suggests that the Amicon Ultra 0.5 mL does not work as expected to obtain the desired results for measuring the binding of the other metabolites in samples. Free concentration measurements in cell culture medium using Centrifree Centrifugal units have yielded results only for the free concentration of 4-hydroxytamoxifen with a maximum of 11.7 ng/mL, suggesting the need for additional ultrafiltration devices for the other metabolites.
Synthesis and Characterization of Microemulsion and Nanoparticles for Use as Topical Drug Delivery Vehicles
Tangorra, E; Hass, M
This research project is focused on the development of microemulsions and nanoparticle technologies for use as topical delivery vehicles of prodrugs and co-drugs derived from mycophenolic acid (MPA), methotrexate (MTX) and aminolevulinic acid (ALA). The work on these topical formulations is part of a broader project to develop novel topical combination therapies for treating psoriasis and other inflammatory skin diseases. The aims of this project are to prepare and characterize microemulsions (ME) and nanoparticles (NP) by particle size, polydispersity index (PDI) and zeta potential, to assess the drug load of the prepared ME and NP using prodrugs of MPA (methyl mycophenolate (MPA-ME), MTX (methotrexate dimethylester, MTX-DME) and ALA (benzylaminolevulinate, ALA-BE) and to evaluate e ciency of delivery of these formulated prodrugs to the viable skin layers using an ex vivo porcine skin model. To date, various oil and lipid components (i.e., Capmul, IPM) combinations with surfactants and aqueous media have been prepared and assessed for thermodynamic stability and characterized using the Winsor system. Phase diagrams have been constructed to document the suitability of each combination as a topical vehicle. Winsor type 4 combination have been assessed for particle size, polydispersity and zeta potential using the Malvern Zeta Sizer 90 instrument. The drug load capacity of each of these m=ME and NP will be evaluated using reversed phase high performance liquid chromatography (RP-HPLC) using a Shimadzu HPLC system. Finally, loaded ME and/or NP will be used to assess the delivery of the MPA-ME into the viable skin layers using an ex vivo porcine skin model.
Breast
Cancer with ESR1 Mutation in Crosstalk of Fibroblast and Adipocyte
Tran, K
Estrogen receptor positive breast cancer is a prevalent disease traditionally treated with hormone therapy which relies on the expression of the estrogen receptor to starve the tumor of growth signaling due to estradiol. While hormone therapy generally has great outcomes, the development of endocrine resistance remains a huge hurdle, with poorer outcomes and less e ective therapeutic options. Endocrine resistant tumors have been found to uniquely contain point mutations in the ligand binding domain of the Estrogen Receptor 1 (ESR1) coding region, predominantly Y537S and D538G. A growing body of literature has shown the stromal involvement in tumorigenesis, which supports resistance, survival, proliferation and eventual metastasis. In previous experiments, secreted factors from 3 di erent genome edited MCF-7 harboring Y537S and D538G ESR1 cells, along with wild-type were identified. Complement components Adipsin and C3 were found to upregulated in ESR1 mutant cells in crosstalk with adipocyte. In addition, we found that CXCL1 is upregulated in ESR1 mutant cells in crosstalk with fibroblast. We hypothesize that a crosstalk between two induced stromal cells (adipocyte and fibroblast) by ESR1 mutant cells via. adipsin, C3, and CXCL1 plays a critical role in ESR1mutant breast cancer cell growth, migration, and therapeutic resistance. We have designed the following aims, 1) Identification of secreted factors in the crosstalk between fibroblast and adipocyte induced by ESR1 mutant cells. 2) Elucidate the mechanism of adipsin, C3, CXCL1 and novel secreted factor in crosstalk between fibroblast and adipocyte induced by ESR1 mutant cells. We predict that we would successfully find a secreted factor of interest that is highly expressed in fibroblast and adipocyte in crosstalk with ESR1 mutant cells. An elucidated mechanism will serve as the foundation for clinical research of factor-specific immune checkpoint therapy in treatment resistant and metastatic breast cancers.
E ects
of Historic Redlining in Albany, New York: Presence of Stressors Surrounding Elementary Schools Across Seasonality
Stewart, K; Pettigrew, S
Home Owners' Loan Corporation (HOLC) maps were first created in the 1930s and implemented discriminatory practices to determine which neighborhoods were able to receive home loans. HOLC maps made it di cult for individuals to move out of low-graded neighborhoods, which prominently e ect low-income individuals and people of color. Redlining has had many long-term e ects on neighborhoods and individuals including income, housing, and education. Previous research has shown a positive correlation between economic disadvantage and stress levels. In addition, previous studies have determined that there is an association between stress as a child and negative health outcomes.
Albany, New York was one of many urban areas where redlining was implemented. Neighborhoods in Albany that were previously redlined continue to have lower incomes and more racially and ethnically diverse communities.
This project examined the prevalence of stressors surrounding elementary schools in historically redlined neighborhoods compared to elementary schools in non-redlined neighborhoods, and di erences in stressors based o seasonality (winter, summer, and spring).
Observational data outside schools were collected to determine what stressors children experienced daily outside of the schools. Stationary stressors were also mapped to evaluate what children are seeing on their way to and from school. Di erences were tested using the Kruskall-Wallis on-way variance.
A statistically significant di erence was observed in the number of stressors present at schools in redlined neighborhoods compared to non-redlined neighborhoods (p-value = 0.0021) over all seasons.
Children living in formerly redlined neighborhoods may experience additional stress on their way to and from school.
