bluuebunny/crossref_metadata_2025 huggingface dataset:
Transient Receptor Potential Vanilloid 4 (TRPV4) Regulates Fibroblast Differentiation in vitro and D. farinae ‐Induced Airway Remodeling in Asthma in vivo via Modulation of Matrix Synthesis and Matrix Degradation Mechanisms
Authors: Gombedza Farai Colin , Kondeti Vinay , Al‐Azzam Nosayba , Thodeti Charles , Paruchuri Sailaja
Published: November 2023

Asthma is a chronic progressive disorder of the airways characterized by inflammation and lung tissue fibrosis. Major features of tissue fibrosis include fibroblast differentiation to contractile myofibroblasts, fibroblast proliferation, and increased synthesis and accumulation of collagen and other extracellular matrix (ECM) proteins. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel expressed in lung fibroblasts, responds to changes in ECM stiffness and mechanical forces. Previous studies have shown that, TRPV4 plays a role in the differentiation of cardiac fibroblasts to myofibroblasts. In the present study, we show that TRPV4 mediates lung fibroblast differentiation and aim to elucidate the signaling mechanism(s) involved. We found that TRPV4 was functionally expressed in normal human lung fibroblasts (NHLF) and that knocking‐down of TRPV4 inhibited TGF‐β1 induced fibroblast differentiation, as determined by decreased α‐SMA expression. Similarly, TRPV4 antagonist RN1734 (RN) also significantly inhibited TGF‐β1 induced expression of α‐SMA and fibronectin protein, as well as pro‐fibrotic genes. TRPV4 regulates TGFβ1‐mediated matrix accumulation via two pathways, both dependent on Rho/MRTF‐A. One pathway enhances fibrotic gene expression and matrix deposition via p38 MAPK, while the other pathway activates PAI‐1 and slows down matrix degradation. We also compared TGF‐β1 induced expression levels of these fibrotic markers in NHLF and diseased human lung fibroblasts (DHLF) and found elevated levels in DHLF. To confirm the role of TRPV4 in lung fibroblast differentiation in vivo, we employed a Dermatophagoides farinae (D. farinae) induced asthma model in wild‐type (WT) and TRPV4 knock‐out (TRPV4 KO) mice. Following exposure to D. farinae, remodeling and inflammatory responses were enhanced in WT mice while they were completely attenuated in TRPV4 KO mice. Altogether, these findings highlight a major role for TRPV4 in lung fibroblast differentiation and D. farinae induced asthma in mice.
Identification of TRPP2 binding partners in mice tissues
Authors: Ng Courtney , Yu Yong
Published: November 2023

Mutations in transient receptor potential polycystin‐2 (TRPP2 or PC2) and its related protein, polycystin‐1 (PKD1 or PC1) result in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 and PKD1 form a receptor/ion channel complex, which has been proposed to function in coupling extracellular stimuli to intracellular Ca2+ signals. The study of these proteins is essential to understanding the characteristics of this channelopathy and developing specific treatments. Our focus has been working with TRPP2 in order to gain insight on the molecular mechanisms of its structural assembly, and channel function and regulation. TRP channels are localized in a variety of tissues and perform specific roles in sensory physiology. Specifically, TRPP2 is known to express in the human heart, brain, kidney, lung, testis, and ovary. It is also known to have direct binding to a number of proteins, many of which are involved in cytoskeletal association, trafficking and ion channel modulation. The intracellular C‐terminus of TRPP2 has been shown to be involved in the association of some of these binding partners. We believe that the C‐terminus of TRPP2 has major implications in diversifying its role in different tissue types. The approach taken here was to identify novel TRPP2 C‐terminal binding partners present in different tissue types. We purified the C‐terminus of human TRPP2 by expressing it in E. coli, and used it to pull‐down these possible binding partners in several mice tissues, including the brain, kidneys, heart, lungs, and liver. Potential partners were isolated and mass spectrometry was used to identify these proteins for further analysis and functional study. The results of this study will shed light on the specific physiological function of TRPP2 in particular organs.

Support or Funding Information

This research is funded NIH Grant DK102092.
Hydrogen Sulfide Oxidation by Myoglobin
Authors: Bostelaar Trever Michael
Published: November 2023

Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from amino acids cysteine and homocysteine. Since it is toxic at elevated concentrations, cells are equipped to clear H2S. A canonical sulfide oxidation pathway operates in mitochondria, converting H2S to thiosulfate and sulfate. We have recently discovered the ability of ferric hemoglobin to oxidize sulfide to thoisulfate and iron‐bound hydropolysulfides. In this study, we report that myoglobin exhibits a similar capacity for sulfide oxidation. We have trapped and characterized iron‐bound sulfur intermediates using cryo‐mass spectrometry and X‐ray absorption spectroscopy. Further support for the postulated intermediates in the chemically challenging conversion of H2S to thiosulfate and iron‐bound catenated sulfur products is provided by EPR and resonance Raman spectroscopy.

Support or Funding Information

This work was supported in part by grants from the National Institutes of Health (GM112455 to R.B. and DK068139 to T.L.S.) and the American Heart Association (14POST18760003 to P.K.Y.). We thank Dr. Markus Ruetz for help with the EPR studies, Andrew Hunt for help with the resonance Raman studies, and Dr. David Ballou for his assistance with the O2 consumption assays.
Induction of apoptosis in carcinoma cells: Perhaps it is right way to kill the metastatic process
Authors: Basu Subhash , Basu Manju , Ma Rui , Moskal Joseph R
Published: November 2023

The normal cells in their life cycle die through apoptosis (programmed cell death or degradation of genomic DNA). On the other hand, metastatic cancer cells die mostly by Necrosis where toxic gene products come out through the holes on the cancer cell surface to induce necrotic death‐effect on normal cells. In cancer cells, the following chemical could inhibit blocked steps for apoptosis: cis‐platin (commonly used drug for testicular cancer treatment), Betulinic acid (a naturally occurring triterpene), L‐PPMP, D‐PDMP (inhibitor of GlcT [ceramide: glucosyltransferase]), Tamoxifen (commonly used drug for breast cancer treatment), and Melphalan (Golgi bodies scrambler). When three clones of metastatic breast cancer cells (SKBR‐3, MCF‐7 and MDA468) were treated with these apoptotic reagents separately, they induced apoptosis [activated Caspases (‐3, ‐8, or ‐9)] and down‐regulated the genes for cancer markers like sialo‐LeX (determined by DNA‐microarray studies). Targeted application of apoptotic chemicals to kill the cancer cells would be an ideal way for breast cancer therapy.

Support or Funding Information

Supported by NIH‐CA‐14764 and NIH‐NS‐18005
Genetic Characterization of Programmed Cell Death in Aneuploid Yeast Cells
Authors: Sanborn Matthew , Timmel Victoria , Barrios Jessie , McBride Trevor , Pinches Robert Seth , Austriaco Nicanor
Published: November 2023

Aneuploidy is the genetic state of a cell that has a chromosomal number that is not an exact multiple of the haploid complement. It is a leading cause of spontaneous abortions and of mental retardation in humans, and is a characteristic defect in cancer. Yeast cells that are aneuploid manifest a diversity of phenotypes including cell cycle defects, genomic instability, protein imbalance, chaperone stress and proteotoxicity. We are investigating the links between aneuploidy on the budding yeast, Saccharomyces cerevisiae and programmed cell death. We have shown that aneuploid yeast cells are more sensitive to ethanol‐induced cell death. In addition, we have also shown that caloric restriction protects aneuploid yeast from programmed cell death.

Support or Funding Information

In addition to funding from the RI‐INBRE Program, our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.
The Effect of Compound L19 on Human Colorectal Cells (DLD‐1)
Authors: Mohammadhosseinpour Sepideh , Clack Beatrice A.
Published: November 2023

Colorectal cancer (CRC) is the third most common type of cancer in the world and the second leading cause of human deaths due to cancer (Siegel, et al. 2014). Rumex crispus extracts, a source of anticancer compounds, have demonstrated the induction of apoptosis in colorectal cancer cells (DLD‐1). Bhandari (2015) screened water soluble compounds from roots and leaves identifying those that induced apoptosis in DLD‐1 cells. L19 is a compound that will be studied in more detail in this project. In this research, specific genes involved in apoptosis induction as a result of treatment of the DLD‐1 cells with L19 are identified using methods already utilized by Bhandari (2015) with addition of qRT‐PCR analysis. Accelerated solvent extraction (ASE) followed by high performance liquid chromatography (HPLC) was utilized to obtain larger amounts of L19. GC‐Mass spectrometry was used to identify the isolated compound. Apoptosis was measured by detecting the levels of caspase 3 and 7 using the APO‐one assay (Promega, Corp.) after treating the DLD‐1 cells with varying concentrations of L19. Furthermore, the mechanism of apoptosis has been determined using qRT‐PCR and gene specific primers for caspase 8,10 (extrinsic pathway), 9 (instrinsic), 4, 5 and 12 (ER stress).

Support or Funding Information

Office of Research and Sponsored Projects, SFASU, Ed and Gwen Cole
Understanding the role of BCL‐2 Proteins in Hyperglycemia‐induced Apoptosis in Cardiomyocytes
Authors: Miller Stefanie , Del Gaizo Moore Victoria
Published: November 2023

Chronic high glucose, or hyperglycemia, is a common marker for type‐2 diabetes and has been linked to diabetic cardiomyopathy. Specifically, when a secondary stressor is introduced, such as oxidative stress, increased cardiomyocyte apoptosis results leading to a larger area of dysfunction. Apoptosis is controlled by the BCL‐2 family of proteins, which consists of both pro‐apoptotic and anti‐apoptotic proteins that either facilitate or restrict cell death, respectively. BCL‐2 proteins interact at the mitochondria and changes to the balance of anti‐and pro‐apoptotic proteins can induce apoptosis. However, there are times during certain types of cellular stress where apoptosis does not occur but rather anti‐apoptotic proteins are sensitized, known as mitochondrial priming. Priming decreases the anti‐apoptotic reserve of the cell, thereby making it easier to antagonize apoptosis upon any further stress. We hypothesize that heart cells chronically exposed to high glucose levels leads to mitochondrial priming. Using a rat cardiomyocyte line, H9C2 cells, a hyperglycemia model was established where cells are cultured in low glucose media and then switched to high glucose media for various amounts of time. To investigate priming, a secondary stressor of diluted H2O2 is then used to induce oxidative stress. The amount of apoptosis in different treatment groups (low glucose, low glucose + H2O2, high glucose, and high glucose + H2O2) was quantified by flow cytometry using Annexin V‐FITC (FL1‐A) and Propidium Iodide (FL3‐A), for early apoptosis and late apoptosis, respectively. Increased levels of apoptosis occurred in cells treated with high glucose media for 72 hours followed by H2O2 (67.8% for FL1‐A and 51.1% for FL3‐A), but not high glucose media alone (9.1% for FL1‐A and 3.0% for FL3‐A). In fact, cells cultured in high glucose media were comparable to those in low glucose with H2O2 (12.2% for FL1‐A and 3.8% for FL3‐A) and without H2O2 (12.9% for FL1‐A and 3.9% for FL3‐A). The levels of apoptosis for the high glucose + H2O2 trial also increased as the time treated with high glucose increased from 48 hours to 72 hours, with values for FL1‐A of 30.6% and 67.8%, respectively. These results demonstrate that high glucose alone does not induce apoptosis, but rather primes cells for apoptosis so when a secondary stressor occurs, cells have an increased propensity to undergo apoptosis.

Support or Funding Information

The authors would like to recognize the following sources of funding: NC Biotech 2013‐IDG‐1019 (VDGM), Elon University Honors Program (SM), Elon U. Undergraduate Research Program (SM and VDGM), and Elon U. FR&D (VDGM).
UV irradiation/cold shock‐induced bubbling death is Ca2+‐dependent
Authors: Tsai Cheng‐Chang , Chen Yan‐Wei , Chang Nan‐Shan
Published: November 2023

Bubbling cell death is a non‐apoptotic event that each cell generates a single bubble from the nucleus in response to UV irradiation and/or cold shock. UV energy is first absorbed by the nucleus, followed by rapid relocation of cytosolic WWOX, p53, NOS2, TRAF2, and many others to the nucleus to generate a nitric oxide‐containing bubble in the nucleus. The single enlarging bubble merges with cell membrane and then blasts off from the cell to cause cell death. Unlike apoptosis, bubbling‐induced death is irreversible. The death event is caspase‐independent and devoid of DNA fragmentation. Tumor necrosis factor receptor‐associated factor 2 (TRAF2) inhibits bubbling death, while tumor suppressors p53 and WWOX do the opposite. Exosome release occurs during bubbling death, but not in apoptosis. The entire nucleoli may dump into the bubble. In certain cells, damaged nucleoli release the nucleolar contents directly to the bubble. Notably, housekeeping proteins, such as α‐tubulin, are significantly down‐regulated in L929 and COS7 fibroblasts and other cell types. When Ca2+ was depleted from environment, UV‐induced nuclear bubbling was blocked. A likely mechanism is that without Ca2+, executor proteins such as p53, NFκB‐p50, TRAF2 and NOS2 do not translocate to nucleus. The filamentary structure of α‐tubulin is disrupted upon exposure to UV/cold shock. Gene expression profiling in L929 cells revealed that mRNA processing, mRNA metabolic process and RNA splicing are down‐regulated, whereas sensory perception of smell, sensory perception of chemical stimulus and G‐protein coupled receptor protein signaling pathway are up‐regulated. In conclusion, we determined that UV irradiation/cold shock‐induced bubbling death at low temperature is via Ca2+influx for leading to nuclear translocation of executor proteins along with shut down of RNA processing machinery.

Support or Funding Information

Ministry of Science and Technology, Taiwan
Stay On Target: Deconvoluting Mixed Redox Messages through Precision Redox Targeting
Authors: Poganik Jesse R. , Parvez Saba , Aye Yimon
Published: November 2023

The precisely timed and spatially regulated chemical signals are the essence of biochemical redox signaling. However, defining the precise biological impacts of localized signals that engage with specific protein targets under physiologic conditions has proven to be highly challenging. This abstract presents a unique set of proximity‐directed chemical tools that enables powerful interrogation into functional consequences of specific redox events through precision redox targeting in living systems (see, for example, Parvez et al. Nature Protocols 2016 11 2328; Long et al. 2016 Nature Chemical Biology Accepted). With this in vivo‐validated redox‐targeting toolset, we identify bona fide “first responders” sufficient to drive phenotypic responses at organismal level exemplified by zebrafish and C. elegans models. Our unique chemical biology toolset sets the stage for ruling in gain‐of‐function (or dominant loss‐of‐function) redox modifications and relating them to phenotype in an unbiased experiment that is not hampered by functional redundancy.

Support or Funding Information

The NSF CAREER, NIH Director's New Innovator, Beckman Young Investigator, and Sloan Fellowship programs are acknowledged for research support.
CCN5/WISP‐2 activates estrogen receptor‐α in normal and cancerous breast epithelial cells and sensitizes them to hormonal therapy
Authors: Sarkar Sandipto , Ghosh Arnab , Banerjee Snigdha , Maity Gargi , Das Amlan , Gupta Vijayalaxmi , Haque Inamul , Tawfik Ossama , Larson Melissa , Banerjee Sushanta
Published: November 2023

Background

Estrogen Receptor‐α (ERα)regulation is a complex process and thus it is still unclear why ER‐α islacking in some aggressive breast cancer patients and develops resistance to hormone therapy. It is uncertain if this scenario be reversible by specific genetic manipulation. CCN5 is a member of the CCN (Cyr61, CTGF and NOV) family of matricellular proteins and is an anti‐invasive element in breast cancer progression. CCN5 suppresses the functions of many genes that are linked to various pathological outcomes and aggressive behavior of breast cancer (BC) cells. However, the mechanisms by which CCN5 prevents aggressive phenotypes of breast cancer cells are not entirely clear. CCN5 protein is exclusively expressed in estrogen receptor‐α(ER‐α) positive breast cancer cells, which are weakly invasive/aggressive and rarely metastasize depending on the functional status of ER‐α.

Results

Unique molecular relations between CCN5 and ER‐α has been tentatively established and have been identified as the components of the same signaling pathway that coordinate some metabolic signals associated with the proliferation of ER‐α‐positive breast cancer cells as well as delaying the disease progression from a non‐invasive to an invasivestate. Given the importance of this connection, we sought to determine the role of CCN5 in regulation of ER‐α in different cellular settings and their functional relationship. These studies were carried out in both invitro models and CCN5‐overexpressing transgenic mouse models driven by doxycycline (Dox). Our data shows that CCN5 is sufficient to induce ER‐α expression at the transcription level and functional activity as well as promote and sustain hormonal sensitivity in BC cells. Mechanistically, we have shown that CCN5 upregulates the expression of ER‐alpha through downregulation of AKT activity leading to stabilization and nuclear localization of Foxo3a. Moreover, induction of CCN5/ER‐αsignaling in normal breast epithelial cells in transgenic mice promotes hyperplasia but no carcinogenic growth, suggesting CCN5/ER‐α signaling is a required component to maintain the non‐aggressive niche and hormone sensitivity as well as deferment of the aggressive process.

Conclusion

CCN5 protein promotes ER‐α activity in both normal and cancer cells and enables cancer cells to respond to anti‐estrogen treatment. CCN5 is a positive regulator of ER‐α in both normal and cancer breast cells.

Significance

CCN5 is required to activate ER‐α. Therefore, resistance to hormonal therapy could be overcome by activation of CCN5 signaling.

Support or Funding Information

VA Merit Grant
Attenuation of γ‐secretase mediated activation of Notch signaling induces autophagic cell death in triple negative breast cancer cells
Authors: Das Amlan , Ganguli Arnab , Narayanam Maruthi Kumar , Mukherjee Pritha , Basu Biswarup , Chatterjee Urmi , Banerjee Sushanta , Karmakar Parimal , Kumar Dalip , Chakrabarti Gopal
Published: November 2023

Notch signaling is reported to be deregulated in several malignancies including breast. γ‐secretase, a multi‐subunit enzyme complex, catalyzes the cleavage of Notch receptor to release the Notch intracellular domain (NICD) in the cytosol, which then translocates into nucleus to activate the downstream signaling. Hence pharmacological inhibition of γ‐secretase might lead to the subsequent inhibition of Notch signaling in cancer cells.

In search of novel γ‐secretase inhibitors (GSIs), we screened a series of triazole‐based compounds, for their potential to bind γ‐secretase and we observed that 3‐(3′4′,5′‐Trimethoxyphenyl)‐5‐(N‐methyl‐3′‐indolyl)‐1,2,4‐triazole compound (also known as NMK‐T‐057) can bind to γ‐secretase complex. Very interestingly, NMK‐T‐057 was found to inhibit proleiferation, colony forming ability, motility in various triple negative breast cancer cells such as MDA‐MB‐231, MDA‐MB‐468, 4T1 and also MCF‐7, with a negligible cytotoxicity against non‐cancerous MCF‐10A, PBMC and WI38. Moreover NMK‐T‐057 treatment resulted in the reprogramming of the EMT process in TNBCs, accompanied by the drastic reduction of CD44high/ CD24low population. NMK‐T‐057 also inhibited the spheroid forming ability of TNBCs and reduced the stem cell enriched side population. It was also observed that in NMK‐T‐057 treated cells, Notch‐1 and its downstream targets such as Hes‐1, pAkt, and Cyclin D1 were significantly down regulated. The in silico study revealing the affinity of NMK‐T‐057 towards γ‐secretase, was further validated by fluorescence based γ‐secretase activity assay, which confirmed the inhibition of γ‐secretase activity in NMK‐T‐057 treated TNBC cells.

Autophagy plays a dual role in cancer depending on its role in the survival or death of cancer cells. The relationship between autophagy and Notch signaling is not clear and only a few reports have come across in highly reputed journals in last one year. We observed that NMK‐T‐057 induces significant autophagic responses in TNBCs and administration of the autophagy inhibitor 3‐MA, attenuates NMK‐T‐057 induced cell death. Similar results were observed on the treatment of TNBC cells with commercially available γ‐secretase inhibitor DAPT. Thus we might conclude that NMK‐T‐057 could be potential drug candidate against breast cancer, which can trigger autophagy mediated cell death in breast cancer cells by inhibiting the γ‐secretase – mediated activation of Notch‐signaling.

Support or Funding Information

This work is supported by Dr. D. S. Korhari Postdoctoral Fellowship Scheme, UGC, Govt. of India.
SRC Regulates Proliferation in ER+ Breast Cancer Cells by Stabilizing MYC mRNA
Authors: Abdullah Christopher , Korkaya Hasan , Courtneidge Sara A
Published: November 2023

Estrogen (E2) stimulation promotes proliferation in estrogen receptor‐positive breast cancer (ER+ BC), which accounts for 60–75% of all breast cancer. E2 signals through the ER via three pathways: the classical genomic pathway involving transcriptional activation of the ER, crosstalk between ER and other transcription factors, and non‐genomic signaling cascades, which do not involve the transcriptional activity of the ER. The non‐genomic pathway likely involves SRC family kinases (SFKs) and has been implicated in cell cycle downstream of the ER. While not typically amplified or mutationally cancer, SRC is often highly active in most breast cancers.

To test whether SRC is required for cell cycle progression in ER+ breast cancer cells, we quiesced ER+ MCF7 or ZR75‐1 cells prior to stimulation with E2 and utilized either a selective SFK inhibitor (SU11333) or shRNA‐mediated knockdown. Inhibition of SFK activity blocked progression from G1 to S phase and proliferation in 2D and 3D assays. We additionally tested the clinically available SRC inhibitor, saracatinib, in an orthotopic xenograft mouse model of ER+ BC and observed decreased tumor growth upon treatment with saracatinib.

As MYC is a known target of both SRC and ER and also is known to drive cell cycle progression, we tested whether MYC could be mediating SRC's proliferative effects in this model. Using MYC‐targeting shRNA constructs, we showed that MYC is required for E2‐dependent cell cycle progression and proliferation. Additionally, E2 stimulates MYC mRNA expression dependent upon SRC activity. We also showed that MYC mRNA expression is mediated primarily through SRC‐dependent stabilization of the transcript. We next aimed to understand the mechanism by which non‐genomic ER‐SRC signaling drives proliferation in ER+ BC cells. A known stabilizer of MYC mRNA, the RNA‐binding protein IMP1, has a truncated form (ΔN‐IMP1) expressed in ER+ BC cells. Our data suggests that ΔN‐IMP1 is required for the SRC‐mediated stabilization of MYC mRNA and proliferation. Taken together, these data suggest that SRC mediates cell cycle progression and proliferation through a non‐genomic E2 signaling pathway by stabilizing MYC mRNA.

Support or Funding Information

NIH 1F31CA180740 to CANIH 1R21CA177382 to SAC
Gemcitabine‐Induced Exosome Hypersecretion Increases the Chemoresistance and Migration of Pancreatic Cancer Cells
Authors: Sweeney Ryan , Richards Katherine E. , Hill Reginald
Published: November 2023

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd most common cause of cancer‐related deaths, due to its highly metastatic nature, delayed detection and robust chemoresistance. Our previous study implicated the role of cancer associated fibroblast (CAF)‐derived exosomes in the promotion of pancreatic cancer proliferation and chemoresistance. However, no studies have been conducted on the influence of cancer cell‐derived exosomes on the migration and chemoresistance of other cancer cells. This study examines the ability of exosomes secreted by pancreatic cancer cells upon treatment with gemcitabine, the current standard‐of‐care chemotherapy, to affect chemoresistance and migration. We have previously shown that upon gemcitabine treatment, pancreatic cancer cells hypersecrete exosomes, and that these exosomes exhibit increased expression of Snail and mir‐146a. Snail is a transcription factor known to promote epithelial‐to‐mesenchymal transition (EMT), chemoresistance, and metastasis, and miR‐146a is one of its known downstream targets. We hypothesized that the uptake of cancer cell‐derived exosomes would lead to increased resistance to gemcitabine and increased migration of the cancer cells as a whole. Upon knocking down exosome secretion with the drug GW4869, we found that AsPC1, MiaPaca, and Panc1 cancer cell lines were less resistant to gemcitabine. Moreover, treatment with GW4869 markedly reduced AsPC1 and Panc1 migration measured via a wound‐healing assay. Taken together, we have shown that pancreatic cancer cell‐derived exosomes promote the chemoresistance and migration of surrounding cancer cells. These findings suggest that exosomes play an important role in the communication between cancer cells within the tumor that is induced by gemcitabine treatment. Therapeutic strategies aimed at blocking chemotherapy‐induced hypersecretion of exosomes should be considered in order to develop more effective treatments for PDAC patients.
USING A CRISPR/CAS9 KNOCKOUT TO EVALUATE THE ROLE OF FURIN IN THE INTOXICATION PATHWAY OF PSEUDOMONAS EXOTOXIN A
Authors: Sanford Jack , Zhu Yuyi , Weldon John
Published: November 2023

Derivatives of Pseudomonas exotoxin A (PE) known as recombinant immunotoxins (RITs) are chimeric proteins being investigated as potential cancer therapies to target and kill cancer cells. RITs gain access to target cells via receptor mediated endocytosis, and traffic through the trans‐Golgi network to the endoplasmic reticulum. From there, RITs gain access to the cytoplasm where they inhibit translation elongation factor 2 (EF2) and halt protein synthesis. The subtilisin/kexin‐like proprotein convertase furin (PCSK3) has been implicated in the PE trafficking pathway as a proteolytic activator of the toxin, but circumstantial evidence suggests that furin may assist intracellular trafficking or have a combination of both cleavage and trafficking functions.

This project aims to use the CRISPR/Cas9 methodology to create a furin‐deficient cell line in order to explore the role of furin in the intoxication pathway of RITs. Thus far, a targeting guide RNA has been used to direct the Cas9 endonuclease to the furin gene in HEK293 cells. The resulting cells were clonally expanded and evaluated for knockout mutations resulting from repair of the Cas9 cleavage events. Several HEK293 subclones without furin expression were identified. Initial RIT‐based cytotoxicity assays have shown a difference in susceptibility between the furin knockout and wild type HEK293 cell lines. We plan to introduce furin variants with altered function into the furin‐deficient cell line to quantifiably characterize the role of furin in the RIT intoxication pathway.

Support or Funding Information

I would like to thank the Fisher College of Science and Mathematics (FCSM) as well as the Towson Office of Undergraduate Research (TOUR) for their financial support of this project. I would also like to thank all of the members of Dr. Weldon's lab for their help with this research.
Manipulating the Bone Marrow Microenvironment to Prevent Survival of AML Cells
Authors: Sterner Rosalie M. , Kremer Kimberly N. , Dudakovic Amel , Westendorf Jennifer J. , van Wijnen Andre J. , Hedin Karen E.
Published: November 2023

The bone marrow microenvironment acts as a protective niche for malignant cells. Within the bone marrow microenvironment is the endosteal niche, which is the area where the ossified surface of the bone and marrow cavity interact. The endosteal niche provides the opportunity for hematological malignancies such as acute myeloid leukemia (AML) of the bone marrow to interact with osteoblasts from the bone. To study the interaction between AML cells and osteoblasts, our lab developed a co‐culture model of osteoblasts (MC3T3 osteoblast cell line) and AML cells (KG1a cell line or patient samples from bone marrow aspirates). AML cells expressing high levels of CXCR4 were cultured alone or with osteoblasts and then challenged with SDF‐1, the ligand to CXCR4 and a chemokine readily abundant in the bone marrow microenvironment that our lab has previously shown actually induces apoptosis in AML cells expressing high levels of CXCR4 and that osteoblasts are able to protect AML cells from SDF‐1 induced apoptosis. Our lab now shows that treating osteoblasts with cyclosporine (CSA) prevents their ability to effectively protect AML cells from SDF‐1 induced cell death as assayed by annexin‐V staining of AML cells by flow cytometry. CSA treatment of osteoblasts decreased protein levels of tissue non‐specific alkaline phosphatase (ALPL), a phosphatase necessary for osteoblast mineralization, compared to vehicle control. As such, osteoblasts were treated with control siRNA or ALPL siRNA. CXCR4 expressing AML cells were then co‐cultured with the transfected osteoblasts and challenged with SDF‐1. siRNA knockdown of ALPL in osteoblasts was sufficient to prevent protection of AML cells from the apoptotic stimuli. These studies indicate that osteoblast differentiation plays a role in mediating the protection of AML cells. Therefore, targeting specific differentiation factors in osteoblasts may help to make to bone marrow microenvironment a more hostile environment for AML cells and aid in better malignancy eradication.

Support or Funding Information

This study was supported by NIH R21 CA194217 (to K.E.H.). R.M.S. is supported by the Mayo Clinic Medical Scientist Training Program Robert L. Howell Physician‐Scientist Scholarship.
ATG5 knockout leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2
Authors: Lee Michael , Hwang Sung‐Hee
Published: November 2023

We previously demonstrated that modulators of autophagy might be used beneficially as adjunctive therapeutic agents during the treatment of cancers with Src tyrosine kinase specific inhibitor PP2. Here, we investigated the role of autophagy in cell growth by using established ATG5 knockout (KO) cell line with CRISPR/Cas9 system. The conversion of non‐autophagic LC3‐I to autophagic LC3‐II and RT‐PCR confirmed the functional gene knockout. Interestingly, the growth of ATG5 KO cells was significantly enhanced compared to wild type (WT) cells. In particular, autophagy‐deficiency in NIH 3T3 cells enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and soft agar colony formation assay. Furthermore, ATG5 KO cells were more resistant to PP2 treatment than were WT cells. Additionally, the proportion of apoptotic cells significantly decreased when treated with PP2 in ATG5 KO cells compared with WT cells. Interestingly, we found that expression levels of p53 were much stronger in ATG5 KO cells, which showed lower apoptosis rates than did WT cells. ATG5 knockout is thought to be lead to a compensatory increase in p53 proteins due to a decreased apoptosis rate. We also found the marked up‐regulation of p‐ERK and p‐MEK in ATG5 KO cells, suggesting that the promotion of PP2 resistance by ATG5 knockout was associated with elevated levels of p‐MEK and p‐ERK. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to anti‐cancer therapy.

Support or Funding Information

Grant Funding Source: Supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2016R1D1A1B03930193)
(A3)Beating Cancer: Impact of APOBEC3B on 5‐Fluorouracil Treatment
Authors: Fine Sydney R
Published: November 2023

The APOBEC3 family of cytosine deaminases consists of seven members that function as part of the innate immunity. One family member, APOBEC3B (A3B), has been implicated as an endogenous source of mutation in cancer cells, specifically in head/neck, lung, cervical and breast cancers. A3B increases the rate of mutation in DNA through its ability to deaminate cytosine into uracil. The aim of this study was to determine if A3B over‐expression synergizes with 5‐Fluorouracil (5‐FU), an FDA‐approved chemotherapeutic agent, to promote cell death. 5‐FU promotes cell death through two mechanisms. First, 5‐FU inhibits thymidylate synthase, an enzyme that converts deoxyuridylate to deoxythymidylate. Second, 5‐FU becomes incorporated into DNA, which causes significant DNA damage. To investigate the hypothesis, A3B was over‐expressed in a mammary epithelial cell line, MCF10A. Then, the cells were treated with various concentrations of 5‐FU. Cell viability in response to 5‐FU treatment was assessed by clonogenic survival assays, which measure cells' ability to proliferate. It was determined that up‐regulation of A3B had little to no effect on the efficacy of 5‐FU. This research has implications in regards to personalized treatment in cancer patients with A3B‐high tumors because treating these tumors with 5‐FU may widen the therapeutic window and allow for more effective disease treatment.

Support or Funding Information

The Harris lab is supported by grants from the NIH. Dr. Harris is an Investigator of the Howard Hughes Medical Institute. The NIH provides funding for the Cancer Research Education & Training Experience.
Evaluation of the immune response induced in mice by a recombinant form of SPO‐1, a Schistosoma mansoni modulatory protein
Authors: de Oliveira Santos Bernardes Wilma Patricia , Alves Clarice Carvalho , Pereira Rosiane Aparecida , Fonseca Cristina Toscano
Published: November 2023

The S. mansoni tegument (Smteg) represents an important source of antigens to vaccine formulations. However the tegument is also composed of molecules that are involved in the modulation of the host immune system to favour parasite survival. Using immune proteomic tools we have identified proteins from the tegument that were recognized by sera from Smteg‐immunized mice and thus might be involved in the induction of the protective immunity observed in these animals. One of these proteins is SPO‐1, also known as Sm16. This protein has been described as a potent anti‐inflammatory response inducer in the host. The aim of this study was to characterize SPO‐1 expression in the parasite and the immune response trigger by the recombinant form of SPO‐1(rSPO‐1) in mice. To obtain the rSPO‐1, the gene sequence coding for amino acid 23‐90 were sub cloned into the expression vector Pet21a. Escherichia coli BL21 strain were transfected with this construction and the expression was induced using 1mM of Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG). A recombinant protein of approximately 12 kDa fused with a histidine tag was obtained and purified using a niquel column. Using an approved protocol from Brazilian Ethical Committee (LW25/15), Balb‐c mice were immunized with rSPO‐1 and their sera were obtained to be used in Western Blot assay to determine SPO‐1 expression levels through parasite life stage. Protein extracts of the different parasite life stage were also obtained. The results showed that SPO‐1 is mostly expressed in cercariae, 3‐hour schistosomulum and 7‐day schistosomulum. To evaluate the immune response induced in mice by rSPO‐1, 7 mice were intraperitoneally inoculated with 25 μg of rSPO‐1 and one week later their spleen were collected to determine lymphocyte activation status and subtypes. A lower frequency of IL‐10‐producer B cells (p<0.02) and activated CD8+T cells (p<0.02) in SPO‐1‐inoculated animals was observed in comparison to saline‐inoculated mice. Moreover, there was no difference in the frequency of regulatory T cells (CD4+CD25+Foxp3+) or activated CD4+ cells (CD4+CD25+) between groups. An increased percentage of CD4+ cells producing interferon gamma and IL‐4 was observed after in vitro SPO‐1 stimulation in both groups. However, rSPO‐1 was unable to trigger IL‐10 production by CD4 T cells. In conclusion, despite being described in the literature as a regulatory protein, Spo‐1 was unable to induce this profile in the adaptive immunity. Thus, it is important to characterize the effect of SPO‐1 inoculation in myeloid lineage cells.

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Fapemig, CNPq, Capes, CPqRR, Proep/CPqRR
Polar Lipid in Human Blood Regulates Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum
Authors: Gerdt Joseph P. , Brancucci Nicolas M.B. , Wang Charley , Adapa Swamy R. , Zhang Min , Adams John H. , Jiang Rays H.Y. , Marti Matthias , Clardy Jon
Published: November 2023

For Plasmodium falciparum to infect hundreds of millions of people annually, it must first be transmitted by a mosquito vector. Only the parasite cells in human blood that have matured into sexual precursor cells (gametocytes) are capable of transferring to mosquitos. Therefore, understanding the regulation of gametocyte development in humans will provide avenues to halt the spread of malaria. Toward this end, we developed a new in vitro gametocytogenesis assay and used it to test the hypothesis that environmental factors exist in human blood that regulate P. falciparum sexual commitment. Our hypothesis was confirmed when we identified a polar lipid extracted from human blood that is sufficient to block gametocytogenesis at physiological concentrations. Fluorescence microscopy and metabolomics analysis revealed that the parasites readily uptake and metabolize this lipid. We furthermore found that parasites that are starved of this lipid upregulate alternative metabolic pathways to restore it. Therefore, we conclude that asexual blood stage P. falciparum senses its environment in the human host; under lipid starvation, the parasite converts to the transmissible gametocyte stage. This finding can be leveraged to identify molecular targets against the spread of malaria.

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J.P.G. was supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award F32 GM116205 from NIGMS. This research was also supported by NIGMS award R01 GM086258 to J.C.
Anti‐fluoroquinolone resistance activity of E558: A natural product
Authors: Iyanda‐Joel Wisdom Oluwayemi , Omonigbehin Emmanuel Adedayo , Iweala Emeka Eze J. , Chinedu Shalom Nwodo
Published: November 2023

Fluoroquinolone resistance (FQR) has paralleled the abuse of this class of antibiotics in Nigeria ever since the patent rights expired in 2003. However, nature has a potent way of uncoupling unfavourable synergies targeted against the serenity of the biosphere. In this preliminary study, we examined the antibacterial activity of E558, a medicinal plant product against multidrug‐resistant gram‐negative isolates from immunocompromised patients with acute respiratory infections. The agar‐well diffusion method was employed to screen for antibacterial activity against strains of Klebsiella pneuminiae, Escherichia coli and Pseudomonas aeruginosa. The organisms were initially subcultured on McConkey agar followed by streaking standard innoculum of the bacterial strains on Mueller‐Hinton agar plates. A known concentration of crude extract was loaded into the well bored on the plates and then incubated at 37 °C for 24 hours. Antibiotic susceptibility testing (AST) was carried out to monitor resistance of isolates. Protein profiling with sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as well as restriction endonuclease mapping with agarose gel electrophoresis was employed to study alterations in the genomic DNA of selected organisms. A reverse mutation was observed in the morphology of organisms exposed to E558 compared to the organism without exposure. AST result further revealed that strains in the reverted zone, which were originally resistant to fluoroquinolones and other antibiotics had become susceptible with very wide zone of inhibition (≥ 40 mm; CLSI‐sensitive). Restriction mapping and SDS‐PAGE respectively revealed there are major differences in the genomic composition and protein profile of the reverted strains when compared to the original organisms. It can therefore be deduced that E558 possibly contains bioactive compound(s) with potent antitumour and anti‐fluoroquinolone resistance activity.

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Organization for Women in Science for the Developing World (OWSD), Trieste, Italy.

Covenant University, Ota, Nigeria
Genesis of Antibiotic Resistance (AR) XXX‐Mitigation of AR Pandemic (ARP) through Rational Measures.
Authors: Blackmon Sarah , Francisco Martinez , Montoya Hector , Flores Jorge , Anaya Edson , Martinez Juan , Calzoncit Melissa , Picazo Antonio , Jauregui Suzzette , Lechler Raul , Beautnagel Joanathan , Gutierrez Sonia , Johnson Dennie , Kannan Subburaj
Published: November 2023

Comprehensive and bolstered analysis of data available in domestic and global quantitative data bases provides the following reasonable and implementable measures that have the potential to mitigate Antibiotic Resistance Pandemic (ARP). The establishment of a centralized national and global data base and infrastructure that will: i. Monitor the amount of antibiotics manufactured per year, ii. Require periodic reporting (Quarterly/semi‐annual/annual) of the volume of antibiotics utilized on a regional, national and global scale, iii. Evaluate the volume of prescription antibiotics recovered by the “National Drug Take Back Program” sponsored cooperatively by the United States Drug Enforcement Agency, Department of Justice, and the Food and Drug Administration (DEA, DOJ, and FDA), iv. Survey and audit the process of disposal of unused prescription antibiotics by the DEA, DOJ, and FDA to ensure that employed destructive practices are conventional and appropriate. Particular oversight and monitoring must be given to incineration and solid waste facilities, v. Detection and evaluative analysis of metabolic breakdown, intermediate, and mother formulation of antibiotics in ground water, portable water systems, raw sewage and waste water, water captured from bio‐remediation processes, rivers, standing storage water reservoirs, storm runoff, and bio‐solids, vi. Collect and maintain epidemiological data on morbidity and mortality rates attributed to AR pathogenic induced infectious diseases on regional, national, and global scales, vii. Collection of data concerned with the effectiveness of antibiotics timeout (ATO) from private practice physicians, institutional reports from mental health facilities and hospitals, both private and public, nursing homes, hospice care, and infant and adult day care facilities, viii. Arrange and contribute to periodic updates concerned with ARP threats discovered or supposed by the United States Department of Health and Human Services Centers for Disease Control and Prevention (CDCP), ix. Research, collection, and reporting of data attentive to the hoarding of antibiotics and partitioning data to regional, national, and global levels, x. Data surveillance of the efficacy of antibiotics, and xi. Elimination of ineffective antibiotics(antibiotics with an increased morbidity and mortality rate) from the pharmacopeia and clinical practice. Taken together, effective universal adoption and implementation of aforesaid measures in health care facilities, and regulatory agencies across the globe, would potentially mitigate the ARP.

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Professional development funds to Subburaj Kannan
Transformations of antibiotics in water systems and the impact on specific microbes
Authors: Beavan Abigail , Bhatnagar Ashita , Henkes Zoe , Peller Julie , Dick Sara
Published: November 2023

Penicillin (Pen G) and Bactrim (sulfamethoxazole and trimethoprim) are two of the most commonly prescribed antibiotics used to target gram positive and gram negative bacteria, respectively. Usage of these medications often results in un‐metabolized antibiotics being discharged into water systems. Bacteria in those water systems can gain resistance from being exposed to low concentrations of these antibiotics. To better understand the lifetimes of antibiotics in water systems and the corresponding impacts on microbes, the decomposition and oxidation of Pen G and Bactrim in water solutions were studied. The bacteria selected for the study were Staphylococcus aureus (gram positive) and Escherichia coli (gram negative) due to their abundance and pathogenic capabilities. Antibiotic resistance in these species of bacteria is especially of concern to human health, since these bacteria have the ability to transfer resistance genes to other cells. The decomposition of 50 uM antibiotic solutions were studied in pure water and in solutions representing natural water: 10 ppm Dissolved Organic Material (DOM), 5mM NaHCO3, and both DOM and NaHCO3. The concentration of the antibiotics was monitored weekly. In separate experiments under the same conditions, the antibiotics were oxidized via hydroxyl radicals using gamma radiolysis. To determine compound concentrations, solutions were analyzed using Ultra High Performance‐Liquid Chromatography‐Mass Spectrometry (UHPLC). Simultaneously to the UHPLC analysis, the solutions were plated on lawns of each bacteria. Following overnight incubation, bacteria susceptibility ring measurements were performed to determine the effects of these solutions on the behavior of S. aureus and E. coli. The UHPLC data showed that Pen G in pure water decomposed completely within two weeks. The rate of decomposition of Pen G in the DOM solutions was substantially slower, as Pen G remained in solution after thirteen weeks, where the concentration was measured as 15uM. The bacteria plates displayed susceptibility ring sizes that corresponded to the decreasing concentration of the Pen G, whether by natural decomposition or hydroxyl radical‐mediated oxidation. In both cases, the fully decomposed/oxidized Pen G was ineffective as an antibiotic. Pen G resistant colonies of the bacteria were found on almost all the plates tested. The Bactrim solutions are currently being analyzed and the results will be presented.