bluuebunny/crossref_metadata_2025 huggingface dataset:
Population genomics and molecular surveillance for Plasmodium falciparum genetic diversities in an endemic area
Authors: Dokunmu Titilope Modupe , Oladejo David Oladoke , Adjekukor Cynthia Ufoma
Published: June 2021

Current malaria control strategies in endemic areas target eradication and elimination of the disease as well as improving artemisinin combination treatments (ACTs) and vector control. In this era of reducing transmission, molecular surveillance and bioinformatics tools offer great insights for understanding transmission dynamics. This study aims to determine prevalence rates and molecular diversities in Plasmodium falciparum in an endemic area. Polymerase chain reaction was used to determine presence of malaria infection, and presence of mutation in 2 select gene markers of drug resistance was determined by sequencing amplified products in few isolates. The sequence data was mapped unto the plasmodium genome to detect mutations in the genes using 3D7 strain (PF3D7_1343700.1 and PF3D7_0523000.1) as reference sequence. A high asymptomatic parasite rate (43%) was detected with a low frequency of SNPs in Kelch 13, which is not associated with drug resistance. There is a high rate of infection in this area with some mutations which may not directly modulate drug responses but the influence on transmission and impact on parasite's survival remains to be determined.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of UGT2A1 by miR‐196a & miR‐196b and Its Implications for Lung Cancer Risk
Authors: Sutliff Aimee Kristina , Chen Gang , Watson Christy J.W. , Lazarus Philip
Published: June 2021

Polycyclic aromatic hydrocarbons (PAH) are a class of highly lipophilic and mutagenic carcinogenic compounds found in tobacco smoke and incomplete combustion reactions. Once inside the cell, PAHs are oxidized by cytochrome P450s 1C1 and 1B1 to form reactive species that can form DNA adducts. PAHs are primarily detoxified by the UDP‐glucuronosyltransferase (UGT) family of enzymes, which comprise a major family of phase II metabolism responsible for the detoxification of a variety of endogenous and exogenous compounds including steroids, many drugs and chemotherapeutic agents, and carcinogens. UGT2A1 is an aero‐digestive tract‐expressing enzyme shown to be highly selective toward a broad range PAHs and is expressed in tobacco target tissues, including lung. microRNA (miRNA) are short, noncoding RNA sequences that play a role in translational regulation of gene expression by binding primarily to the 3′ untranslated regions (UTR) of mRNA. Significant (P<0.05 and P<0.001) decreases in luciferase activity were observed in cells over‐expressing heterologous vectors containing the 3′‐untranslated region (UTRs) of UGT2A1 with the miR‐196a and miR‐196b mimics, respectively. Pull‐down assays revealed miR‐196a and miR‐196b bound to the 3′ UTR of UGT2A1 at 3.4‐and 5.3‐fold higher levels, respectively, compared to the coding region control sequence. In the lung cancer cell lines, H1944 and H146, a significant trend is observed when cells were transfected with increasing levels of miR‐196a and miR‐196b (Ptrend=0.0065 and Ptrend=0.0114 for H1944, and Ptrend=0.0219 and Ptrend=0.0091 for H146, respectively). These data indicate that miR‐196a and miR‐196b may be important in regulating UGT2A1 expression in human tissues and may be potential biomarkers for cancer risk in smokers.

Support or Funding Information

National Institute of Environmental Health Sciences (NIEHS) of the National Institute of Health (NIH) to Dr. Philip Lazarus (Grant R01‐ES025460) and the Health Sciences and Services Authority of Spokane, WA (Grant WSU002292).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Role for Protein Kinase C in Gill Lateral Cell Activity of Crassostrea virginica
Authors: Ernest Krystle , Johnson Alexcia , Carroll Margaret A. , Catapane Edward J.
Published: June 2021

Gill lateral cell cilia of Crassostrea virginica are controlled by serotonergic‐dopaminergic innervations from their ganglia. Dopamine is the neurotransmitter causing cilio‐inhibition and serotonin causing cilio‐excitation. Gill lateral cell dopamine postsynaptic receptors involved in cilio‐inhibitory actions are D2‐like (D2DR). The D2DR signaling pathway involves steps that include inhibition of adenylyl cyclase, which has been well studied, and activation of phospholipase C (PLC). PLC activation generates second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 binds to an IP3 receptor which causes the release of intracellular calcium stores. Collectively, DAG and calcium ions are required to activate protein kinase C (PKC). While high cytoplasmic calcium levels have been shown to slow beating rates of gill lateral cell cilia, the role of PKC activation on gill lateral cell cilia has not been studied. We hypothesize PKC activation, which is downstream in the D2DR signaling pathway, is involved in the cilio‐inhibitory response of gill lateral cell cilia caused by dopamine in C. virginica. To study this we conducted acute experiments testing the PKC activator SC9 [N‐(6‐Phenylhexyl)‐5‐chloro‐1‐naphthalenesulfonamide], and the PKC inhibitor rottlerin. Ciliary activity of gill lateral cells was measured by stroboscopic microscopy and expressed as beats/sec ± sem. Statistical analysis was performed by ANOVA. Neither SC9 (10−6 – 10−3M) or rottlerin (10−6 – 10−4M) when applied directly to excise gill altered the gill lateral cell cilia beating rates, nor did they alter the cilio‐excitatory actions of applying serotonin (10−5M) to the gill. However, rottlerin (10−5M) treated gill did not respond normally to dopamine (10−6 – 10−3M) application to the gill, the cilia did not slow down their beating rates. Applying SC9 did not alter the cilio‐inhibitory effects of dopamine. These finding indicate that PKC is involved in the dopamine induced slowing of gill lateral cell cilia in C. virginica. Inhibiting PKC prevents the action of dopamine, while activating PKC by itself did not significantly alter the cilia beating rates. The study suggests that PKC plays a role in cilio‐inhibitory response caused by dopamine, but alone PKC activation is insufficient in generating the cilio‐inhibitory response and therefore other aspects of the D2DR signaling transduction pathway also must be involved. These findings are helpful in furthering the understanding of the D2DR mechanism of gill lateral cells and provides a foundation for further research.

Support or Funding Information

Supported by 2R25GM06003 of the Bridge Program of NIGMS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification/Optimization of Novel Small Molecule Inhibitors of Zika Virus Infection
Authors: Khachatoorian Ronik , Micewicz Ewa D. , Buzzanco Anthony S. , Riahi Rana , Jin Daphne , Ruchala Piotr , French Samuel W.
Published: June 2021

The rate of Zika virus (ZIKV) infection has been increasing since the original 2014 re‐emergence of the virus in Brazil, and the World Health Organization has warned against potential ZIKV epidemics. Furthermore, ZIKV, a flavivirus family member, has been identified as the cause of microcephaly and other developmental neuroimmunopathologies in fetuses of pregnant women and Guillain‐Barre Syndrome in adults, and to date, no therapeutics or vaccines are available against ZIKV infection. In this study, we screened a limited proprietary library of diverse organic small molecules, and identified a number of leads with significant antiviral activity. Briefly, the anti‐ZIKV activity of the compounds was assayed through plaque‐forming assays in A549 cells. Subsequently, dose response assays were performed for the active compounds using the same experimental setup. Furthermore, we performed structure/activity relationship (SAR) analyses and managed to improve the anti‐ZIKV activity with the most active analogs showing activity in low picomolar range. Finally, we constructed a general model of the overall structure of these anti‐ZIKV compounds which is based on the structural similarities of all active hits. These results identify excellent template(s) with potential for further optimization of antiviral activity through rigorous SAR analyses which may be utilized for the development of inexpensive and orally available anti‐ZIKV drugs. These compounds may also be beneficial for treatment of other viruses in the same family including Dengue virus.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Prodrug Approaches for Long‐Acting Nanoformulations of Emtricitabine‐Based Regimens
Authors: Al‐khouja Amer , Hobson James J. , Henriquez Stephanie , Meyers David , Curley Paul , Siccardi Marco , Owen Andrew , Flexner Charles , Rannard Steven , Freel Meyers Caren L.
Published: June 2021

Most current antiretroviral (ARV) medications for HIV treatment and prevention necessitate lifelong daily dosing, and require high levels of patient adherence to be effective. Long‐acting injectable (LAI) ARVs would allow less frequent administration, improving adherence. Technologies currently used to produce LAI‐ARVs are incompatible with the nucleos(t)ide reverse transcriptase inhibitors (NRTIs) due to their high water solubility. It is critical to overcome this challenge to development of LAI NRTI strategies as most current ARV combination therapies include NRTI backbone therapies. We synthesized a series of carbamate‐based emtricitabine (FTC) prodrugs to increase hydrophobicity and improve compatibility for solid drug nanoparticle (SDN) formation. Prodrug activation kinetics were assessed in vitro via HPLC in human muscle, plasma, and liver fractions. Prodrugs were then screened for SDN compatibility using an emulsion templated/freeze drying method. Hits from these screens are defined by the following properties: Z‐average diameter <1000 nm, a polydispersity index of <0.5, and the ability to disperse in water at a concentration of 1 mg/mL. Prodrug release from SDNs and subsequent enzymatic conversion to FTC was quantified by HPLC in human muscle fractions in vitro. Carbamates bearing longer alkyl chains (C7 and C8) were most efficiently activated in all conditions. Hydrolysis rates in liver were higher than those in plasma or muscle.

Carbonate moieties at the 5′‐position were hydrolyzed more efficiently than the carbamate moiety under all conditions. Nanoparticle screening at 10 wt% prodrug demonstrated a correlation between calculated logP and the number of hits obtained for carbamate/carbonate prodrugs (39/42), but not for carbamate‐only prodrugs (4/42). Formation of nanoparticles with higher drug loadings was also demonstrated with a hit for an octyl carbamate/carbonate FTC prodrug SDN achieved at 70 wt%. Assessments of in vitro prodrug release from SDNs monitoring FTC formation over time suggested that prodrug release is rate‐limiting under conditions of high SDN concentration. Taken together, this work suggests that carbamate‐based prodrug approaches offer a promising starting point for development of LAI FTC toward a complete LAI‐ARV regimen to improve adherence.

Support or Funding Information

NIH 1F31AI129549

NIH 5R01AI114405

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lansoprazole inhibits adipocyte differentiation via LXR‐independent mechanism
Authors: Benchamana Ameena , Soodvilai Sunhapas
Published: June 2021

Liver X receptor (LXR), a ligand‐activated transcription factor, belongs to the nuclear receptor superfamily. It is believed to regulate adipocyte differentiation and lipid metabolism. Lansoprazole (LPZ), an effective FDA‐approved drug used in acid relate disorders, recently is known to be LXR agonist. However, the effect of LPZ on adipocyte differentiation is still unknown. In this study, we investigated anti‐adipogenesis effect of LPZ in 3T3‐L1 pre‐adipocyte and elucidated its possible underlying mechanisms. To determine whether LPZ inhibit adipogenesis, 3T3‐L1 pre‐adipocyte was treated with LPZ (10, 50 and 100 μM) for 48 hours during differentiation induction followed by determination of intracellular lipid accumulation by Oil Red O staining. Our findings showed that LPZ significantly reduced intracellular lipid accumulation only at dose 100 μM without cytotoxicity effect. Co‐treatment the cells with LPZ and LXR antagonists (fenofibrate 10 μM and SR9238 1 μM) for 24 hours failed to attenuate the effect of LPZ on adipocyte differentiation. This result indicated that LPZ inhibited adipocyte differentiation via LXR‐independent mechanism. Expressions of adipocyte differentiation related proteins; peroxisome proliferator‐activated receptor‐γ (PPARγ), CCAAT‐enhancer‐binding protein‐α (C/EBPα), and fatty acid synthase (FAS) were significantly reduced following LPZ treatment. Taken together, our results demonstrated that LPZ inhibits adipocyte differentiation through downregulate PPARγ, C/EBPα, and FAS expression and this inhibition was LXR‐independent pathway. Our results point towards the new indication of LPZ as an alternative therapeutic agent for obesity treatment.

Support or Funding Information

This work was carried out by the support from the Office of the Higher Education Commission of Thailand (Grant to Ameena Benchamana).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of c‐Cbl Antagonists to Promote Corneal Epithelial Wound Healing
Authors: Ceresa Brian
Published: June 2021

Ligand mediated desensitization is a key negative regulator of cell surface receptors. In the human cornea, epidermal growth factor receptor (EGFR) desensitization, due to the relatively high levels of endogenous EGF in the tear fluid, limits the repair and restoration of the corneal epithelium. The E3 ubiquitin ligase, c‐Cbl is a critical modulator of EGFR desensitization, as it mediates the ubiquitylation of the activated receptor and targets it to the lysosome for degradation. Ultimately, this terminates receptor signaling. We have previously demonstrated that knock down of c‐Cbl results in an enhancement of EGFR‐mediated cell migration that is necessary for healing of corneal epithelial wounds. The goal of these experiments was to identify novel compounds that antagonize c‐Cbl activity and could be developed to enhance EGFR‐mediated corneal epithelial wound healing. Using the published crystal structures of the EGFR:c‐Cbl complex, we identified the point of interaction between the two proteins. We subsequently performed an in silico screen of ~25,000,000 compounds (from the ZINC15 library) to identify compounds that are predicted to antagonize c‐Cbl binding to and ubiquitylation of the EGFR. The top 50 candidates were screened using a biochemical assay for their ability to bind c‐Cbl using Differential Scanning Fluorimetry (Thermofluor™). From that screen, six compounds were identified as c‐Cbl binders and were tested in immortalized corneal epithelial cells to determine which compounds could inhibit c‐Cbl function. Two of the six compounds blocked EGF‐mediated EGFR ubiquitylation, albeit at relatively high concentrations (IC50 were ~50 μM and ~5 μM). Further, these compounds exhibited some cytotoxicity at 100 μM. These compounds represent two lead compounds that can be chemically optimized to enhance their affinity for c‐Cbl and become more potent inhibitors of c‐Cbl activity. The long‐term goal is to use these compounds as therapeutic agents to promote the restoration of damaged corneal epithelium, to minimize patient discomfort, reduce ocular infections, and minimize the likelihood of blindness.

Support or Funding Information

Research was supported by NIH/NEI grant EY027032.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Predicting Resistance To Investigational Microbicide MIV‐150 Using Structure‐based methods and Fluorescence Enzyme Inhibition
Authors: Muwonge Alecia Nicole , Azeem Syeda Maryam , Frey Kathleen M.
Published: June 2021

Microbicides may be an alternative option in HIV prevention for women affected in sub‐Saharan Africa. Many microbicides are non‐nucleoside reverse transcriptase inhibitors (NNRTIs) that target HIV reverse transcriptase (RT). Resistance mutations result in NNRTI treatment failure. These mutations may also affect the efficacy of chemically similar NNRTIs used as microbicides. The objective of this study was to predict mutations in RT that may cause resistance to investigational microbicide NNRTI MIV‐150. A computational, structure‐based approach was used to predict resistance mutations for MIV‐150. This method calculates changes in affinity (ΔA) and stability (ΔS) imparted by the mutation. The ΔA + ΔS values for K101P predict that this mutation confers high‐level resistance to MIV‐150. Based on this prediction, we analyzed the models and used Molecular Dynamics (MD) to compare the interactions of MIV‐150 with RT wild type (WT) and with RT (K101P). From MD, we found key interactions were lost with K101P. To experimentally validate our findings, we conducted a fluorescence‐based reverse transcription assay for MIV‐150 with RT (WT) and RT (K101P). Our IC50 values showed a 170‐fold change in potency. The assays results validate that the K101P mutation confers resistance to MIV‐150. Our results are consistent with antiviral data reported in the literature. From these results, we believe that this approach is effective for predicting resistance to microbicide NNRTIs.

Support or Funding Information

Research is supported by Dr. Kathleen Frey at Long Island University, Brooklyn.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Inert, Injectable Sustained Release Formulation for Recombinant Human TIMP‐2 in peptide hydrogels
Authors: Chowdhury Ananda , Yamada Yuji , Schneider Joel P. , Stetler‐Stevenson William G.
Published: June 2021

Tissue Inhibitor of Metalloprotease 2 (TIMP‐2), is a soluble endogenous 22 kDa metalloprotease inhibitor, that exhibits anti‐tumor, anti‐metastatic and anti‐angiogenic activities both in vitro and in vivo. These effects are mediated via several reported mechanisms including direct MMP inhibition, as well as MMP‐independent processes. Recently we have reported bioprocess development for enhanced production, as well as biochemical and biologic characterization, of recombinant, human TIMP‐2. Thus, allowing pre‐clinical testing of rhTIMP‐2 in multiple murine tumor models. Here we report the development of inert, injectable peptide hydrogel matricies for encapsulation and sustained release of rhTIMP‐2. Additionally, we describe comparison of biophysical attributes and pharmacological properties of rhTIMP‐2 released over time from these biogels.

rhTIMP‐2 (with 6X His epitope tag) was produced and characterized as described. Three types of b‐hairpin peptide gels, with varying net charges at physiological pH, exhibited effective encapsulation/retention of up to 4 mg/ml of rhTIMP‐2 (maximal tested concentration). All gels were subjected to oscillatory shear rheology and demonstrate shear‐thin recovery properties post‐incorporation of rhTIMP2. A slow and linear rhTIMP‐2 release profile was obtained from the anionic AcVES3 (net charge: −5) formulation up to 28 days post formulation. Regression equations allow extrapolation of released rhTIMP‐2 based on formulation dose and time elapse. Released rhTIMP‐2 exhibits identical secondary structure (circular dichroism spectra) as well as no detectable change in molecular mass (ESI‐MS) compared with the unformulated rhTIMP‐2. Biogel‐released rhTIMP‐2 inhibits MMP‐2 enzymatic activity and suppresses rhEGF mediated A549 lung cancer cell proliferation in‐vitro similar to unformulated rhTIMP‐2. These findings suggest that these peptide biogels gels could be useful as an injectable vehicle for systemic delivery of rhTIMP‐2 in vivo that would be useful in subsequent dosing experiments studying the anti‐tumorigenic activities of rhTIMP‐2 in murine cancer models.

Support or Funding Information

The authors acknowledge National Institutes of Health Intramural Grants ZIA‐SC‐009179 and ZIA‐BC‐011204 (NCI/CCR Project 8386620) to W.G.S.‐S.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
THE ANTI‐INFLAMMATORY EFFECTS OF ADELMIDROL + SODIUM HYALURONATE (VESSILEN®) IN PRECLINICAL AND CLINICAL STUDIES
Authors: Cuzzocrea Salvatore , Peritore Alessio , Impellizzeri Daniela , Cordaro Marika , Siracusa Rosalba , Fusco Roberta , Gugliandolo Enrico , Crupi Rosalia , Di Paola Rosanna
Published: June 2021

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic bladder condition characterized by frequent urination, bladder inflammation and pain. It is a particular challenging disease and a clear unmet medical need in terms of identifying new therapeutic strategies. Adelmidrol, the diethanolamide derivative of azelaic acid, represents an effective treatment for human inflammatory disorders. The aim of study was first, to evaluate the anti‐inflammatory effects of intravesical Vessilen® administration (a new formulation of 2% adelmidrol + 0.1% sodium hyaluronate) in acute and chronic animal models of cystitis induced by a single or repetitive intraperitoneal injection of cyclophosphamide (CYP) and then, in patients with IC/PBS treated once weekly by endo‐bladder instillation of Vessilen® for 8 weeks.

CYP instillation caused macroscopic and histological bladder alterations, inflammatory infiltrate, increased mast cell number and bladder pain that were reduced by Vessilen® treatment. In addition, an increased expression of nitrotyrosine, a marker of oxidative stress and a decreased expression of endothelial tight junction zonula occludens‐1 were found in CYP‐injected animals. Vessilen® diminished nitrotyrosine and increased zonula occludens‐1 formation. To characterize the inflammatory state and possible reactive oxygen species‐induced pathways involved during chronic cystitis, we also analyzed the nuclear factor‐κB pathway and expression of inducible nitric oxide synthase, interleukin‐1β and monocyte chemoattractant protein‐1. Vessilen® inhibited nuclear factor‐κB pathway and inflammatory mediator levels. Moreover, an increase in nerve growth factor was also found in chronic CYP‐induced cystitis triggering inflammatory response and mechanical hyperalgesia. In that regard, Vessilen® treatment reduced mechanical allodynia and nerve growth factor levels. Interestingly, a reduction in symptom intensity and a significant improvement in the assessment of quality of life were also evident in IC/PBS patients treated with Vessilen®.

Thus, in conclusion, we demonstrate that Vessilen® treatment could be a new therapeutic approach in the treatment of human cystitis.

Support or Funding Information

no funding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
BPC 157 Therapy Heals Tendon Muscle Junction in Rats
Authors: Japjec Mladen , Staresinic Mario , Sebecic Bozidar , Pavlov Katarina Horvat , Drmic Domagoj , Vidovic Tinka , Vlainic Josipa , Seiwerth Sven , Sikiric Predrag
Published: June 2021

Aim

After tendon was separated from muscle in the right quadriceps muscle in rats, we focused on the tendon‐muscle junction healing and therapy with the stable gastric pentadecapeptide BPC 157. Noteworthy, BPC 157 is an original antiulcer peptide, stable in human gastric juice, used in ulcerative colitis and now multiple sclerosis trials), with particular wound healing ability: healing of transected or crushed muscle, transected or detached tendon, and transected ligament, demonstrated macro/microscopically, biomechanically and functionally (i.e., absent leg contracture), with parenteral and peroral therapy application (Curr Pharm Des. 2017;23(27):4012–4028; J Orthop Res. 2006 May;24(5):1109–17; J Orthop Res. 2006 May;24(5):982–9; J Orthop Res. 2003 Nov;21(6):976–83).

Methods

Under deep rat anesthesia tendon muscle junction of right quadriceps muscle was presented, and without direct transection of tendon muscle junction, the tendon was gently separated from muscle fibres. Medication was BPC 157 (10 ug or 10 ng/kg/day, intraperitoneally or in drinking water (0.16ug or 0.16ng/ml; 12ml/rat/day) while controls received an equivolume of saline (5 ml/kg intraperitoneally) or drinking water only. Macro/microscopical, biomechanical assessment was at 1st, 2nd, 4th, and 6th week after surgery, while functional presentation (walking patterns, leg contracture) was daily assessed.

Results

Gross assessment reveals in quadriceps muscle a huge defect between tendon and muscle sustainably progressing throughout the experiment and failed healing. By contrast, in rats underwent tendon muscle junction separation that received BPC 157, the healing was continuously presented. Since very beginning, BPC 157 rats presented defect markedly less and finally grossly reestablished tendon muscle junction (Fig. 1). Also, controls exhibited severe leg contracture, and tottering walk, which were fully counteracted by BPC 157 therapy. These results are completely confirmed by microscopy. Illustratively, at 2nd week controls showed edema of the tendon with moderate lymphocytic infiltrate and formation of granulation tissue, while BPC 157 rats exhibited only minimal lymphocytic infiltrate with no granulation tissue formation in all pentadecapeptide BPC 157 treated animals and with better already reestablished myotendinous junction structures (Fig. 2). Likewise, biomechanic assessment demonstrated only a poor recovery of muscle strength in controls, while a full recovery ensues in BPC 157 treated rats.

Conclusion

In addition to the therapy of the injured muscle and tendon, BPC 157 therapy could also specifically benefit the injuries of tendon muscle junction.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of BPC 157 on Tracheocutaneous Fistula Healing in Rat
Authors: Madzarac Goran , Cesarec Vedran , Djakovic Zeljko , Becejac Tomislav , Hirsl Dorian , Drmic Domagoj , Seiwerth Sven , Stancic‐Rokotov Dinko , Sikiric Predrag
Published: June 2021

Introduction

One of the common late complications of a tracheotomy is tracheocutaneous fistula. The surgical procedure is needed to close such fistulas. BPC 157, anti‐ulcer peptide (GEPPPGKPADDAGLV, M.W. 1419) successful in trials for inflammatory bowel disease, wound treatment, effective alone without a carrier. BPC 157 interferes with NO‐system in vitro/in vivo on different models and different animal species. The impact of BPC 157 and NO‐system on gastrocutaneous, colocutaneous and oesophagocutaneous fistulas healing process indicates potential effect of BPC 157 in tracheocutaneous fistula healing process, which has not yet been investigated.

Methods

Albino Wistar male rats were used in this experiment, 220–280 g. Tracheocutaneous fistula, 4 mm trachea and skin defect, was surgically made under anaesthesia. Animals were treated according to the experiment protocol: a) drinking water p.o., b) BPC 157 (10 μg/kg, 12 ml/rat/day) p.o., c) BPC 157 (10 ng/kg, 12 ml/rat/day) p.o., d) saline 5ml/kg/day i.p., e) BPC 157 (10μg/kg, 5ml/kg/day) i.p., f) BPC 157 (10 ng/kg, 5 ml/kg/day) i.p., g) L‐NAME (5 mg/kg/day) i.p., h) L‐arginine (100 mg/kg/day) i.p., i) L‐NAME L‐arginine i.p., j) L‐NAME BPC 157 (μg) i.p., k) L‐arginine BPC 157 (μg) i.p., l) L‐NAME L‐arginine BPC 157 (μg) i.p., m) L‐NAME BPC 157 (ng) i.p., n) L‐arginine BPC 157 (ng) i.p., o) L‐NAME L‐arginine BPC 157 (ng) i.p.. Third, fifth and seventh postoperative day animals were euthanized. Fistula specimens were harvested and macroscopic and histological analysis was made.

Results

A consistent counteracting beneficial effect was shown in all animals treated with BPC 157, alone and with (L‐NAME) and/or L‐arginine in a 7‐day interval. BPC 157 accelerated healing of tracheocutaneous fistulas and showed macroscopic and histological healing improvements. Tracheocutaneous fistulas healing was improved speedily (BPC 157 completely counteracted L‐NAME effects (L‐NAME+BPC 157 and L‐NAME+L‐arginine+BPC 157 groups), or with delay and to less extent (L‐arginine) or aggravated, rapidly and prominently (L‐NAME). L‐arginine reduces aggravation by NOS‐blockade (L‐NAME) to the control levels. Also, BPC 157 more than nullifies the effect of L‐NAME.

Conclusion

BPC 157 improved healing of both tracheal and skin defects and mediated fistula closing. This effect was shared by L‐arginine, with an opposite effect seen by L‐NAME, thereby portraying NO‐system involvement in the healing of tracheocutaneous fistula.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influence of liver‐specific ablation of gp78 E3‐ligase on hepatic cytochrome P450‐ dependent drug metabolism: Clinical implications
Authors: Kwon Doyoung , Kim Sung‐Mi , Correia Maria Almira
Published: June 2021

The autocrine motility factor receptor (AMFR)/gp78 is a membrane‐anchored E3 ligase involved in ubiquitin (Ub)‐dependent proteasomal degradation (UPD). We have previously documented that gp78 is involved in the UPD of two clinically relevant hepatic cytochromes P450 Cyp2E1 and Cyp3A4 engaged in therapeutic drug and/or xenobiotic metabolism. To determine the role of gp78 in the UPD of other hepatic P450s, we generated a liver specific gp78 knockout (KO) mouse model. Cultured primary hepatocytes from KO and wild type (WT) mice were untreated or treated with P450 inducer β‐naphthoflavone (Cyp1a2), phenobarbital (Cyps2a, 2b and 2c), isoniazid (Cyp2e1) or dexamethasone (Cyp3a). Immunoblotting and selective functional marker analyses revealed that the functional content of Cyps1a2, 2a, 2c, 2e1 and 3a was increased, but that of Cyp2b or Cyp2d was decreased/unchanged in KO relative to WT. To determine the potential clinical relevance of these findings, the in culture metabolism of tamoxifen, a breast cancer chemotherapeutic prodrug, to its active metabolite endoxifen was monitored by LC/MS. Upon 8 h of tamoxifen incubation, the levels of Cyp3a‐generated metabolites N‐desmethyltamoxifen and endoxifen were significantly increased in KO over WT hepatocytes, but the Cyp2d‐generated metabolite 4‐hydroxytamoxifen was unchanged, consistent with the corresponding functional levels of hepatic Cyps3a and 2d. Collectively, our findings reveal that hepatic gp78 deficiency significantly increased the functional levels of several drug‐metabolizing P450s most likely by impairing their UPD. These increases in turn enhanced the hepatic metabolic activation of a clinically relevant anti‐cancer drug. Because gp78 genetic polymorphisms exist, and gp78 is overexpressed in certain malignant tumors and human cancers (i.e. breast and colon) that correlates well with their progression and poor prognosis, our findings suggest that gp78 be considered a significant modulator of P450‐mediated metabolism of clinically relevant/chemotherapeutic drugs and associated drug‐drug interactions.

Support or Funding Information

Supported by NIH grants GM44037, DK26506 and DK26743.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Pharmacokinetic Compatibility of XueBiJing Injection with Antibiotics Co‐administered in Management of Sepsis
Authors: Li Chuan
Published: June 2021

Sepsis is life‐threatening organ dysfunction caused by a dysregulated host response to infection. XueBiJing, an intravenous injection approved by the China FDA in 2004, has been incorporated into conventional sepsis management, which relies on timely and appropriate antibiotics and resuscitation therapies. In China, about 800,000 patients use XueBiJing injection each year and 80% of them are patients with sepsis or septic shock. The injection is prepared from a combination of Carthamus tinctorius flowers (Honghua in Chinese), Paeonia lactiflora roots (Chishao), Ligusticum chuanxiong rhizomes (Chuanxiong), Angelica sinensis roots (Danggui), and Salvia miltiorrhiza roots (Danshen). Many clinical studies in China suggested that adding XueBiJing injection to conventional management of sepsis reduces patients' 28‐day mortality and incidence of complications, improves their Acute Physiology and Chronic Health Evaluation II scores and prognosis, and shortens their stay in intensive care units, with low incidence of side effects. Despite the promising results of clinical studies, success of such combination drug therapies containing XueBiJing injection also relies on PK compatibility, i.e., exhibiting limited pharmacokinetic interaction problems that can counteract the drugs' synergistic or additive pharmacodynamic effects, as well as their safety. In this presentation, the speaker will report evaluation, by his lab, of pharmacokinetic compatibility of XueBiJing injection with antibiotics co‐administered in management of sepsis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synergistic Effect of Combined Inhibition of the Checkpoint kinase 1 and G9a Methyltransferase Pathways in Pancreatic Cancer
Authors: Urrutia Guillermo , Salmonson Ann , Lomberk Gwen
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer‐related deaths in the United States with a devastating overall five‐year survival rate of less than five percent. Consequently, there is an urgent need to further improve our understanding of molecular mechanisms underlying PDAC development to identify innovative therapeutic targets. Our laboratory is focused on utilizing epigenetic inhibitors in novel therapeutic approaches. Our experimental design involves harnessing the capacity of cell cycle inhibitors to arrest cells in G2/M while combining them with epigenetic inhibitors, which by altering chromatin structure can give rise to an aberrant mitotic checkpoint response leading to rapid death (Mol Cancer Res; 15(8): 984–97). In the present study, we have optimized and tested an experimental combinatorial therapy for pancreatic cancer using the CHK1 inhibitor LY2606368 and the G9a (Eukaryotic Histone Methyltransferase 2) inhibitor BRD4770. Using MTS assay, we show that the growth of pancreatic cancer cells grown in monolayer is reduced by both LY2606368 and BRD4770 individually, while their combination achieves a synergistic effect, as calculated using the Chou and Talalay method. In addition, this synergistic effect is also observed using pancreatic cancer cells grown as 3D spheroids. Moreover, the combination of LY2606368 with either of two additional G9a inhibitors, BIX01294 or UNC0631, reveals similar synergy, further supporting the specificity of this effect. FACS analysis of cells treated with the LY2606368 and BRD4770 combination shows an increased population in G2/M at 24 hours and an apoptotic pre‐G1 signal. The apoptotic response coincides with the activation of caspases 3 and 7. In vivo studies demonstrate that targeting these pathways reduces pancreatic tumor growth in a subcutaneous pancreatic cancer xenograft model. In summary, these data establish that targeting the epigenetic regulator G9a in the context of cell cycle inhibition holds promise as a novel combinatorial therapy for pancreatic cancer.

Support or Funding Information

NIH R01CA178627; Advancing a Healthier Wisconsin Endowment

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Searching for a new cancer drug: Kentucky Hemp‐induced modulation of ovarian cancer cell proliferation and total cell death
Authors: Duff Adam , Wang Annie , Biela Sara , Sumanasekera Wasana
Published: June 2021

Marijuana (cannabis sativa), a schedule 1 drug has been recently approved by some of states in the US for its therapeutic benefit. Although few reports discuss about its anti‐cancer potential, currently it has been used mainly for epilepsy and to alleviate pain associated with many diseases. However, due to marijuana's addictive potential there are restrictions for its use. Hemp, which belongs to the same genus and species as marijuana shows similar therapeutic benefits with much less addictive potential due to the low level of the addictive component, tetrahydrocannabinol (THC). Both marijuana and hemp are rich in therapeutically valuable cannabidiol (CBD) and cannabidiolic acid (CBD‐A). The major objective of the current study is to investigate KY hemp's therapeutic potential against ovarian cancer. Our laboratory is interested in searching for alternative therapies for ovarian cancer. As a candidate drug we have investigated the anti‐cancer potential of KY grown hemp using two different epithelial ovarian cancer cell (OCC) lines, which are A2780 and MES‐OV. KY Hemp extracts were made using supercritical CO2 extraction method by KY Commonwealth Extracts Company. Following High Performance Liquid Chromatography (HPLC) analysis by MRX laboratories we have obtained this extract to study its anti‐cancer potential. There are no reports on anti‐cancer activity of KY hemp against ovarian cancer or any type of cancer yet. Therefore, this study provides novelty.

Methods

Following serum starvation, OCCs were treated with vehicle (negative control), 80 μM doxorubicin and cisplatin, and different concentrations of hemp extracts that contain 8.5–335 μM CBD. Hemp‐induced modulation of ovarian cancer cell viability was assessed spectrophotometrically by MTT cell proliferation assay and Calcein AM assay. The MTT assay detects formazan product formed due to mitochondrial reductase enzyme present in viable cells. The Calcein assay detects the esterase enzyme activity present in viable cells. Ethidium homodimer dye (ETHD‐1) was used to detect total cell death. ETHD‐1 penetrates only damaged cell membranes and binds different molecules such as cell debris, single stranded DNA, double stranded DNA, and peptides. Calcein AM and ETHD‐1 fluorescence was also detected microscopically. KY Hemp extract has significantly decreased OCC proliferation and caused total cell death. KY Hemp extract has shown anti‐cancer properties comparable to doxorubicin and cisplatin. Based on the data here we conclude that KY Hemp has significant anti‐cancer potential against ovarian cancer

Support or Funding Information

Funding is provided by the Sullivan University System via a faculty development grant awarded to Dr. Sumanasekera. Grant number: RG‐1‐PS‐2017‐04
Structure‐Activity Analysis of Bisphenol‐Z Derivatives for Anticancer Activity
Authors: Stang Christopher R.T. , Inbody Laura R. , Stitzlein Lea M. , Dick Matt J. , Eichel Amanda M. , Orahood Cassandra B. , Mohamed Ahmed H. , Dudley Richard W. , Schneider Ryan A.
Published: June 2021

Much of the research surrounding bisphenol derivatives focuses on their use as polymers and plasticizers for industry. Research has demonstrated that bisphenol‐A (BPA) adversely affects humans via endocrine disruption through hormonal modulation. Despite the recent explosion in BPA research, little effort has been devoted to researching the biological activities of other bisphenol derivatives, including bisphenol‐Z (BPZ). Initial screening of BPZ revealed moderate in vitro anticancer activity against four diverse human cancer cell lines (A375 malignant melanoma, A‐172 glioblastoma, HT‐29 colorectal adenocarcinoma, and MCF7 breast adenocarcinoma) with IC50 values of approximately 65 μM following 24 hour treatments. In an attempt to enhance the activity of BPZ, seven analogs with varying degrees of polarity, lipophilicity, and sterics on the cyclohexane ring were synthesized. Screening of these analogs revealed that polar substitutions resulted in a loss of activity, while a simple methyl substitution had similar activity to BPZ. Anticancer activity was enhanced relative to BPZ following substitution with an ethyl, isopropyl, or N‐palmitoyl group at the 4‐ position. The most active compounds from this structure‐activity study were the N‐palmitoyl (LS4) and isopropyl (LS6) derivatives with 24 hour IC50 values ranging from 25 to 53 μM and 33 to 42 μM respectively.

Given the increased activity of LS4 and LS6, additional studies were performed including a trypan blue exclusion assay and a Caspase‐Glo® 3/7 assay. The results of these studies revealed exclusion of trypan blue at supra‐IC50 concentrations six hours after treatments, suggestive of a non‐necrotic mechanism of death. To determine if caspase‐dependent apoptosis was involved, we performed a Caspase‐Glo® 3/7 assay. Functional screening of executioner caspase‐3 and ‐7 revealed significant increases in activity following a 12 hour treatment with LS4 in all cells lines. This effect was least evident in the MCF7 cells, potentially due to a mutation wherein these cells lack expression of caspase‐3. In contrast, LS6 failed to activate executioner caspases‐3 and ‐7 in these same cell lines.

In conclusion, this structure‐activity study revealed that nonpolar substitution at the 4‐ position of BPZ enhanced anticancer activity. Additionally, membrane integrity remained intact following exposure to LS4 and LS6. While LS4 activated executioner caspases‐3/7, LS6 failed to activate this hallmark pathway of apoptotic death. Further investigations are ongoing to elucidate the mechanisms of cell death, identify putative cellular targets, and determine potential selectivity against normal human cell lines.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of the anticancer activity of a newly synthetic combretastatine analogue against hepatocellular carcinoma.
Authors: El‐Moselhy Mohamed A. , Aboubakr Esam Mohamed , Aly Omar Mohamed , Taye Ashraf
Published: June 2021

Combretastatin A‐4 has been emerged as a promising vascular disrupting agent, which displayed potent cytotoxic activities against a wide range of human cancer cell lines. Therefore, the present study was conducted to evaluate the anticancer activity of a newly synthesized combretastatin A‐4 analogue (1‐(3,4‐Dimethoxyphenyl)‐N‐(2‐methoxyphenyl)‐5‐(3,4,5‐trimethoxyphenyl)‐1H‐1,2,4‐triazole‐3‐carboxamide) against hepatocellular carcinoma (HCC) both in vitro and in vivo. In vitro study used HepG2 cell lines to evaluate the cytotoxic (antitumour) activity, tubulin protein inhibition and cell cycle analysis. While, in vivo study used DENA + CCl4 as a well‐established model for HCC induction in rats. Animal groups were divided as following: Normal non‐treated group, DENA + CCl4 group, CA4‐P treated group and CA4‐P analogue treated group. The following parameters were determined: hepatic relative weight, number of hepatic nodules, relative volume of hepatic nodules, hepatic tissue content of tubulin, histopathological examination of the hepatic tissue, hepatic tissue lipid peroxidation, hepatic tissue carbonyl content, serum ALT, serum AST, hepatic tissue TNF‐α and hepatic AFP concentrations. CA4‐P analogue significantly decreased liver relative weight, number of hepatic nodules and there relative volumes, tubulin content of the hepatic tissue, GSH and AFP which were more pronounced than CA4‐P group. Whereas a marked increase in MDA, carbonyl content and TNF‐a inside hepatic tissue were observed in the CA4‐P analogue treated group, which were higher than CA4‐P group. In conclusion CA4‐P analogue has a potential anti‐cancer activity against hepatocellular carcinoma and this effect was more noticeable than its parent drug (CA4‐P).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SHetA2 and its Analogs Induce Growth Inhibition in Human Glioblastoma Cells
Authors: Chan Allen , Liu Shengquan , Rajagopalan Vanishree
Published: June 2021

SHetA2, a flexible heteroarotinoid and new cancer‐preventative drug candidate, has exhibited growth inhibition in many human cancer cell lines such as lung, kidney, ovarian, and colon and in animal models of cancer. Because SHetA2 has high lipophilicity (Log P of 7.09), indicating low bioavailability, this suggests that SHetA2 is not an ideal drug candidate (Ideal Log P between 0 and 5). In order to address this issue, modifications were done to the thiochroman ring, resulting in SL‐analogs with lower Log P values. In this study, we examined the growth inhibitory effects of SHetA2 and three thiochroman analogs, SL‐01‐39, SL‐01‐30, and SL‐01‐18 on T98‐G cells, a human glioblastoma cell line.

After T98‐G cells were treated for 48 hours with SHetA2 and its analogs at 0.5, 1, 5, 10, and 20 μM, MTS assays were performed to measure cell proliferation and calculate their GI50 values (drug concentration which causes 50% inhibition of cell growth). In addition, trypan blue exclusion assays were performed, using a Vi‐Cell XR cell counter, to determine percent cell viability and relative cell counts of T98‐G cells after treatment with SHetA2 or its analogs. GraphPad Prism statistical software was used to generate a non‐linear regression model from the MTS assay readings to calculate the GI50 values. Results from trypan blue exclusion were analyzed, using Microsoft Excel, to determine the trend in cell viability and viable cell counts with respect to drug concentrations.

GI50 values of SHetA2, SL‐01‐39, SL‐01‐30, and SL‐01‐18 were calculated to be 5.57 μM, 5.38 μM, 6.41 μM, and 5.31 μM, respectively, using a one‐phase decay model. With trypan blue exclusion assay, percent cell viability remained relatively unaffected in T98‐G cells treated with SHetA2 and its analogs, despite increasing concentrations. On the contrary, trypan blue exclusion assay results showed a significant decrease in relative cell counts in cells treated with SHetA2 or its analogs when compared to the untreated cells.

This is the first report showing the growth inhibitory effects of SHetA2 in T98‐G human glioblastoma cells. The GI50 value of SHetA2 in T98‐G cells is in low micromolar range (~5 μM), comparable to its GI50 values in other cell lines published in literature. In addition, the GI50 values of the three analogs, SL‐01‐39, SL‐01‐30, and SL‐01‐18, were comparable to that of SHetA2. SHetA2 and its analogs decreased the relative cell number of T98‐G cells without significantly affecting cell viability, suggesting that these agents primarily inhibit cell growth of T98‐G cells without inducing cytotoxic effects. Further investigation on the mechanism of growth inhibition is ongoing.

Support or Funding Information

Touro CA COP‐MS Program for funding this research.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.