10-fold Reduction Research Articles

MAPK pathway and NIS in B-CPAP human papillary thyroid carcinoma cells treated with resveratrol

BackgroundResveratrol, a herbal phytoalexin, is known to have anti-tumor effects in several tumors including thyroid cancer cells. AimThe aim of this study was to determine the effects of resveratrol on the expression of BRAF, ERK and NIS mRNA levels and protein expression in B-CPAP human thyroid papillary cancer cell line. MethodsB-CPAP cells were treated with resveratrol at concentrations of 10–100 μM for 24–48–72 h. Cell viability was assessed by XTT Cell Proliferation Assay. BRAF, ERK and NIS mRNA levels were evaluated by rt-PCR method. Protein expressions were evaluated by Western Blot method. ResultsResveratrol was found to inhibit cell proliferation in a time and dose dependent manner. The IC50 values of resveratrol were 18.7 μM and 56.8 μM after 48 h and 72 h respectively. Resveratrol treatment of B-CPAP cells resulted in up to 1.5-fold reduction in BRAF mRNA and up to 5.5 fold reduction in ERK mRNA levels. NIS mRNA levels showed up to 3-fold increase. Western Blot studies confirmed the rt- PCR results with a decrease in BRAF and ERK, and increase in NIS protein expressions. ConclusionThis study demonstrated that resveratrol inhibits thyroid papillary carcinoma cell proliferation and reduces poor prognostic BRAF and ERK mRNA and protein expressions, while increasing NIS mRNA and protein expression suggesting a redifferentiating effect. More studies are needed to evaluate resveratrol as a novel therapeutic agent in the treatment of papillary thyroid cancer.

Launching triple-hit chemo attack on TNBC through nanoparticle-mediated codelivery of cisplatin-chlorambucil conjugate and venetoclax.

The BRCA1 dysfunction and HR deficiency in TNBC are responsible for high effectiveness of DNA-damaging agents in TNBC treatment. Preclinical and clinical studies confirmed the effectiveness of cisplatin in TNBC treatment. Nevertheless, the clinical utility of cisplatin is inadequate due to severe systemic side effects and resistance development. Dual-action cisplatin (IV) prodrugs provide an excellent opportunity to improve anticancer activity, reduce toxicities and minimize chance of resistance development. Therefore, in this investigation we have synthesized cisplatin-chlorambucil (CP-CBL) prodrug and loaded it with venetoclax (VTX) in phenylboronic acid conjugated TPGS-lactide nanoparticles (TNPs) to achieve tumor-targeted drug delivery thereby reducing the therapeutic dose as well as increasing the efficacy of free cisplatin, chlorambucil and venetoclax. The TNPs possessed particle size of 143nm, PDI 0.186 and entrapment efficiency of 63.5% and 56.4% for VTX and CP-CBL. The TNPs followed Higuchi release kinetic model and represented biphasic release behaviour with early burst release of drug within 2h succeeded by sustained drug release till 72h. Further, the TNPs showed∼42 folds and∼19 folds reduction in the IC50 values compared to free CP. Also, higher cellular uptake and therefore greater apoptotic index was observed for the TNPs in comparison to the untargeted nanoparticles. The TNPs further showed higher ROS generating potential, enhanced mitochondrial membrane depolarization, higher intensity of nuclear condensation and highest level of caspase-3 expression. Moreover, a noteworthy decrease in the tumor volume was noticed in the mice treated with TNPs along with lower serum toxicity biomarker levels compared to the free drugs. Overall, the developed TNPs proved to be a promising and safer strategy for inducing triple-hit action in TNBC management.

Multichannel Quantum Defect Theory with a Frame Transformation for Ultracold Atom-Molecule Collisions in Magnetic Fields.

We extend the powerful formalism of multichannel quantum defect theory combined with a frame transformation to ultracold atom-molecule collisions in magnetic fields. By solving the coupled-channel equations with hyperfine and Zeeman interactions omitted at short range, the extended theory enables a drastically simplified description of the intricate quantum dynamics of ultracold molecular collisions in terms of a small number of short-range parameters. We apply the formalism to ultracold Mg+NH collisions in a magnetic field, achieving a 10^{4}-fold reduction in computational effort.

Intracellular bactericidal activity and action mechanism of MDP1 antimicrobial peptide against VRSA and MRSA in human endothelial cells.

Staphylococcus aureus is a prominent cause of postoperative infections, often persisting within host cells, leading to chronic infections. Conventional antibiotics struggle to eliminate intracellular S. aureus due to poor cell penetration. Antimicrobial peptides are a new hope for tackling intracellular bacteria. Accordingly, this study examines the antimicrobial peptide MDP1, derived from melittin, for its efficacy against intracellular S. aureus. In this study, the physiochemical properties (Prediction of three-dimensional structure, circular dichroism and helical wheel projection analysis) were investigated. Extracellular antibacterial activity and cytotoxicity of MDP1 were also assessed. The mechanism of interaction of MDP1 with S. aureus was evaluated by molecular dynamic simulation, atomic force and confocal microscopy. Bacterial internalization into an endothelial cell model was confirmed through culture and transmission electron microscopy. The effect of the peptide on intracellular bacteria was investigated by culture and epi-fluorescence microscopy. 3D structural prediction proved the conformation of MDP1 as an α-helix peptide. Helical-wheel projection analysis indicated the proper orientation of hydrophobic amino acid residues for membrane interaction. CD spectroscopy of MDP1 showed that MDP1 in SDS 10 and 30 mM adopted 87 and 91% helical conformation. Atomic force and confocal microscopy assessments as well as molecular dynamics studies revealed the peptide-bacterial membrane interaction. MDP1, at the concentration of 0.32 μg mL-1, demonstrated a fold reduction of 21.7 ± 1.8, 1.7 ± 0.2, and 7.3 ± 0.8 in intracellular bacterial load for ATCC, VRSA, and MRSA, respectively. Molecular dynamics results demonstrate a preferential interaction of MDP1 with POPG/POPE membranes, primarily driven by electrostatic forces and hydrogen bonding. In POPC systems, two out of four MDP1 interacted effectively, while all four MDP1 engaged with POPG/POPE membranes. Gathering all data together, MDP1 is efficacious in the reduction of intracellular VRSA and MRSA proved by culture and epi-fluorescent microscopy although further studies should be performed to increase the intracellular activity of MDP1.

An enantioselective and modular platform for C4ʹ-modified nucleoside analogue synthesis enabled by intramolecular trans-acetalizations

C4ʹ-modified nucleoside analogues continue to attract global attention for their use in antiviral drug development and oligonucleotide-based therapeutics. However, current approaches to C4ʹ-modified nucleoside analogues still involve lengthy (9–16 steps), non-modular routes that are unamenable to library synthesis. Towards addressing the challenges associated with their syntheses, we report a modular 5-step process to a diverse collection of C4ʹ-modified nucleoside analogues through a sequence of intramolecular trans-acetalizations of readily assembled polyhydroxylated frameworks. Overall, the 2–3 fold reduction in step-count compares favorably to even recently reported biocatalytic approaches and should ultimately enable new opportunities in drug design around this popular chemotype.

Maleimide conjugated PEGylated liposomal antibiotic to combat multi-drug resistant Escherichia coli and Klebsiella pneumoniae with enhanced wound healing potential

Antibiotic resistance is a significant threat, leaving us vulnerable to bacterial infections. Novel strategies are needed to combat bacterial resistance beyond discovering new antibiotics. This research focuses on using maleimide conjugated PEGylated liposomes (Mal-PL-Ab) to individually encapsulate a variety of antibiotics (ceftriaxone, cephalexin, doxycycline, piperacillin, ampicillin, and ceftazidime) and enhance their delivery against multi-drug resistant (MDR) bacteria like Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). Mal-PL-Ab, with an average size of 84.2 nm ± 4.32 nm, successfully encapsulated these antibiotics with an encapsulation efficiency of 37.73 ± 3.19%. Compared to non-PEGylated liposomes (L-Ab), Mal-PL-Ab exhibited reduced toxicity in human dermal cells, emphasizing the importance of PEGylation in minimizing adverse effects. Mal-PL-Ab significantly decreased the minimum inhibitory concentration (MIC) values against both E. coli and K. pneumoniae by 9.33-fold and eightfold reduction (compared to non-PEGylated liposomes with 2.33-fold and 2.33fold reduction), respectively, indicating enhanced efficacy against MDR strains. Furthermore, in vitro scratch assay and gene expression analysis of human dermal fibroblast revealed that Mal-PL-Ab promoted cell proliferation, migration, and wound healing through upregulation of cell cycle, DNA repair, and angiogenesis-related genes. Harnessing the power of encapsulation, Mal-PL-Ab presents a novel avenue for enhanced antibiotic delivery and wound healing, potentially transcending the limitations of traditional options.

A map of canine sequence variation relative to a Greenland wolf outgroup.

For over 15years, canine genetics research relied on a reference assembly from a Boxer breed dog named Tasha (i.e., canFam3.1). Recent advances in long-read sequencing and genome assembly have led to the development of numerous high-quality assemblies from diverse canines. These assemblies represent notable improvements in completeness, contiguity, and the representation of gene promoters and gene models. Although genome graph and pan-genome approaches have promise, most genetic analyses in canines rely upon the mapping of Illumina sequencing reads to a single reference. The Dog10K consortium, and others, have generated deep catalogs of genetic variation through an alignment of Illumina sequencing reads to a reference genome obtained from a German Shepherd Dog named Mischka (i.e., canFam4, UU_Cfam_GSD_1.0). However, alignment to a breed-derived genome may introduce bias in genotype calling across samples. Since the use of an outgroup reference genome may remove this effect, we have reprocessed 1929 samples analyzed by the Dog10K consortium using a Greenland wolf (mCanLor1.2) as the reference. We efficiently performed remapping and variant calling using a GPU-implementation of common analysis tools. The resulting call set removes the variability in genetic differences seen across samples and breed relationships revealed by principal component analysis are not affected by the choice of reference genome. Using this sequence data, we inferred the history of population sizes and found that village dog populations experienced a 9-13 fold reduction in historic effective population size relative to wolves.

Assessment of Air Quality Index of Kolkata, Howrah, and 24 Parganas North during COVID- 19 Lockdown

In the course of COVID- 19 pandemic, a nationwide lockdown in India has been imposed from 22nd March 2020 extended to June end, thereafter a partial lockdown was enforced as a precautionary measure to confine the spread of COVID- 19. The anthropogenic activity was restricted during lockdown therefore the pollution level across many cities in India reduced drastically. This eventually led us to analyze the air quality scenario of Kolkata metropolitan city along with suburban areas of North 24 Parganas and Howrah in the state of West Bengal, India. In the present research, the air quality parameters like PM10, PM2.5, SO2, NO2, CO, O3, and NH3 were analyzed the during lockdown phase of 23rd March 2020 to 30th June 2020. A comparative study was done with the air quality data of the pre-lock down phase (March 2019 to 4th March 2020) and an Air Quality Index (AQI) was prepared. During lockdown coal/ fuel consumption was low due to the restriction of vehicle movement and road transport thus carbon emission in the air was less. AQI improved enormously with reduction of SO2 content to <80 %; among other pollutants NO2 and NH3 reduced to <54 % and <91 % in Kolkata, Howrah, and 24 Parganas North. P.M 2.5 drastically reduced in the regions of Kolkata, Howrah, and 24 Parganas North of West Bengal effectively to <81%, <91 %, and <95% respectively. P.M 10 reduced to <75%, <79%, and < 81 % in Kolkata, Howrah, and 24 Parganas North. In Kolkata there was 55% reduction in the level of CO. There was 4 folds reduction of P.M2.5 and P.M10 in Kolkata compared to March- June 2019. About 50 % to 60 % improvement in air quality was identified within 7days of the lockdown. Improvement of air quality during lockdown has benefited the environment and ecology.

Acquisition of Resistance to PEGylated Branched Polyethylenimine Increases Pseudomonas Aeruginosa Susceptibility to Aminoglycosides.

PEGylated branched polyethylenimine (PEG-BPEI) has antibacterial and antibiofilm properties. Exposure to PEG-BPEI through serial passage leads to resistant P. aeruginosa strains. The minimum inhibitory concentration (MIC) of 600 Da BPEI and PEGylated 600 Da BPEI (PEG-BPEI) in the wild-type PAO1 strain is 16 μg/ml while, after 15 serial passages, the MIC increased to 1024 μg/mL. An additional 15 rounds of serial passage in the absence of BPEI or PEG-BPEI did not change the 1024 μg/mL MIC. Gentamicin, Neomycin, and Tobramycin, cationic antibiotics that inhibit protein synthesis, have a 16-32 fold reduction of MIC values in PEG350-BPEI resistant strains, suggesting increased permeation. The influx of these antibiotics occurs using a self-mediated uptake mechanism, suggesting changes to the outer membrane Data show that resistance causes changes in genes related to outer membrane lipopolysaccharide (LPS) assembly. Mutations were noted in the gene coding for the polymerase Wzy that participates in the assembly of the O-antigen region. Other mutations were noted with wbpE and wbpI of the Wbp pathway responsible for the enzymatic synthesis of ManNAc(3NAc)A in the LPS of P. aeruginosa. These changes suggest that an altered gene product could lead to PEG-BPEI resistance. Nevertheless, the increased susceptibility to aminoglycosides could prevent the emergence of PEG-BPEI resistant bacterial populations.

Unveiling the earthworm-associated preferential remediation of emerging organic pollutants and heavy metals in MSW-based vermicomposting systems: Insights through the lens of multivariate techniques and novel empirical models

Studies on the efficacies of vermicomposting and composting in countering the toxic impacts of pollutant cocktails in municipal solid waste (MSW) are scarce. Moreover, further research is needed to explore earthworms' remediation preferences for various pollutants in heterogeneous vermicomposting feedstocks, such as MSW. Therefore, removal dynamics of pesticides (chlorpyrifos, cypermethrin, and carbofuran), pharmaceuticals (diclofenac and carbamazepine), and heavy metals (Pb, Zn, Cu, and Mn) in MSW-based vermicomposting (Eisenia fetida and Eudrilus eugeniae) and composting systems were evaluated through multivariate analytical techniques (principal component (PCA) and multi-factor (MFA)) on the R-platform. Both earthworms satisfactorily increased their population and augmented NPK (nitrogen, phosphorous, and potassium) availability, cation exchange, microbial biomass C&N, and their metabolic activity 2–3 folds more than composting, accompanied by a 3–4 folds reduction of organic C, pH, and bulk density. Correspondingly, heavy metals, pesticides, and pharmaceuticals decreased by 8-10-folds via earthworm's significant pollutant removal efficiencies that subsided MSW-driven ecological risks by 60–90%. PCA and MFA revealed that N, P, and K-availability, organic C, and microbial activity were the indicative attributes for heavy metal and emerging organic micropollutant (EOMP)-removal during biocomposting; however, earthworms remove pesticides faster than pharmaceuticals and heavy metals. PCA-based novel empirical models demonstrated that in MSW-only feedstock, earthworm-mediated pollutant detoxification followed the order of pesticides > pharmaceuticals > heavy metals. However, in MSW combined with cow dung (1:1 ratio) feedstock, the detoxification order shifted to pharmaceuticals > heavy metals > pesticides. Therefore, this study provides fresh insights into pollutant-focused feedstock optimization for vermicomposting through model-based approaches, advancing the eco-friendly valorization of toxic MSW.

Lactoferrin, chitosan double-coated oleosomes loaded with clobetasol propionate for remyelination in multiple sclerosis: Physicochemical characterization and in-vivo assessment in a cuprizone-induced demyelination model.

Multiple sclerosis is a chronic inflammatory demyelinating disorder of the CNS characterized by continuous myelin damage accompanied by deterioration in functions. Clobetasol propionate (CP) is the most potent topical corticosteroid with serious side effects related to systemic absorption. Previous studies introduced CP for remyelination without considering systemic toxicity. This work aimed at fabrication and optimization of double coated nano-oleosomes loaded with CP to achieve brain targeting through intranasal administration. The optimized formulation was coated with lactoferrin and chitosan for the first time. The obtained double-coated oleosomes had particle size (220.07 ± 0.77 nm), zeta potential (+30.23 ± 0.41 mV) along with antioxidant capacity 9.8 μM ascorbic acid equivalents. Double coating was well visualized by TEM and significantly decreased drug release. Three different doses of CP were assessed in-vivo using cuprizone-induced demyelination in C57Bl/6 mice. Neurobehavioral tests revealed improvement in motor and cognitive functions of mice in a dose-dependent manner. Histopathological examination of the brain showed about 2.3 folds increase in corpus callosum thickness in 0.3 mg/kg CP dose.Moreover, the measured biomarkers highlighted the significant antioxidant and anti-inflammatory capacity of the formulation. In conclusion, the elaborated biopolymer-integrating nanocarrier succeeded in remyelination with 6.6 folds reduction in CP dose compared to previous studies.

High capacity clinical SARS-CoV-2 molecular testing using combinatorial pooling

BackgroundThe SARS-CoV-2 pandemic led to unprecedented testing demands, causing major testing delays globally. One strategy used for increasing testing capacity was pooled-testing, using a two-stage technique first introduced during WWII. However, such traditional pooled testing was used in practice only when positivity rates were below 2%.MethodsHere we report the development, validation and clinical application of P-BEST - a single-stage pooled-testing strategy that was approved for clinical use in Israel.ResultsP-BEST is clinically validated using 3636 side-by-side tests and is able to correctly detect all positive samples and accurately estimate their Ct value. Following regulatory approval by the Israeli Ministry of Health, P-BEST was used in 2021 to clinically test 837,138 samples using 270,095 PCR tests - a 3.1fold reduction in the number of tests. This period includes the Alpha and Delta waves, when positivity rates exceeded 10%, rendering traditional pooling non-practical. We also describe a tablet-based solution that allows performing manual single-stage pooling in settings where liquid dispensing robots are not available.ConclusionsOur data provides a proof-of-concept for large-scale clinical implementation of single-stage pooled-testing for continuous surveillance of multiple pathogens with reduced test costs, and as an important tool for increasing testing efficiency during pandemic outbreaks.

Pre-clinical safety and efficacy evaluation of sodium chloride nanoparticles for treating non-muscle invasive bladder cancer.

e14591 Background: Non-muscle invasive bladder cancer (NMIBC) presents a significant therapeutic challenge. Current treatment modalities such as BCG and intravesical chemotherapy given after transurethral resection of bladder tumors (TURBT), while effective, are associated with high cost burdens and are difficult for patients to tolerate due to side effects. This ultimately limits treatment regimen duration and decreases the effectiveness of the therapy which frequently leads to local recurrence. Sodium Chloride Nanoparticles (NaCl-NPs) have previously shown promise in preclinical models of intra-tumoral injection of various tumor types but has not been tested in more clinically relevant models of bladder cancer. In this study, we evaluate the safety and efficacy of NaCl-NPs as a local intravesical therapy for preventing tumor development in a preclinical murine model of bladder cancer. Methods: We conducted a dose escalation experiment via intravesical instillation of increasing doses of NaCl-NPs (300 ug/ml-2700 ug/ml, n=3 mice per group) to identify the maximum tolerated dose. Mice were treated on a weekly dosing schedule and toxicity was evaluated at day 35 as a composite of: evaluation of bladder histology with Hematoxylin and Eosin (H&E) staining by a board-certified pathologist, blood chemistry measurements, complete blood counts, as well as body weight measurement. Secondly, we assessed the efficacy of the nanoparticles through a tumor prevention experiment where MB49 bladder cancer cells were instilled in the murine bladder and shortly after treated with intravesical: 1.) Saline, 2.) Gemcitabine, or 3.) NaCl-NPs for 1 hour (n=18 per group). Mice were monitored for tumor development with 3D bladder ultrasound. In additional, overall survival (OS) was assessed. Results: The dose escalation study showed that there was no local or systemic toxicity in the NaCl-NP treated mice, even at the highest concentration (2700ug/ml). The tumor prevention study showed that mice treated with NaCl-NPs had a significant 2.55 fold reduction in tumors that developed at 4 weeks compared to saline (22% vs 56% respectively, p <0.05). This reduction was similar to that seen with intravesical gemcitabine, which is clinically, a standard of care intravesical chemotherapy. We also found an improvement in overall survival in the NaCl NP group with 70% of mice alive at 60 days compared to the other treatment groups (65% gemcitabine; 40% saline). Conclusions: Our findings suggest that NaCl-NPs could provide a safe and effective alternative therapy for preventing recurrence of NMIBC following transurethral resection of bladder tumors. This opens avenues for safer treatment protocols that can mitigate the side effects commonly seen in the management of early-stage bladder cancer.

Newly synthesized acridone derivatives targeting lung cancer: A toxicity and xenograft model study.

AKT is one of the overexpressed targets in nonsmall cell lung cancer (NSCLC) and plays an important role in its progression and offers an attractive target for the therapy. The PI3K/AKT/mTOR pathway is upregulated in NSCLC. Acridone is an important heterocycle compound which treats cancer through various mechanisms including AKT as a target. In the present work, the study was designed to evaluate the safety profile of three acridone derivatives (AC-2, AC-7, and AC-26) by acute and repeated dose oral toxicity. In addition to this, we also checked the pAKT overexpression and its control by these derivatives in tumor xenograft model. The results from acute and repeated dose toxicity showed these compounds to be highly safe and free from any toxicity, mortality, or significant alteration in body weight, food, and water intake in the rats. In the repeated dose toxicity, compounds showed negligible variations in a few hematological parameters at 400 mg/kg. The histopathology, biochemical, and urine parameters remained unchanged. The xenograft model study demonstrated AC-2 to be inhibiting HOP-62 induced tumor via reduction in p-AKT1 (Ser473) expression significantly. In immunofluorescence staining AC-2 treated tissue section showed 2.5 fold reduction in the expression of p-AKT1 (Ser473). Histopathology studies showed the destruction of tumor cells with increased necrosis after treatment. The study concluded that AC-2 causes cell necrosis in tumor cells via blocking the p-AKT1 expression. The findings may provide a strong basis for further clinical applications of acridone derivatives in NSCLC.

Microwave-assisted pyrolysis in biomass and waste valorisation: Insights into the life-cycle assessment (LCA) and techno-economic analysis (TEA)

Microwave-assisted pyrolysis (MAP) has been perceived as a promising technology for biomass and waste conversion due to its distinctive features, including fast, even, and precise heating. This results in higher energy efficiency when compared to conventional pyrolysis via thermal heating. However, the scaling up of MAP of biomass and waste poses challenges, with investigations ongoing to uncover not only the technological aspect, but also both the environmental impacts and economic feasibility associated with this process/technology. The possible environmental impacts associated with MAP processes can be analyzed through systematic life-cycle assessment (LCA), while the economic feasibility can be evaluated via techno-economic analysis (TEA). This paper presents an overview of the current research trend in MAP and the products produced, as well as the LCA and TEA of the pyrolysis technologies. The LCA study reported a 2.5 folds reduction in energy consumption and up to 62% reduction in global warming potential. TEA study revealed that conventional pyrolysis has a greater profit for long-term assessment due to a higher maturity and less complexity technology; however, MAP may be more economically feasible in the future owing to the increased maturity and more established technology. Finally, the challenges and future perspectives for LCA and TEA in MAP are elucidated.

Modification of Staphylococcal aureus quorum sensing and virulence factor expression by cold plasma treatment

Abstract Staphylococcal aureus (S. aureus) resides in biofilms and produces virulence factors. Cold atmospheric plasma treatment can be used to kill and reduce bacteria virulence expression. This study will observe the effects of cold plasma treatment on S. aureus killing, biofilm disruption, virulence factor, and quorum sensing gene expression in vitro and in vivo. S. aureus biofilms were grown in vitro on discs and then treated with plasma (J-Plasma; Apyx Medical Inc.) CFU counts, Crystal Violet Staining, and SEM were conducted to determine biofilm disruption and killing. Gene expression was determined by qPCR for Superoxide dismutase (SodA), Panton-Valentine Leukocidin (luk-PV), alpha-hemolysin (Hla), and AgrB and SarA. A similar study was performed on muscles infected in a rat model, treated and untreated, at Days 0, 4 and 14. Cold plasma-treated biofilms on discs had significantly less bacterial CFU counts, lower crystal violet absorbance, and visible disturbances of biofilm. qPCR showed a significant reduction of virulence factor and quorum signaling genes. In vivo, all day 4 and 14 muscle samples had significantly higher CFU counts than 6-hour samples. Results showed a fold change reduction after plasma treatment in virulence and quorum sensing gene expression. Cold plasma treatment disrupts S. aureus biofilm formation and gene expression, both in vivo and in vitro, highlighting the potential of cold plasma treatment to aid in the clearance of S. aureus infections.

NFAT5 upregulates osmolyte transporters to protect IMCD3 cells against hyperosmolarity

The kidney inner medulla is one of the only places in the body exposed to extremely high osmolarity, reaching up to 1300 mOsm, while the iso-osmotic cortex and plasma are ≍300m mOsm. Renal epithelial cells need a mechanism to address the stress that comes with hyperosmolarity. One possible mechanism is regulation of osmolyte transporters to increase intracellular osmolyte concentration and reduce osmotic stress. NFAT5 is an important factor in regulating renal epithelial cell responses to high osmolarity. We hypothesize that NFAT5 is protective in inner medullary collecting duct cells (IMCD3) via upregulation of key transporters. To test this hypothesis, we used CRISPR to delete NFAT5 in IMCD3 cells. IMCD3 -NFAT5 null (NFAT5 null) and control (Ctl, IMCD3) cells were used and cell death assessed. Cells were plated at the same concentrations, in media (320mmol) with additional osmolarities of: urea (160mmol), mannitol (160mmol) and salt (40 and 80 mmol). Cell death was measured over 5 days using Janus Green B, which permeates living cell membranes. Cells were incubated with Janus Green B, lysed and then absorbance measured with a spectrophotometer to determine concentration. Data were analyzed as mean ± SEM; cell confluence, two-way ANOVA, n=3-5. Total mRNA was also isolated for qPCR analysis and data expressed as a fold change (DCT) over housekeeping (PPIA). At iso-osmolarity (»300mOsm), no differences were observed between NFAT5 null and Ctl cells. However, by day 3, NFAT5 null cells had reduced cell growth in all hyperosmolar conditions: urea 160 mmol (1.192 ± 0.225 vs. 1.953 ± 0.167 Wt ****p<0.0001, n=4), mannitol 160 mmol (0.852 ± 0.135 vs. 1.683 ± 0.283 Wt ****p<0.0001, n=3-4), NaCl 40 mmol (1.745 ± 0.210 vs. 2.020 ± 0.252 Wt ****p<0.0001, n=4-5), NaCl 80 mmol (0.385 ± 0.059 vs. 1.089 ± 0.191 Wt ****p<0.0001, n=4-5). Interestingly, NFAT5 null cells recovered growth patterns similar to Ctl cells by day 5. This suggests that although NFAT5 is essential in the cellular response hyperosmolarity, other mechanisms are present that allow for adaptation over time. To address a possible mechanism, we performed qPCR investigating common osmo-protective genes. We hypothesized that expression is lower in the NFAT5 null cells as compared to Wt. When cultured with 40 mmol of NaCl (n=2), we found that the NFAT5 null cells showed a 3.31 fold reduction for SMIT1, a 2924.98 fold reduction for TauT, and a 4.91 fold reduction for BGT1 as compared to Wt, each of which are osmolyte transporters. Additionally, we saw a 161.46 fold reduction for AQP1, a water channel and a 4.91 fold reduction for Akr1b3, which influences water homeostasis, as compared to Wt. Together, these results suggest that NFAT5 is a critical regulator of both osmolyte and transporters. Here, we show that loss of NFAT5 reduces IMCD cell viability in the face of hyperosmotic stress, possibly through a reduction in key osmolyte transporters. These data suggest that NFAT5 is key in the renal epithelial cell response to, and survival in, the hyperosmolar environment of the inner medulla. Undergraduate Research Opportunity Program grant NIH NIDDK DK115660-01A1 University of Utah DOIM, UCares. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2508: EMP2 is a targetable biomarker in GBM tumors resistant to anti-angiogenic therapies

Abstract Background: Glioblastoma (GBM) is characterized as the most aggressive and lethal primary brain tumor. One of the hallmarks of GBM tumor growth and progression lies within the proangiogenic tumor microenvironment. Current anti-angiogenic drugs such as the anti-VEGF antibody take effect by inducing tumor cell starvation, leading to a functional normalization of the tumor vasculature. However, the survival benefits of anti-angiogenic drugs have been very limited. To this end, we hypothesized that the tetraspan Epithelial Membrane Protein-2 (EMP2) may help mediate resistance. Objective: We hypothesize that Epithelial Membrane Protein-2 may serve as a targetable biomarker for anti-VEGF resistance. We further explored the effectiveness of combination therapy in SB28 and GL261 GBM mouse models using anti-VEGF drug, Aflibercept, with a novel anti-EMP2 monoclonal antibody (mAb). Results: Tumor tissue was collected prospectively from patients undergoing surgery for suspected glioblastoma and stained for EMP2 and HIF2a using standard immunohistochemistry. Patients had banked tumor tissue both prior to and after bevacizumab treatment with 3 months of clinical and radiologic follow-up available. Bevacizumab treatment increased EMP2 protein expression. This increase in EMP2 correlated with reduced mean survival time post-bevacizumab therapy. Similarly, HIF-2a showed increased expression post-bevacizumab treatment and showed expression in both tumor and stromal cells. Limited animal models exist for anti-VEGF therapy for GBM. To establish syngeneic models, 10 week-old mice were subcutaneously implanted with murine SB28 or GL261 GBM cells. Treatment with either polyclonal sera to VEGF or Aflibercept failed to reduce tumor load or provide a therapeutic benefit. To next investigate if this model could recapitulate the response obtained patients tumors, formalin fixed paraffin embedded tissue was analyzed for both EMP2 and HIF-2a expression. Both markers were significantly upregulated in Aflibercept treated samples compared to the control.To next investigate if combination treatment could produce a significant reduction in tumor load, animals were treated with a combination treatment with Aflibercept and anti-EMP2 mAb. The combination therapy significantly reduced tumor volume and weight in comparison to control-treated tumors. Combination treatment in SB28 tumors resulted in a two fold reduction in tumor load and 50% reduction in tumor weight. Moreover, tumors showed reduced Ki67 expression. Similarly, treated GL261 tumors produced a 2.3 fold reduction in tumor size and ~2.7 fold reduction in tumor weight. Our results suggest that EMP2 can be a promising new target therapeutic target that can be combined with anti-VEGF treatment to potentially increase overall survival in GBM patients. Citation Format: Brian Aguirre, Mahlet Mekonnen, Riddhi Mehta, Meera Suresh, Anubhav Chandla, Karam Han, Isaac Yang, Madhuri Wadehra. EMP2 is a targetable biomarker in GBM tumors resistant to anti-angiogenic therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2508.

Circulating tumor-associated antigen-specific IFNγ+4-1BB+ CD8+ T cells as peripheral biomarkers of treatment outcomes in patients with pancreatic cancer.

CD8+ T cells affect the outcomes of pancreatic ductal adenocarcinoma (PDAC). Using tissue samples at pre-treatment to monitor the immune response is challenging, while blood samples are beneficial in overcoming this limitation. In this study, we measured peripheral antigen-specific CD8+ T cell responses against four different tumor-associated antigens (TAAs) in PDAC using flow cytometry and investigated their relationships with clinical features. We analyzed the optimal timing within the treatment course for effective immune checkpoint inhibition in vitro. We demonstrated that the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells was correlated with a fold reduction in CA19-9 before and after neoadjuvant therapy. Moreover, patients with TAA-specific IFNγ+4-1BB+ CD8+ T cells after surgery exhibited a significantly improved disease-free survival. Anti-PD-1 treatment in vitro increased the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells before neoadjuvant therapy in patients, suggesting the importance of the timing of anti-PD-1 inhibition during the treatment regimen. Our results indicate that peripheral immunophenotyping, combined with highly sensitive identification of TAA-specific responses in vitro as well as detailed CD8+ T cell subset profiling via ex vivo analysis, may serve as peripheral biomarkers to predict treatment outcomes and therapeutic efficacy of immunotherapy plus neoadjuvant chemotherapy.

The Effect of short-term hyper-ammonia exposure in Indiancatfish, Clarias magur (Hamilton, 1822) on haematological parameters, tissues structure and gene expression profiles

In the present study, pattern of changes in blood physiological parameters, structuralchanges in gills, liver and kidnney as well as gene expression patterns for ornithine-ureacycle (OUC) were studied, under hyper-ammonia stress (25 mM NH4Cl) for 1, 3, 6 and 9 hand 24 h in normal water condition after 9 h exposure (9h+N), in Clarias magur (Hamilton,1822). Analysis showed increase in blood ammonia upto 3 h, with decreasing trend (upto2 folds reduction) subsequently. It was accompanied by simultaneous increase in urealevels upto 6 folds at 9 h exposure. However, there were no significant changes in bloodparameters, except for serum protein, serum glutamic pyruvic transaminase (SGPT) andlactate dehydrogenase (LDH) levels after 1 h exposure. Histological analysis of gills revealedhighest (88%) percentage of damaged gills with congested blood capillaries, treated for 9 h,but declined by 65% at 9h+N. Histological alterations in liver and kidney were prominentonly at 9 h exposure, with nuclear hypertrophy and necrosis of hepatic cells in liver tissuesand distension as well as degeneration of Bowman’s capsule in kidneys. Significantup-regulation of cpsIII and otc genes at 9 h and 9h+N, points to activation of OUC in liver at6 h exposure and unique physiological characteristics of turning towards ureotelism fromammoniotelism, after short term exposure to high ammonia level. Keywords:Ammonium chloride; Clarias magur, Congested capillaries, Gills, Hyperplasia, Ornithine urea cycle, Urea