TIN-X version 3: update with expanded dataset and modernized architecture for enhanced illumination of understudied targets.

TIN-X (Target Importance and Novelty eXplorer) is an interactive visualization tool for illuminating associations between diseases and potential drug targets and is publicly available at newdrugtargets.org. TIN-X uses natural language processing to identify disease and protein mentions within PubMed content using previously published tools for named entity recognition (NER) of gene/protein and disease names. Target data is obtained from the Target Central Resource Database (TCRD). Two important metrics, novelty and importance, are computed from this data and when plotted as log(importance) vs. log(novelty), aid the user in visually exploring the novelty of drug targets and their associated importance to diseases. TIN-X Version 3.0 has been significantly improved with an expanded dataset, modernized architecture including a REST API, and an improved user interface (UI). The dataset has been expanded to include not only PubMed publication titles and abstracts, but also full-text articles when available. This results in approximately 9-fold more target/disease associations compared to previous versions of TIN-X. Additionally, the TIN-X database containing this expanded dataset is now hosted in the cloud via Amazon RDS. Recent enhancements to the UI focuses on making it more intuitive for users to find diseases or drug targets of interest while providing a new, sortable table-view mode to accompany the existing plot-view mode. UI improvements also help the user browse the associated PubMed publications to explore and understand the basis of TIN-X's predicted association between a specific disease and a target of interest. While implementing these upgrades, computational resources are balanced between the webserver and the user's web browser to achieve adequate performance while accommodating the expanded dataset. Together, these advances aim to extend the duration that users can benefit from TIN-X while providing both an expanded dataset and new features that researchers can use to better illuminate understudied proteins.

Natural compounds solasonine and alisol B23-acetate target GLI3 signaling to block oncogenesis in MED12-altered breast cancer.

Breast cancer remains to be the second leading cause of cancer deaths worldwide thereby highlighting the critical need to find superior treatment strategies for this disease. In the current era of cancer treatment, personalized medicine is garnering much attention as this type of treatment is more selective thereby minimizing harmful side effects. Personalized medicine is dependent upon knowing the underlying genetic landscape of the initial tumor. In our study, we focused our efforts on a specific subset of breast cancer that harbors genetic alterations in the Mediator subunit 12 (MED12). Our results show that loss of MED12 leads to enhanced cellular proliferation and colony formation of breast cancer cells through a mechanism that involves activation of GLI3-dependent SHH signaling, a pathway that is central to breast development and homeostasis. To find a personalized treatment option for this subset of breast cancer, we employed a natural compound screening strategy which uncovered a total of ten compounds that selectively target MED12 knockdown breast cancer cells. Our results show that two of these ten compounds, solasonine and alisol B23-acetate, block GLI3-dependent SHH signaling which leads to a reversal of enhanced cellular proliferation and colony formation ability. Thus, our findings provide promising insight into a novel personalized treatment strategy for patients suffering from MED12-altered breast cancer.

Therapeutic potential of co-signaling receptor modulation in hepatitis B.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

Bioprospecting of endophytic fungi from semidesert candelilla (Euphorbia antisyphilitica Zucc): Potential for extracellular enzyme production.

Endophytic microbial communities colonize plants growing under various abiotic stress conditions. Candelilla (Euphorbia antisyphilitica Zucc.) is a shrub that develops functionally in arid and semi-arid zones of Mexico; these conditions generate an association between the plant and the microorganisms, contributing to the production of enzymes as a defense mechanism for resistance to abiotic stress. The objective of this research was to isolate and identify endophyte fungi of candelilla and bioprospection of these endophytic fungi for enzyme production using candelilla by-products. Fungi were isolated and identified using ITS1/ITS4 sequencing. Their potency index (PI) was evaluated in producing endoglucanase, xylanase, amylase, and laccase. Fermentation was carried out at 30°C for 8 days at 200 rpm, with measurements every 2 days, using candelilla by-products as substrate. All fungi exhibited higher cellulase, amylase, and laccase activities on the 2nd, 6th, and 8th day of fermentation, respectively, of fermentation. The fungus Aspergillus niger ITD-IN4.1 showed the highest amylase activity (246.84 U/mg), the genus Neurospora showed the highest cellulase activity, reaching up to 13.45 FPU/mg, and the strain Neurospora sp. ITD-IN5.2 showed the highest laccase activity (3.46 U/mg). This work provides the first report on the endophytic diversity of E. antisyphilitica and its potential role in enzyme production.

Neurophysiological and neuroimaging markers of repetitive transcranial magnetic stimulation treatment response in major depressive disorder: A systematic review and meta-analysis of predictive modeling studies.

Predicting repetitive transcranial magnetic stimulation (rTMS) treatment outcomes in major depressive disorder (MDD) could reduce the financial and psychological risks of treatment failure. We systematically reviewed and meta-analyzed studies that leveraged neurophysiological and neuroimaging markers to predict rTMS response in MDD. Five databases were searched from inception to May 25, 2023. The primary meta-analytic outcome was predictive accuracy pooled from classification models. Regression models were summarized qualitatively. A promising marker was identified if it showed a sensitivity and specificity of 80% or higher in at least two independent studies. Searching yielded 36 studies. Twenty-two classification modeling studies produced an estimated area under the summary receiver operating characteristic curve of 0.87 (95% CI = 0.83-0.92), with 86.8% sensitivity (95% CI = 80.6-91.2%) and 81.9% specificity (95% CI = 76.1-86.4%). Frontal theta cordance measured by electroencephalography is closest to proof of concept. Predicting rTMS response using neurophysiological and neuroimaging markers is promising for clinical decision-making. However, replications by different research groups are needed to establish rigorous markers.

Lack of Highly Pathogenic Avian Influenza H5N1 in the South Shetland Islands in Antarctica, Early 2023.

In January 2023, an active surveillance initiative was undertaken in the South Shetland Islands, Antarctica, with the specific objective of ascertaining evidence for the presence of avian influenza, and specifically the highly pathogenic avian influenza virus subtype H5N1 (HPAIV H5N1). The investigation encompassed diverse locations, including Hanna Point (Livingston Island), Lions Rump (King George Island), and Base Escudero (King George Island), with targeted observations on marine mammals (southern elephant seals), flying birds (the kelp gull, snowy sheathbill and brown skua), and penguins (the chinstrap penguin and gentoo penguin). The study encompassed the examination of these sites for signs of mass mortality events possibly attributable to HPAIV H5N1, as well as sampling for influenza detection by means of real-time RT-PCR. Two hundred and seven (207) samples were collected, including 73 fecal samples obtained from the environment from marine mammals (predominantly feces of southern elephant seals), and 77 cloacal samples from penguins of the genus Pygoscelis (predominantly from the gentoo penguin). No evidence of mass mortality attributable to HPAIV H5N1 was observed, and all the collected samples tested negative for the presence of the virus, strongly suggesting the absence of the virus in the Antarctic territory during the specified period. This empirical evidence holds significant implications for both the ecological integrity of the region and the potential zoonotic threats, underscoring the importance of continued surveillance and monitoring in the Antarctic ecosystem.

Effect of Melatonin on Chemoresistance Exhibited by Spheres Derived from Canine Mammary Carcinoma Cells.

Mammary cancer is a frequent disease in female dogs, where a high proportion of cases correspond to malignant tumors that may exhibit drug resistance. Within the mammary tumor microenvironment, there is a cell subpopulation called cancer stem cells (CSCs), which are capable of forming spheres in vitro and resisting anti-tumor treatments, partly explaining the recurrence of some tumors. Previously, it has been described that spheres derived from canine mammary carcinoma cells CF41.Mg and REM 134 exhibit stemness characteristics. Melatonin has shown anti-tumor effects on mammary tumor cells; however, its effects have been poorly evaluated in canine mammary CSCs. This study aimed to analyze the effect of melatonin on the chemoresistance exhibited by stem-like neoplastic cells derived from canine mammary carcinoma to cytotoxic drugs such as doxorubicin and mitoxantrone. CF41.Mg and REM 134 cells were cultured in high-glucose DMEM supplemented with fetal bovine serum and L-glutamine. The spheres were cultured in ultra-low attachment plates in DMEM/F12 medium without fetal bovine serum and with different growth factors. The CD44+/CD24-/low phenotype was analyzed by flow cytometry. The viability of sphere-derived cells (MTS reduction) was studied in the presence of melatonin (0.1 or 1 mM), doxorubicin, mitoxantrone, and luzindole. In addition, the gene (RT-qPCR) of the multidrug resistance bombs MDR1 and ABCG2 were analyzed in the presence of melatonin. Both cell types expressed the MT1 gene, which encodes the melatonin receptor MT1. Melatonin 1 mM does not modify the CD44+/CD24-/low phenotype; however, the hormone reduced viability (p < 0.0001) only in CF41.Mg spheres, without inducing an additive effect when co-incubated with cytotoxic drugs. These effects were independent of the binding of the hormone to its receptor MT1, since, by pharmacologically inhibiting them, the effect of melatonin was not blocked. In CF41.Mg spheres, the relative gene expression of ABCG2 and MDR1 was decreased in response to the hormone (p < 0.001). These results indicate that melatonin negatively modulates the cell survival of spheres derived from CF41.Mg cells, in a way that is independent of its MT1 receptor. These effects did not counteract the resistance to doxorubicin and mitoxantrone, even though the hormone negatively regulates the gene expression of MDR1 and ABCG2.

Instantaneous effects of prefrontal transcranial magnetic stimulation on brain oxygenation: A systematic review.

This systematic review investigates how prefrontal transcranial magnetic stimulation (TMS) immediately influences neuronal excitability based on oxygenation changes measured by functional magnetic resonance imaging (fMRI) or functional near-infrared spectroscopy (fNIRS). A thorough understanding of TMS-induced excitability changes may enable clinicians to adjust TMS parameters and optimize treatment plans proactively. Five databases were searched for human studies evaluating brain excitability using concurrent TMS/fMRI or TMS/fNIRS. Thirty-seven studies (13 concurrent TMS/fNIRS studies, 24 concurrent TMS/fMRI studies) were included in a qualitative synthesis. Despite methodological inconsistencies, a distinct pattern of activated nodes in the frontoparietal central executive network, the cingulo-opercular salience network, and the default-mode network emerged. The activated nodes included the prefrontal cortex (particularly dorsolateral prefrontal cortex), insula cortex, striatal regions (especially caudate, putamen), anterior cingulate cortex, and thalamus. High-frequency repetitive TMS most consistently induced expected facilitatory effects in these brain regions. However, varied stimulation parameters (e.g., intensity, coil orientation, target sites) and the inter- and intra-individual variability of brain state contribute to the observed heterogeneity of target excitability and co-activated regions. Given the considerable methodological and individual variability across the limited evidence, conclusions should be drawn with caution.

Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium.

In vitro gamete derivation has been proposed as an interesting strategy for treatment of infertility, improvement of genetic traits, and conservation of endangered animals. Spermatogonial stem cells (SSCs) are primary candidates for in vitro gamete derivation; however, recently, mesenchymal stem cells (MSCs) have also been proposed as candidates for germ cell (GCs) differentiation mainly due to their transdifferentiating capacity. The objective of the present study was to compare the potential for GC differentiation of bovine peripheral blood-derived MSCs (PB-MSCs) and SSCs under the effect of conditioned medium (CM) derived from Sertoli cells (SCs/CM). Samples were collected every 7 days for 21 days and analyzed for pluripotent, GC, and MSC marker expression. The absence of OCT4 and the increased (p < 0.05) expression of NANOG seems to play a role in SSC differentiation, whereas the absence of NANOG and the increased expression (p < 0.05) of OCT4 may be required for PB-MSC differentiation into GCs. SSCs cultured with SCs/CM increased (p < 0.05) the expression of PIWIL2 and DAZL, while PB-MSCs cultured under the same condition only increased (p < 0.05) the expression of DAZL. Overall, the patterns of markers expression suggest that PB-MSCs and SSCs activate different signaling pathways after exposure to SCs/CM and during differentiation into GCs.

Complex alterations in inflammatory pain and analgesic sensitivity in young and ageing female rats: involvement of ASIC3 and Nav1.8 in primary sensory neurons.

OBJECTIVE AND DESIGN: Our aim was to determine an age-dependent role of Nav1.8 and ASIC3 in dorsal root ganglion (DRG) neurons in a rat pre-clinical model of long-term inflammatory pain. METHODS: We compared 6 and 24 months-old female Wistar rats after cutaneous inflammation. We used behavioral pain assessments over time, qPCR, quantitative immunohistochemistry, selective pharmacological manipulation, ELISA and in vitro treatment with cytokines. RESULTS: Older rats exhibited delayed recovery from mechanical allodynia and earlier onset of spontaneous pain than younger rats after inflammation. Moreover, the expression patterns of Nav1.8 and ASIC3 were time and age-dependent and ASIC3 levels remained elevated only in aged rats. In vivo, selective blockade of Nav1.8 with A803467 or of ASIC3 with APETx2 alleviated mechanical and cold allodynia and also spontaneous pain in both age groups with slightly different potency. Furthermore, in vitro IL-1ß up-regulated Nav1.8 expression in DRG neurons cultured from young but not old rats. We also found that while TNF-α up-regulated ASIC3 expression in both age groups, IL-6 and IL-1ß had this effect only on young and aged neurons, respectively. CONCLUSION: Inflammation-associated mechanical allodynia and spontaneous pain in the elderly can be more effectively treated by inhibiting ASIC3 than Nav1.8.

Grape Pomace-Advances in Its Bioactivity, Health Benefits, and Food Applications.

From a circular economy perspective, the appropriate management and valorization of winery wastes and by-products are crucial for sustainable development. Nowadays, grape pomace (GP) has attracted increasing interest within the food field due to its valuable content, comprising nutritional and bioactive compounds (e.g., polyphenols, organic and fatty acids, vitamins, etc.). Particularly, GP polyphenols have been recognized as exhibiting technological and health-promoting effects in different food and biological systems. Hence, GP valorization is a step toward offering new functional foods and contributing to solving waste management problems in the wine industry. On this basis, the use of GP as a food additive/ingredient in the development of novel products with technological and functional advantages has recently been proposed. In this review, we summarize the current knowledge on the bioactivity and health-promoting effects of polyphenolic-rich extracts from GP samples. Advances in GP incorporation into food formulations (enhancement of physicochemical, sensory, and nutritional quality) and information supporting the intellectual property related to GP potential applications in the food industry are also discussed.

Bovine adipose tissue-derived mesenchymal stem cells self-assemble with testicular cells and integrates and modifies the structure of a testicular organoids.

Mesenchymal stem cells (MSC) display self-renewal and mesodermal differentiation potentials. These characteristics make them potentially useful for in vitro derivation of gametes, which may constitute experimental therapies for human and animal reproduction. Organoids provide a spatial support and may simulate a cellular niche for in vitro studies. In this study, we aimed at evaluating the potential integration of fetal bovine MSCs derived from adipose tissue (AT-MSCs) in testicular organoids (TOs), their spatial distribution with testicular cells during TO formation and their potential for germ cell differentiation. TOs were developed using Leydig, Sertoli, and peritubular myoid cells that were previously isolated from bovine testes (n = 6). Thereafter, TOs were characterized using immunofluorescence and Q-PCR to detect testicular cell-specific markers. AT-MSCs were labeled with PKH26 and then cultured with testicular cells at a concentration of 1 × 106 cells per well in Ultra Low Attachment U-shape bottom (ULA) plates. TOs formed by testicular cells and AT-MSCs (TOs + AT-MSCs) maintained a rounded structure throughout the 28-day culture period and did not show significant differences in their diameters. Conversely, control TOs exhibited a compact structure until day 7 of culture, while on day 28 they displayed cellular extensions around their structure. Control TOs had greater (P < 0.05) diameters compared to TOs + AT-MSCs. AT-MSCs induced an increase in proportion of Leydig and peritubular myoid cells in TOs + AT-MSCs; however, did not induce changes in the overall gene expression of testicular cell-specific markers. STAR immunolabelling detected Leydig cells that migrated from the central area to the periphery and formed brunches in control TOs. However, in TOs + AT-MSCs, Leydig cells formed a compact peripheral layer. Sertoli cells immunodetected using WT1 marker were observed within the central area forming clusters of cells in TOs + AT-MSCs. The expression of COL1A associated to peritubular myoids cells was restricted to the central region in TOs + AT-MSCs. Thus, during a 28-day culture period, fetal bovine AT-MSCs integrated and modified the structure of the TOs, by restricting formation of branches, limiting the overall increase in diameters and increasing the proportions of Leydig and peritubular myoid cells. AT-MSCs also induced a reorganization of testicular cells, changing their distribution and particularly the location of Leydig cells.

Interleukin-17 signaling influences CD8+ T cell immunity and tumor progression according to the IL-17 receptor subunit expression pattern in cancer cells.

IL-17 immune responses in cancer are controversial, with both tumor-promoting and tumor-repressing effects observed. To clarify the role of IL-17 signaling in cancer progression, we used syngeneic tumor models from different tissue origins. We found that deficiencies in host IL-17RA or IL-17A/F expression had varying effects on the in vivo growth of different solid tumors including melanoma, sarcoma, lymphoma, and leukemia. In each tumor type, the absence of IL-17 led to changes in the expression of mediators associated with inflammation and metastasis in the tumor microenvironment. Furthermore, IL-17 signaling deficiencies in the hosts resulted in decreased anti-tumor CD8+ T cell immunity and caused tumor-specific changes in several lymphoid cell populations. Our findings were associated with distinct patterns of IL-17A/F cytokine and receptor subunit expression in the injected tumor cell lines. These patterns affected tumor cell responsiveness to IL-17 and downstream intracellular signaling, leading to divergent effects on cancer progression. Additionally, we identified IL-17RC as a critical determinant of the IL-17-mediated response in tumor cells and a potential biomarker for IL-17 signaling effects in tumor progression. Our study offers insight into the molecular mechanisms underlying IL-17 activities in cancer and lays the groundwork for developing personalized immunotherapies.

At-sea distribution patterns of the Peruvian diving petrel Pelecanoides garnotii during breeding and non-breeding seasons.

At-sea distributions of seabird species are strongly associated with the distribution patterns of their prey, which are influenced by physical oceanic features. During breeding and non-breeding seasons, seabirds move extraordinary distances among different environments. However, foraging site fidelity by seabirds appears to be high in areas of known high productivity, such as frontal zones and upwellings. Here, we present a tracking study for the Peruvian diving-petrel Pelecanoides garnotii, an endemic seabird of the highly productive Humboldt Current System, to assess whether adults use the same foraging areas throughout the year, combining data from nest monitoring and global location sensors (GLS) deployed on 12 individuals between two breeding seasons (2013-2014 and 2014-2015), in Choros Island (29°15'S; 71°32'W), Chile. Two main foraging areas were registered. During the breeding season, adults moved in the northern direction, between 60 to 144 km away from their colony, foraging in areas with high primary productivity. During the non-breeding period, they moved to southern latitudes (~ 1200 km). Adults spent 37% and 63% of their time in flight/land and on/underwater activities, respectively. We determined that birds move northward from their colony during breeding, where prey availability seems more predictable throughout the year. However, during the non-breeding period, it is likely that other environmental factors influence the distribution pattern of the Peruvian diving-petrel.

Outcomes of Hospital-Acquired Hypernatremia.

BACKGROUND: Hospital-acquired hypernatremia is highly prevalent, overlooked, and associated with unfavorable consequences. There are limited studies examining the outcomes and discharge dispositions of various levels of hospital-acquired hypernatremia in patients with or without CKD. METHODS: We conducted an observational retrospective cohort study, and we analyzed the data of 1,728,141 patients extracted from the Cerner Health Facts database (January 1, 2000, to June 30, 2018). In this report, we investigated the association between hospital-acquired hypernatremia (serum sodium [Na] levels >145 mEq/L) and in-hospital mortality or discharge dispositions with kidney function status at admission using adjusted multinomial regression models. RESULTS: Of all hospitalized patients, 6% developed hypernatremia after hospital admission. The incidence of in-hospital mortality was 12% and 1% in patients with hypernatremia and normonatremia, respectively. The risk of all outcomes was significantly greater for serum Na >145 mEq/L compared with the reference interval (serum Na, 135-145 mEq/L). In patients with hypernatremia, odds ratios (95% confidence interval) for in-hospital mortality, discharge to hospice, and discharge to nursing facilities were 14.04 (13.71 to 14.38), 4.35 (4.14 to 4.57), and 3.88 (3.82 to 3.94), respectively ( P < 0.001, for all). Patients with eGFR (Chronic Kidney Disease Epidemiology Collaboration) 60-89 ml/min per 1.73 m 2 and normonatremia had the lowest odds ratio for in-hospital mortality (1.60 [1.52 to 1.70]). CONCLUSIONS: Hospital-acquired hypernatremia is associated with in-hospital mortality and discharge to hospice or to nursing facilities in all stages of CKD.

Toxicology knowledge graph for structural birth defects.

BACKGROUND: Birth defects are functional and structural abnormalities that impact about 1 in 33 births in the United States. They have been attributed to genetic and other factors such as drugs, cosmetics, food, and environmental pollutants during pregnancy, but for most birth defects there are no known causes. METHODS: To further characterize associations between small molecule compounds and their potential to induce specific birth abnormalities, we gathered knowledge from multiple sources to construct a reproductive toxicity Knowledge Graph (ReproTox-KG) with a focus on associations between birth defects, drugs, and genes. Specifically, we gathered data from drug/birth-defect associations from co-mentions in published abstracts, gene/birth-defect associations from genetic studies, drug- and preclinical-compound-induced gene expression changes in cell lines, known drug targets, genetic burden scores for human genes, and placental crossing scores for small molecules. RESULTS: Using ReproTox-KG and semi-supervised learning (SSL), we scored >30,000 preclinical small molecules for their potential to cross the placenta and induce birth defects, and identified >500 birth-defect/gene/drug cliques that can be used to explain molecular mechanisms for drug-induced birth defects. The ReproTox-KG can be accessed via a web-based user interface available at https://maayanlab.cloud/reprotox-kg . This site enables users to explore the associations between birth defects, approved and preclinical drugs, and all human genes. CONCLUSIONS: ReproTox-KG provides a resource for exploring knowledge about the molecular mechanisms of birth defects with the potential of predicting the likelihood of genes and preclinical small molecules to induce birth defects.

While birth defects are common, for most birth defects there are no known causes. During pregnancy, developing babies are exposed to drugs, cosmetics, food, and environmental pollutants that may cause birth defects. However, exactly how these environmental factors are involved in producing birth defects is difficult to discern. Also, birth defects can be a consequence of the genes inherited from the parents. We combined general data about human genes and drugs with specific data previously implicating genes and drugs in inducing birth defects to create a knowledge graph representation that connects genes, drugs, and birth defects. This knowledge graph can be used to explore new links that may explain why birth defects occur, particularly those that result from a combination of inherited and environmental influences.

Mind the Curve: Dose-Response Fitting Biases the Synergy Scores across Software Used for Chemotherapy Combination Studies.

Drug combinations are increasingly studied in the field of anticancer agents. Mathematical models, such as Loewe, Bliss, and HSA, are used to interpret drug combinations, while informatics tools help cancer researchers identify the most effective combinations. However, the different algorithms each software uses lead to results that do not always correlate. This study compared the performance of Combenefit (Ver. 2.021) and SynergyFinder (Ver. 3.6) in analyzing drug synergy by studying combinations involving non-steroidal analgesics (celecoxib and indomethacin) and antitumor drugs (carboplatin, gemcitabine, and vinorelbine) on two canine mammary tumor cell lines. The drugs were characterized, their optimal concentration-response ranges were determined, and nine concentrations of each drug were used to make combination matrices. Viability data were analyzed under the HSA, Loewe, and Bliss models. Celecoxib-based combinations showed the most consistent synergistic effect among software and reference models. Combination heatmaps revealed that Combenefit gave stronger synergy signals, while SynergyFinder produced better concentration-response fitting. When the average values of the combination matrices were compared, some combinations shifted from synergistic to antagonistic due to differences in the curve fitting. We also used a simulated dataset to normalize each software's synergy scores, finding that Combenefit tends to increase the distance between synergistic and antagonistic combinations. We conclude that concentration-response data fitting biases the direction of the combination (synergistic or antagonistic). In contrast, the scoring from each software increases the differences among synergistic or antagonistic combinations in Combenefit when compared to SynergyFinder. We strongly recommend using multiple reference models and reporting complete data analysis for synergy claiming in combination studies.