SPACe (Swift Phenotypic Analysis of Cells): an open-source, single cell analysis of Cell Painting data.

Phenotypic profiling by high throughput microscopy has become one of the leading tools for screening large sets of perturbations in cellular models. Of the numerous methods used over the years, the flexible and economical Cell Painting (CP) assay has been central in the field, allowing for large screening campaigns leading to a vast number of data-rich images. Currently, to analyze data of this scale, available open-source software ( i.e. , CellProfiler) requires computational resources that are not available to most laboratories worldwide. In addition, the image-embedded cell-to-cell variation of responses within a population, while collected and analyzed, is usually averaged and unused. Here we introduce SPACe ( S wift P henotypic A nalysis of Ce lls), an open source, Python-based platform for the analysis of single cell image-based morphological profiles produced by CP experiments. SPACe can process a typical dataset approximately ten times faster than CellProfiler on common desktop computers without loss in mechanism of action (MOA) recognition accuracy. It also computes directional distribution-based distances (Earth Mover's Distance - EMD) of morphological features for quality control and hit calling. We highlight several advantages of SPACe analysis on CP assays, including reproducibility across multiple biological replicates, easy applicability to multiple (∼20) cell lines, sensitivity to variable cell-to-cell responses, and biological interpretability to explain image-based features. We ultimately illustrate the advantages of SPACe in a screening campaign of cell metabolism small molecule inhibitors which we performed in seven cell lines to highlight the importance of testing perturbations across models.

Cancer-associated fibroblast-derived acetate promotes pancreatic cancer development by altering polyamine metabolism via the ACSS2-SP1-SAT1 axis.

The ability of tumour cells to thrive in harsh microenvironments depends on the utilization of nutrients available in the milieu. Here we show that pancreatic cancer-associated fibroblasts (CAFs) regulate tumour cell metabolism through the secretion of acetate, which can be blocked by silencing ATP citrate lyase (ACLY) in CAFs. We further show that acetyl-CoA synthetase short-chain family member 2 (ACSS2) channels the exogenous acetate to regulate the dynamic cancer epigenome and transcriptome, thereby facilitating cancer cell survival in an acidic microenvironment. Comparative H3K27ac ChIP-seq and RNA-seq analyses revealed alterations in polyamine homeostasis through regulation of SAT1 gene expression and enrichment of the SP1-responsive signature. We identified acetate/ACSS2-mediated acetylation of SP1 at the lysine 19 residue that increased SP1 protein stability and transcriptional activity. Genetic or pharmacologic inhibition of the ACSS2-SP1-SAT1 axis diminished the tumour burden in mouse models. These results reveal that the metabolic flexibility imparted by the stroma-derived acetate enabled cancer cell survival under acidosis via the ACSS2-SP1-SAT1 axis.

Characterization of flavonoids with potent and subtype-selective actions on estrogen receptors alpha and beta.

The initial step in estrogen-regulated transcription is the binding of a ligand to its cognate receptors, named estrogen receptors (ERα and ERß). Phytochemicals present in foods and environment can compete with endogenous hormones to alter physiological responses. We screened 224 flavonoids in our engineered biosensor ERα and ERß PRL-array cell lines to characterize their activity on several steps of the estrogen signaling pathway. We identified 83 and 96 flavonoids that can activate ERα or ERß, respectively. While most act on both receptors, many appear to be subtype-selective, including potent flavonoids that activate ER at sub-micromolar concentrations. We employed an orthogonal assay using a transgenic zebrafish in vivo model that validated the estrogenic potential of these compounds. To our knowledge, this is the largest study thus far on flavonoids and the ER pathway, facilitating the identification of a new set of potential endocrine disruptors acting on both ERα and ERß.

eEF1A2 promotes PTEN-GSK3ß-SCF complex-dependent degradation of Aurora kinase A and is inactivated in breast cancer.

The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3ß, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.

The MUC1-HIF-1α signaling axis regulates pancreatic cancer pathogenesis through polyamine metabolism remodeling.

Dysregulation of polyamine metabolism has been implicated in cancer initiation and progression; however, the mechanism of polyamine dysregulation in cancer is not fully understood. In this study, we investigated the role of MUC1, a mucin protein overexpressed in pancreatic cancer, in regulating polyamine metabolism. Utilizing pancreatic cancer patient data, we noted a positive correlation between MUC1 expression and the expression of key polyamine metabolism pathway genes. Functional studies revealed that knockdown of spermidine/spermine N1-acetyltransferase 1 (SAT1), a key enzyme involved in polyamine catabolism, attenuated the oncogenic functions of MUC1, including cell survival and proliferation. We further identified a regulatory axis whereby MUC1 stabilized hypoxia-inducible factor (HIF-1α), leading to increased SAT1 expression, which in turn induced carbon flux into the tricarboxylic acid cycle. MUC1-mediated stabilization of HIF-1α enhanced the promoter occupancy of the latter on SAT1 promoter and corresponding transcriptional activation of SAT1, which could be abrogated by pharmacological inhibition of HIF-1α or CRISPR/Cas9-mediated knockout of HIF1A. MUC1 knockdown caused a significant reduction in the levels of SAT1-generated metabolites, N1-acetylspermidine and N8-acetylspermidine. Given the known role of MUC1 in therapy resistance, we also investigated whether inhibiting SAT1 would enhance the efficacy of FOLFIRINOX chemotherapy. By utilizing organoid and orthotopic pancreatic cancer mouse models, we observed that targeting SAT1 with pentamidine improved the efficacy of FOLFIRINOX, suggesting that the combination may represent a promising therapeutic strategy against pancreatic cancer. This study provides insights into the interplay between MUC1 and polyamine metabolism, offering potential avenues for the development of treatments against pancreatic cancer.

Metabolomic Investigations into Hypoxia-Mediated Metabolic Reprogramming of Pancreatic Cancer Cells.

Hypoxia is a crucial microenvironmental factor that defines tumor cell growth and aggressiveness. Cancer cells adapt to hypoxia by altering their metabolism. These alterations impact various cellular and physiological functions, including energy metabolism, vascularization, invasion and metastasis, genetic instability, cell immortalization, stem cell maintenance, and resistance to chemotherapy (Li et al. Technol Cancer Res Treat 20:15330338211036304, 2021). Hypoxia-inducible factor-1α (HIF-1α) is known to be a critical regulator of glycolysis that directly regulates the transcription of multiple key enzymes of the glycolysis pathway. Moreover, HIF-1α stabilization can be directly modulated by TCA-derived metabolites, including 2-ketoglutarate and succinate (Infantino et al, Int J Mol Sci 22(22), https://doi.org/10.3390/ijms22115703 , 2021). Overall, the molecular mechanisms underlying the adaptation of cellular metabolism to hypoxia impact the metabolic phenotype of cancer cells. Such adaptations include increased glucose uptake, increased lactate production, and increased levels of other metabolites that stabilize HIF-1α, leading to a vicious circle of hypoxia-induced tumor growth.

Papillary Tumors of Pineal Region: A Single-Center Experience in Management of 11 Cases.

BACKGROUND: Papillary tumors of pineal region (PTPR) comprise a very rare subset of pineal region tumors that have been recently described. Literature on the management and outcome of PTPR is scarce owing to the rarity of these tumors. To address this lacuna, we analyzed our experience in management of PTPR. METHODS: We retrospectively analyzed the outcome of 11 patients with histopathologically proven PTPR who underwent surgical excision at our center. RESULTS: Mean patient age was 33.3 years (range, 12-45 years), and male-to-female ratio was 1.75:1. Headache was the most common presentation followed by visual disturbances, altered sensorium, Perinaud syndrome, and seizures. Cerebrospinal fluid diversion was required in 6 patients. Krause approach was the most common approach used for tumor excision (9/11 cases). There was no perioperative mortality. Two patients were lost to follow-up. In the remaining 9 patients, the average follow-up period was 45 months (range, 12-79 months). On first postoperative magnetic resonance imaging, 8 patients showed no evidence of residual tumor (gross total resection), while 1 patient had small residual tumor (near-total resection) that remained stable during follow-up. Four patients underwent adjuvant chemoradiotherapy. None of the patients developed recurrence during follow-up. CONCLUSIONS: PTPR are a rare subgroup of pineal region tumors with distinct cells of origin but presentation similar to other pineal region tumors. Surgical resection constitutes the mainstay of management, and the extent of resection appears to be the most important determinant of prognosis. The role of adjuvant therapy still needs to be determined.

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is 1) strictly dependent on pyrimidine nucleotide depletion, 2) independent of canonical antigen presentation pathway transcriptional regulators, and 3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Methotrexate-based PROTACs as DHFR-specific chemical probes.

Methotrexate (MTX) is a tight-binding dihydrofolate reductase (DHFR) inhibitor, used as both an antineoplastic and immunosuppressant therapeutic. MTX, like folate undergoes folylpolyglutamate synthetase-mediated γ-glutamylation, which affects cellular retention and target specificity. Mechanisms of MTX resistance in cancers include a decrease in MTX poly-γ-glutamylation and an upregulation of DHFR. Here, we report a series of potent MTX-based proteolysis targeting chimeras (PROTACs) to investigate DHFR degradation pharmacology and one-carbon biochemistry. These on-target, cell-active PROTACs show proteasome- and E3 ligase-dependent activity, and selective degradation of DHFR in multiple cancer cell lines. By comparison, treatment with MTX increases cellular DHFR protein expression. Importantly, these PROTACs produced distinct, less-lethal phenotypes compared to MTX. The chemical probe set described here should complement conventional DHFR inhibitors and serve as useful tools for studying one-carbon biochemistry and dissecting complex polypharmacology of MTX and related drugs. Such compounds may also serve as leads for potential autoimmune and antineoplastic therapeutics.

Vitamin B6 Competition in the Tumor Microenvironment Hampers Antitumor Functions of NK Cells.

Nutritional factors play crucial roles in immune responses. The tumor-caused nutritional deficiencies are known to affect antitumor immunity. Here, we demonstrate that pancreatic ductal adenocarcinoma (PDAC) cells can suppress NK-cell cytotoxicity by restricting the accessibility of vitamin B6 (VB6). PDAC cells actively consume VB6 to support one-carbon metabolism, and thus tumor cell growth, causing VB6 deprivation in the tumor microenvironment. In comparison, NK cells require VB6 for intracellular glycogen breakdown, which serves as a critical energy source for NK-cell activation. VB6 supplementation in combination with one-carbon metabolism blockage effectively diminishes tumor burden in vivo. Our results expand the understanding of the critical role of micronutrients in regulating cancer progression and antitumor immunity, and open new avenues for developing novel therapeutic strategies against PDAC. SIGNIFICANCE: The nutrient competition among the different tumor microenvironment components drives tumor growth, immune tolerance, and therapeutic resistance. PDAC cells demand a high amount of VB6, thus competitively causing NK-cell dysfunction. Supplying VB6 with blocking VB6-dependent one-carbon metabolism amplifies the NK-cell antitumor immunity and inhibits tumor growth in PDAC models. This article is featured in Selected Articles from This Issue, p. 5.

Artificial intelligence: Machine learning approach for screening large database and drug discovery.

Recent research in drug discovery dealing with many faces difficulties, including development of new drugs during disease outbreak and drug resistance due to rapidly accumulating mutations. Virtual screening is the most widely used method in computer aided drug discovery. It has a prominent ability in screening drug targets from large molecular databases. Recently, a number of web servers have developed for quickly screening publicly accessible chemical databases. In a nutshell, deep learning algorithms and artificial neural networks have modernised the field. Several drug discovery processes have used machine learning and deep learning algorithms, including peptide synthesis, structure-based virtual screening, ligand-based virtual screening, toxicity prediction, drug monitoring and release, pharmacophore modelling, quantitative structure-activity relationship, drug repositioning, polypharmacology, and physiochemical activity. Although there are presently a wide variety of data-driven AI/ML tools available, the majority of these tools have, up to this point, been developed in the context of non-communicable diseases like cancer, and a number of obstacles have prevented the translation of these tools to the discovery of treatments against infectious diseases. In this review various aspects of AI and ML in virtual screening of large databases were discussed. Here, with an emphasis on antivirals as well as other disease, offers a perspective on the advantages, drawbacks, and hazards of AI/ML techniques in the search for innovative treatments.

Erratum: Exploring the metabolic landscape of pancreatic ductal adenocarcinoma cells using genome-scale metabolic modeling.

[This corrects the article DOI: 10.1016/j.isci.2022.104483.].

Clinical, Microbiological, Serological and Radiological Profile of Patients With Mild-Moderate and Severe Allergic Bronchopulmonary Aspergillosis (ABPA).

Objective Allergic bronchopulmonary aspergillosis (ABPA) is a complex hypersensitivity reaction to Aspergillus antigen mostly Aspergillus fumigatus that occurs almost exclusively in patients with asthma and cystic fibrosis. ABPA is an underdiagnosed and undertreated disease because of its presentation with various grades of severity in asthma patients. Data available regarding the clinical, serological, and radiological profile of ABPA patients is limited due to lack of consensus on diagnostic criteria and treatment guidelines. Thus ABPA is a significant disease, especially in the Indian population where the incidence of allergic diseases like asthma is on the rise. Methods This prospective study was conducted in the Department of Pulmonary Medicine at one of the tertiary centers of north India. All consecutive patients diagnosed with allergic bronchopulmonary aspergillosis (ABPA) from 1st January 2017 to 30th September 2017 were included in the study. A total of 67 consecutive patients diagnosed with bronchial asthma were included in the study. The diagnosis of ABPA was based upon either criterion given by Rosenberg and Paterson or the International Society of Human and Animal Mycology (ISHAM) criteria. Patients diagnosed with ABPA were finally divided into mild, moderate, and severe. Results The majority of patients showed an obstructive pattern on spirometry and moderate to severe obstruction was the most common pattern observed among patients who had an obstructive pattern on spirometry. Also, all three patients with the mixed pattern on spirometry had severe disease. Serological analysis revealed that patients in the moderate category had a higher level of absolute eosinophil count (AEC), total IgE, and Aspergillus-specific IgE antibodies, especially in patients who had either high attenuation mucus (HAM) or centrilobular nodules on their high-resolution computed tomography (HRCT) scan. Conclusion ABPA is a disease of divergent presentation. We concluded to have alternate or add-on criteria for the classification of ABPA which was not based on the sequelae of chronic inflammatory changes in the lungs.

Analysis and Modeling of Early Estradiol-induced GREB1 Single Allele Gene Transcription at the Population Level.

Single molecule fluorescence in situ hybridization (smFISH) can be used to visualize transcriptional activation at the single allele level. We and others have applied this approach to better understand the mechanisms of activation by steroid nuclear receptors. However, there is limited understanding of the interconnection between the activation of target gene alleles inside the same nucleus and within large cell populations. Using the GREB1 gene as an early estrogen receptor (ER) response target, we applied smFISH to track E2-activated GREB1 allelic transcription over early time points to evaluate potential dependencies between alleles within the same nucleus. We compared two types of experiments where we altered the initial status of GREB1 basal transcription by treating cells with and without the elongation inhibitor flavopiridol (FV). E2 stimulation changed the frequencies of active GREB1 alleles in the cell population independently of FV pre-treatment. In FV treated cells, the response time to hormone was delayed, albeit still reaching at 90 minutes the same levels as in cells not treated by FV. We show that the joint frequencies of GREB1 activated alleles observed at the cell population level imply significant dependency between pairs of alleles within the same nucleus. We identify probabilistic models of joint alleles activations by applying a principle of maximum entropy. For pairs of alleles, we have then quantified statistical dependency by computing their mutual information. We have then introduced a stochastic model compatible with allelic statistical dependencies, and we have fitted this model to our data by intensive simulations. This provided estimates of the average lifetime for degradation of GREB1 introns and of the mean time between two successive transcription rounds. Our approach informs on how to extract information on single allele regulation by ER from within a large population of cells, and should be applicable to many other genes. AUTHOR SUMMARY: After application of a gene transcription stimulus, in this case the hormone 17 ß -estradiol, on large populations of cells over a short time period, we focused on quantifying and modeling the frequencies of GREB1 single allele activations. We have established an experimental and computational pipeline to analyze large numbers of high resolution smFISH images to detect and monitor active GREB1 alleles, that can be translatable to any target gene of interest. A key result is that, at the population level, activation of individual GREB1 alleles within the same nucleus do exhibit statistically significant dependencies which we quantify by the mutual information between activation states of pairs of alleles. After noticing that frequencies of joint alleles activations observed over our large cell populations evolve smoothly in time, we have defined a population level stochastic model which we fit to the observed time course of GREB1 activation frequencies. This provided coherent estimates of the mean time between rounds of GREB1 transcription and the mean lifetime of nascent mRNAs. Our algorithmic approach and experimental methods are applicable to many other genes.

Results of Antibiotic-Impregnated Cement/Polymer-Coated Intramedullary Nails in the Management of Infected Nonunion and Open Fractures of Long Bones.

Background Fractures of long bones can sometimes lead to complications such as infection or nonunion, resulting in significant patient morbidity. Surgical intervention and antibiotics are often necessary to treat these complications. Antibiotic-impregnated cement/polymer-coated intramedullary nails have emerged as an effective surgical treatment for infected nonunion and open fractures. These implants elude high concentrations of antibiotics at the infection site while stabilizing the fracture. Extensive research has shown promising results, with success rates ranging from 60% to 100%. Benefits of these implants include stable fracture fixation, early weight-bearing, and reduced need for prolonged antibiotic therapy. However, concerns remain regarding antibiotic resistance and potential toxicity. This study aims to evaluate the efficacy and safety of these implants in managing infected nonunion and open fractures of the femur and tibia. Methods This prospective hospital-based study aimed to assess the efficacy and safety of antibiotic-impregnated cement/polymer-coated intramedullary nails for managing infected nonunion and open fractures of the femur and tibia. The study included patients aged 18 or older who received treatment with these implants between January 1, 2021 and December 31, 2022. Patients allergic to vancomycin or teicoplanin, with gap nonunion >2 cm, or lost to follow-up were excluded. Data on demographics, fracture details, previous treatment, surgery, antibiotics, and outcomes were collected using a structured proforma. Surgeries involved implant removal, debridement, culture testing, reaming, fracture reduction, and stabilization with an antibiotic-impregnated cement/polymer-coated intramedullary nail. Postoperatively, patients received antibiotics, had wound inspections, and were gradually allowed weight-bearing. Follow-up appointments and radiographic/laboratory assessments were conducted at regular intervals. The primary outcome was successful bone union, and secondary outcomes included time to union, infection rate, nonunion rate, and revision surgery. Results The majority of participants were male, with a mean age of 39.76 years. Most fractures were Gustilo-Anderson grade 3 (46.7%) and involved the tibia (73.3%). The mean bone gap after debridement was 1.3 cm. The median follow-up period was 8.21 months. Infection was controlled in 93.3% of patients, with the tibia being the most common site (70.0%). Successful bone union was achieved in 90.0% of patients, with a mean union rate of 22.13 weeks for tibial fractures and 17.21 weeks for femoral fractures. Among patients with bone union, 60.0% did not require additional procedures. Most patients had excellent bony (76.7%) and functional (70.0%) outcomes. The most common complications were the persistence of bone nonunion, impingement of proximal nail, and debonding of nail cement, each occurring in 10.0% of patients. Conclusion The study concluded that antibiotic-impregnated cement/polymer-coated intramedullary nails are effective in managing infected nonunion and open fractures of the femur and tibia. The procedure demonstrated a high success rate in controlling infections (93.3%) and achieving bone union (90.0%). Paley's criteria showed excellent bony and functional outcomes in the majority of patients. These findings support the use of this treatment option for such fractures.

High throughput microscopy and single cell phenotypic image-based analysis in toxicology and drug discovery.

Measuring single cell responses to the universe of chemicals (drugs, natural products, environmental toxicants etc.) is of paramount importance to human health as phenotypic variability in sensing stimuli is a hallmark of biology that is considered during high throughput screening. One of the ways to approach this problem is via high throughput, microscopy-based assays coupled with multi-dimensional single cell analysis methods. Here, we will summarize some of the efforts in this vast and growing field, focusing on phenotypic screens (e.g., Cell Painting), single cell analytics and quality control, with particular attention to environmental toxicology and drug screening. We will discuss advantages and limitations of high throughput assays with various end points and levels of complexity.

Basic Image Analysis and Manipulation in ImageJ/Fiji.

Microscopy-generated images contain quantitative information that can be extracted using a variety of analysis methods and automated pipelines. In this protocol, basic aspects of image analysis are outlined, including software installation, data import, image processing functions, and analytical tools that can be used to extract information from microscopy data using the open-source software ImageJ/Fiji. Step-by-step protocols for analyzing objects (i.e., nuclei) in a fluorescence image are included, focusing on segmentation routines. © 2023 Wiley Periodicals LLC.

LXR Signaling-Mediated Cholesterol Metabolism Reprogramming Regulates Cancer Cell Metastasis.

Metastasis is a key contributor to mortality in patients with cancer. While many regulators of metastasis have been identified, critical targets to prevent and inhibit metastatic tumor growth remain elusive. A recent study in this issue of Cancer Research by Deng and colleagues compared gene expression signatures between primary esophageal squamous cell carcinoma tumors and metastatic tumors and combined the analysis with genes induced in metastatic cancer cell lines, which identified anoctamin 1 (ANO1) as a key driver of metastasis. ANO1 caused cholesterol accumulation by inhibiting LXR signaling and decreased cholesterol hydroxylation by downregulating the expression of cholesterol hydroxylase CYP27A1. ANO1 also regulated tumor cell-fibroblast cross-talk that contributed to inflammatory cytokine signaling (IL1ß) and metastasis. Through in silico analysis, the study identified a novel small-molecule inhibitor of ANO1 that decreased tumor burden at a metastatic site. These studies provide novel insights into the role of ANO1 in cellular cholesterol metabolism and associated signaling in mediating metastasis. See related article by Deng et al., p. 1851.

A Study of the Anatomy of the Eustachian Tube for Its Surgical Approach in Otorhinolaryngology.

BACKGROUND: The eustachian tube (ET) is a connection between the nasopharynx and the middle ear behind the inferior nasal concha. It plays an important role in regulating air pressure across the tympanic membrane for proper transmission of sound. The pharyngeal opening of the tube is an important landmark for endoscopic evaluation in patients suffering from chronic otitis media and is also an important anatomical landmark for the transnasal approach to the infratemporal fossa. Hence, the study was done to locate the position of the pharyngeal opening of the ET in relation to various important anatomical landmarks. METHODOLOGY: Hundred (50 right and 50 left sides) adult (60-80 years) formalin-fixed sagittal sections of head and neck specimens were taken for the study, which was obtained during the undergraduate teaching program. The shape, size, and position of the pharyngeal opening of the ET were noted. The distance between the pharyngeal opening of the ET and various anatomical landmarks was measured with the help of the digital Vernier caliper. The mean and standard deviation of all the parameters were calculated and tabulated. RESULTS: In the present study, a slit-like shape was the most common shape of the pharyngeal opening, present in 62 out of 100 specimens. The difference between the anteroposterior length and vertical height of the two sides showed a statistically significant difference. CONCLUSION: The present study will help to locate the position of the pharyngeal opening of the ET during otorhinolaryngological evaluation for performing various surgeries in the middle ear.