Multi-Center Assessment of Lymph-Node Density and Nodal-Stage to Predict Disease-Specific Survival in Patients with Bladder Cancer Treated by Radical Cystectomy.

BACKGROUND: Prognostic tools in pathological-node (pN) patients after radical cystectomy (RC) are needed. OBJECTIVES: To evaluate the prognostic impact of lymph node (LN)-density on disease-specific survival (DSS) in patients with bladder cancer (BC) undergoing RC with pelvic lymph node dissection. METHODS: We analyzed a multi-institutional cohort of 1169 patients treated with upfront RC for cT1-4aN0M0 urothelial BCat nine centers. LN-densitywas calculated as the ratio of the number of positive LNs×100% to the number of LNs removed. The optimal LN-density cut-off value was defined by creating a time-dependent receiver operating characteristic (ROC) curve in pN patients. Univariable and multivariable Cox' regression analyses were used to assess the effect of conventional Tumor Nodes Metastasis (TNM) nodal staging system, LN-density and other LN-related variables on DSS in the pN-positive cohort. RESULTS: Of the 1169 patients, 463 (39.6%) patients had LN-involvement. The area under the ROC curve was 0.60 and the cut-off for LN-density was set at 20%, 223 of the pN-positive patients (48.2%) had a LN-density ≥ 20%. In multivariable models, the number of LN-metastases (HR 1.03, p = 0.005) and LN-density, either as continuous (HR 1.01, p = 0.013) or as categorical variable (HR 1.37, p = 0.014), were independently associated with worse DSS, whereas pN-stage was not. CONCLUSIONS: LN-density ≥ 20% was an independent predictor of worse DSS in BC patients with LN-involvement at RC. The integration of LN-density and other LN-parameters rather than only conventional pN-stage may contribute to a more refined risk-stratification in BC patients with nodal involvement.

Upfront versus deferred cytoreductive nephrectomy following targeted or immunotherapy: a population-based propensity score-matched analysis of perioperative complications.

PURPOSE: To evaluate the incidence of postoperative complications after cytoreductive nephrectomy (CN) following first-line treatment for metastatic renal cell carcinoma (mRCC), and to compare it with postoperative complications of upfront CN. METHODS: For this population-based retrospective study, the PearlDiver Mariner database (PearlDiver Technologies, Colorado Springs, CO), a database of insurance billing records was analyzed. Using relevant ICD-9/10 and CPT codes, patients diagnosed with mRCC between 2011 and 2021, who received first-line systemic molecular therapy (SMT), either tyrosine kinase inhibitors (TKI) or immune-checkpoint inhibitors (ICI), were identified. The selected population was stratified into two cohorts according to the timing of CN (deferred: after SMT vs. upfront: before SMT). Propensity-score matching (PSM) was performed as per baseline patients' characteristics to control for potential confounders between the two cohorts. The primary outcome was to compare 30-day postoperative complications rate between patients undergoing upfront vs. deferred CN. RESULTS: After PSM, 162 patients who received upfront CN were compared with 162 patients who underwent deferred CN. The overall rate of 30-day postoperative complications was statistically significantly higher in patients undergoing deferred CN (33.9%), compared to patients treated with upfront CN (21%, p < 0.01). In addition, the rate of both medical (26.5% vs. 14.2%, p < 0.01) and surgical (14.8 vs. 7.4%, p = 0.03) complication rate was statistically significantly higher in deferred vs. upfront CN. Multivariable logistic regression analysis revealed that none of the treatment regimens significantly predicted the occurrence of postoperative complications. CONCLUSION: Patients undergoing deferred CN experience a higher rates of overall, medical, and surgical 30-day postoperative complications compared to those receiving upfront surgery. Findings from this study should be interpreted within the limitations of this type of analysis.

NF-κB inhibitor alpha controls SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.

Colloidal Aggregation Confounds Cell-Based Covid-19 Antiviral Screens.

Colloidal aggregation is one of the largest contributors to false positives in early drug discovery. Here, we consider aggregation's role in cell-based infectivity assays in Covid-19 drug repurposing. We investigated the potential aggregation of 41 drug candidates reported as SARs-CoV-2 entry inhibitors. Of these, 17 formed colloidal particles by dynamic light scattering and exhibited detergent-dependent enzyme inhibition. To evaluate the impact of aggregation on antiviral efficacy in cells, we presaturated the colloidal drug suspensions with BSA or spun them down by centrifugation and measured the effects on spike pseudovirus infectivity. Antiviral potencies diminished by at least 10-fold following both BSA and centrifugation treatments, supporting a colloid-based mechanism. Aggregates induced puncta of the labeled spike protein in fluorescence microscopy, consistent with sequestration of the protein on the colloidal particles. These observations suggest that colloidal aggregation is common among cell-based antiviral drug repurposing and offers rapid counter-screens to detect and eliminate these artifacts.

A systematic review and meta-analysis to evaluate the diagnostic accuracy of PSMA PET/CT in the initial staging of prostate cancer.

BACKGROUND: Positron Emission Tomography-Computed Tomography using Prostate-Specific Membrane Antigen (PSMA PET/CT) is notable for its superior sensitivity and specificity in detecting recurrent PCa and is under investigation for its potential in pre-treatment staging. Despite its established efficacy in nodal and metastasis staging in trial setting, its role in primary staging awaits fuller validation due to limited evidence on oncologic outcomes. This systematic review and meta-analysis aims to appraise the diagnostic accuracy of PSMA PET/CT compared to CI for comprehensive PCa staging. METHODS: Medline, Scopus and Web of science databases were searched till March 2023. Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines were followed to identify eligible studies. Primary outcomes were specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of PSMA PET/CT for local, nodal and metastatic staging in PCa patients. Due to the unavailability of data, a meta-analysis was feasible only for detection of seminal vesicles invasion (SVI) and LNI. RESULTS: A total of 49 studies, comprising 3876 patients, were included. Of these, 6 investigated accuracy of PSMA PET/CT in detection of SVI. Pooled sensitivity, specificity, PPV and NPV were 42.29% (95%CI: 29.85-55.78%), 87.59% (95%CI: 77.10%-93.67%), 93.39% (95%CI: 74.95%-98.52%) and 86.60% (95%CI: 58.83%-96.69%), respectively. Heterogeneity analysis revealed significant variability for PPV and NPV. 18 studies investigated PSMA PET/CT accuracy in detection of LNI. Aggregate sensitivity, specificity, PPV and NPV were 43.63% (95%CI: 34.19-53.56%), 85.55% (95%CI: 75.95%-91.74%), 67.47% (95%CI: 52.42%-79.6%) and 83.61% (95%CI: 79.19%-87.24%). No significant heterogeneity was found between studies. CONCLUSIONS: The present systematic review and meta-analysis highlights PSMA PET-CT effectiveness in detecting SVI and its good accuracy in LNI compared to CI. Nonetheless, it also reveals a lack of high-quality research on its performance in clinical T staging, extraprostatic extension and distant metastasis evaluation, emphasizing the need for further rigorous studies.

Molecular insights into Pashmina fiber production: comparative skin transcriptomic analysis of Changthangi goats and sheep.

Ladakh, one of the highest inhabited regions globally, hosts the unique Changthangi goat, renowned for producing Pashmina, the world's most luxurious natural fiber. In comparison, the fiber derived from Changthangi sheep is considered next only to Pashmina. This research endeavors to compare the skin transcriptome profiles of Changthangi goats and Changthangi sheep, aiming to discern the molecular determinants behind the recognition of Changthangi goats as the source of Pashmina. Drawing upon previously conducted studies, a collective of 225 genes correlated with fiber characteristics were extracted from the differentially expressed genes noticed between the two species (p-value of ≤ 0.05 and a log2 fold change of ≥ 1.5). These genes were analyzed using DAVID software to understand their biological functions and to identify enriched KEGG and Reactome pathways. The protein-protein interaction networks were constructed using Cytoscape, cytoHubba, and STRING to focus on key genes and infer their biological significance. Comparative transcriptome analysis revealed significantly higher expression of genes involved in signaling pathways like Wnt, MAPK, PI3K-Akt, Hedgehog, associated with fiber development and quality in Changthangi goats. These pathways play crucial roles in hair follicle (HF) formation, maintenance of epidermal stem cells, and fiber characteristics. Findings also highlight the enrichment of cell adhesion molecules and ECM-receptor interaction, emphasizing their roles in HF structure, growth, and signaling. This investigation offers an in-depth understanding of the molecular intricacies governing Pashmina production in Changthangi goats, providing valuable insights into their unique genetic makeup and underlying mechanisms influencing the exceptional quality of Pashmina fibers.

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Use of Behavioral Health Treatment Among Parenting Women With Opioid Use Disorder in the United States.

OBJECTIVE: To examine use of mental health treatment, substance use disorder treatment, and perceived barriers to treatment by whether a major depressive episode occurred during the past year among parenting women with opioid use disorder. DESIGN: Secondary analysis of survey data from the National Survey on Drug Use and Health, 2015-2019. SETTING: United States. PARTICIPANTS: Women aged 18 to 44 years with opioid use disorder and at least one child in the household. METHODS: We computed descriptive statistics for demographic characteristics, treatment by major depressive episode status, and barriers to treatment by major depressive episode status. We conducted multinomial logistic regression to examine associations among demographic characteristics, major depressive episode status, and type of treatment. RESULTS: Of the 36% of respondents in our weighted sample (N ≈ 254,300) who experienced major depressive episode, 35% received substance use disorder and mental health treatment, and 27% did not receive any form of treatment. We found that identification as a person of color was significantly associated with a lower relative risk of receiving any type of treatment. Frequently reported barriers to treatment included affordability, access, and stigma. CONCLUSION: Respondents with opioid use disorder and co-occurring major depressive episode did not obtain necessary treatment. Barriers to treatment, including affordability, access to treatment, and stigma, need to be addressed, particularly among women of color.

The association between rurality, dual Medicare/Medicaid eligibility and chronic conditions with telehealth utilization: An analysis of 2019-2020 national Medicare claims.

INTRODUCTION: Telehealth services have the potential to increase healthcare access among underserved populations, such as rural residents and racial/ethnic minority groups. The COVID-19 public health emergency led to unprecedented growth in telehealth utilization, but evidence suggests the growth has not been equitable across all patient populations. This study aimed to explore whether telehealth utilization and expansion changed equitably from 2019 to 2020 among sub-groups of Medicare beneficiaries. METHODS: We conducted an analysis of telehealth utilization among a 20% random sample of 2019-2020 Medicare beneficiaries on a national level. We fit multivariable logistic regression models and calculated average marginal effects (AME) to assess the association between demographic and clinical characteristics on telehealth utilization. RESULTS: We found telehealth utilization was less likely among non-Hispanic Black/African-American (2019: adjusted odds ratio [aOR] = 0.77, AME = -0.15; 2020: aOR = 0.85, AME = -3.50) and Hispanic (2019: aOR = 0.79, AME = -0.13; 2020: aOR = 0.87, AME = -2.89) beneficiaries, relative to non-Hispanic White beneficiaries in both 2019 and 2020, with larger disparities in 2020. Rural beneficiaries were more likely to utilize telehealth than urban beneficiaries in 2019 (aOR = 2.62, AME = 0.84), but less likely in 2020 (aOR = 0.57, AME = -14.47). In both years, dually eligible Medicare/Medicaid beneficiaries were more likely than non-dually eligible beneficiaries to utilize telehealth (2019: aOR = 4.75, AME = 0.84; 2020: aOR = 1.34, AME = 2.25). However, the effects of dual eligibility and rurality changed in both models as the number of chronic conditions increased. DISCUSSION: We found evidence of increasing disparities in telehealth utilization among several Medicare beneficiary sub-groups in 2020 relative to 2019, including individuals of minority race/ethnicity, rural residents, and dually eligible beneficiaries, with disparities increasing among individuals with more chronic conditions. Although telehealth has the potential to address health inequities, our findings suggest that many of the patients in greatest need of healthcare are least likely to utilize telehealth services.

Chronic melatonin treatment improves obesity by inducing uncoupling of skeletal muscle SERCA-SLN mediated by CaMKII/AMPK/PGC1α pathway and mitochondrial biogenesis in female and male Zücker diabetic fatty rats.

Melatonin acute treatment limits obesity of young Zücker diabetic fatty (ZDF) rats by non-shivering thermogenesis (NST). We recently showed melatonin chronically increases the oxidative status of vastus lateralis (VL) in both obese and lean adult male animals. The identification of VL skeletal muscle-based NST by uncoupling of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)- sarcolipin (SLN) prompted us to investigate whether melatonin is a SERCA-SLN calcium futile cycle uncoupling and mitochondrial biogenesis enhancer. Obese ZDF rats and lean littermates (ZL) of both sexes were subdivided into two subgroups: control (C) and 12 weeks orally melatonin treated (M) (10 mg/kg/day). Compared to the control groups, melatonin decreased the body weight gain and visceral fat in ZDF rats of both sexes. Melatonin treatment in both sex obese rats restored the VL muscle skin temperature and sensitized the thermogenic effect of acute cold exposure. Moreover, melatonin not only raised SLN protein levels in the VL of obese and lean rats of both sexes; also, the SERCA activity. Melatonin treatment increased the SERCA2 expression in obese and lean rats (both sexes), with no effects on SERCA1 expression. Melatonin increased the expression of thermogenic genes and proteins (PGC1-α, PPARγ, and NRF1). Furthermore, melatonin treatment enhanced the expression ratio of P-CaMKII/CaMKII and P-AMPK/AMPK. In addition, it rose mitochondrial biogenesis. These results provided the initial evidence that chronic oral melatonin treatment triggers the CaMKII/AMPK/PGC1α axis by upregulating SERCA2-SLN-mediated NST in ZDF diabetic rats of both sexes. This may further contribute to the body weight control and metabolic benefits of melatonin.

Comprehensive evaluation and validation of optimal reference genes for normalization of qPCR data in different caprine tissues.

BACKGROUND: Quantitative real-time PCR (qPCR) is a highly reliable method for validating gene expression data in molecular studies due to its sensitivity, specificity, and efficiency. To ensure accurate qPCR results, it's essential to normalize the expression data using stable reference genes. METHODS: This study aimed to identify suitable reference genes for qPCR studies in goats by evaluating 18 candidate reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) in 10 different caprine tissues (heart, intestine, kidney, liver, lung, muscle, rumen, skin, spleen, and testis). An integrated tool called RefFinder, which incorporates various algorithms like NormFinder, GeNorm, BestKeeper, and ΔCt, was used to assess the stability of expression among these genes. RESULTS: After thorough analysis, ACTB, PPIB, and B2M emerged as the most stable reference genes, while RPL19, RPS15, and RPS9 were found to be the least stable. The suitability of the selected internal control genes was further validated through target gene analysis, confirming their efficacy in ensuring accurate gene expression profiling in goats. CONCLUSION: The study determined that the geometric average of ACTB, PPIB, and B2M creates an appropriate normalization factor for gene expression studies in goat tissues.

TennCare Disenrollment Led To Increased Eviction Filings And Evictions In Tennessee Relative To Other Southern States.

Previous research suggests that enrolling in Medicaid reduces evictions by improving health and providing financial protection. However, previous studies have not examined whether the loss of Medicaid affects eviction outcomes. We analyzed eviction filings and completed evictions after a large, mandatory Medicaid disenrollment in Tennessee in 2005. We conducted a difference-in-differences analysis using data from the Eviction Lab at Princeton University and found that relative to other southern states, the TennCare disenrollment led to a 27.6 percent greater increase in the average annual number of eviction filings at the county level during the period 2005-09 and a 24.5 percent greater increase in the average annual number of completed evictions at the county level during that same period. Our findings have implications for the housing stability of Medicaid recipients today, many of whom are being disenrolled because of the unwinding of the Medicaid continuous enrollment provision that is occurring across the country. To protect housing stability for people disenrolled from Medicaid, policy makers may wish to consider new initiatives aimed at preventing an increase in eviction.

Health Services Use Among Children With Prenatal Substance Exposure or Neonatal Abstinence Syndrome.

OBJECTIVE: Infants with prenatal substance exposure or neonatal abstinence syndrome (NAS) use health services more often than other children; however, little is known about their use of mental health services and psychotropic medication. METHODS: The sample (N=1,004,085) consisted of infants born in 2016 in 38 states who were followed through the fifth year of life and enrolled each year in Medicaid or the Children's Health Insurance Program. Infants with prenatal substance exposure or NAS were identified with ICD-10 diagnosis codes; procedure and revenue codes documented their service use. RESULTS: Rates of any mental health visit and of psychotropic medication use were higher among infants with prenatal substance exposure or NAS compared with infants without either condition; these patterns persisted during most years of the 5-year study. CONCLUSIONS: Infants' elevated mental health services use through their first 5 years of life highlights the importance of early screening and subsequent engagement in school-based mental health interventions.

Assessment of Pregnancy-Related Telehealth Interventions in the United States: A 10-Year Scoping Review.

Introduction: Telehealth use in obstetrics has been demonstrated to improve efficiency, access to care, and pregnancy outcomes. Despite reported successful implementation of these programs, information regarding the program variations and its impact on health care costs and outcomes are scarce. Methods: This is a scoping review of pregnancy-related telehealth studies to understand the current landscape of pregnancy-related telehealth interventions as well as to subset those that are used in high-risk pregnancies. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses framework to guide this review. Results: A total of 70 articles were included in this scoping review. Of those, 53 (75.7%) studies included a pregnant population and 17 (24.3%) studies focused on a rural and/or urban population. Most studies (n = 56; 80%) included some form of synchronous interaction between provider and participant. Patient outcomes included maternal/infant health outcomes (n = 41; 44.1%), patient satisfaction (n = 9; 9.7%), and attendance/compliance (n = 5; 5.4%). Provider-level outcomes included knowledge change (n = 11; 11.8%) and self-efficacy (n = 3; 3.2%). Other outcomes included assessment of costs and patient/provider feasibility and acceptability of the intervention. Overall, there has been a growing trend in articles published on pregnancy-related telehealth studies since 2011, with 2018 having the most publications in a single year. Conclusion: This review suggests a steadily growing body of literature on pregnancy-related telehealth interventions; however, more research is needed to better understand outcomes of telehealth for pregnancy-related care, especially related to patient satisfaction, health disparities, and cost-benefit.

A Walnut-Enriched Diet for 2 Years Changes the Serum Oxylipin Profile in Healthy Older Persons.

BACKGROUND: Oxylipins are products derived from polyunsaturated fatty acids (PUFAs) that play a role in cardiovascular disease and aging. Fish oil-derived n-3 PUFAs promote the formation of anti-inflammatory and vasodilatory oxylipins; however, there are little data on oxylipins derived from α-linolenic acid (C18:3n-3), the primary plant-derived n-3 PUFA. Walnuts are a source of C18:3n-3. OBJECTIVES: To investigate the effect on serum oxylipins of a diet enriched with walnuts at 15% energy (30-60 g/d; 2.6-5.2 g C18:3n-3/d) for 2 y compared to a control diet (abstention from walnuts) in healthy older males and females (63-79 y). METHODS: The red blood cell proportion of α-linolenic acid was determined by gas chromatography as a measure of compliance. Ultra-performance liquid chromatography-tandem mass spectrometry was used to measure serum concentrations of 53 oxylipins in participants randomly assigned to receive the walnut diet (n = 64) or the control diet (n = 51). Two-year concentration changes (final minus baseline) were log-transformed (base log-10) and standardized (mean-centered and divided by the standard deviation of each variable). Volcano plots were then generated (fold change ≥1.5; false discovery rate ≤0.1). For each oxylipin delta surviving multiple testing, we further assessed between-intervention group differences by analysis of covariance adjusting for age, sex, BMI, and the baseline concentration of the oxylipin. RESULTS: The 2-y change in red blood cell C18:3n-3 in the walnut group was significantly higher than that in the control group (P < 0.001). Compared to the control diet, the walnut diet resulted in statistically significantly greater increases in 3 C18:3n-3-derived oxylipins (9-HOTrE, 13-HOTrE, and 12,13-EpODE) and in the C20:5n-3 derived 14,15-diHETE, and greater reductions of the C20:4n-6-derived 5-HETE, 19-HETE, and 5,6-diHETrE. CONCLUSIONS: Long-term walnut consumption changes the serum oxylipin profile in healthy older persons. Our results add novel mechanistic evidence on the cardioprotective effects of walnuts. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT01634841.

Comparative study on thermo-mechanical, structural and functional properties of pectin extracted from immature wasted apples and commercial pectin.

Pectin yield of 22.22 ± 0.98 % (dry basis) was achieved from prematurely dropped Golden Delicious apples, having a light orange hue (hue value: 78.08 ± 0.04) and an overall color difference (ΔE) of 9.92 ± 0.01 compared to commercial pectin (CP). Extracted AP exhibited a lower equivalent weight (725.24 ± 29.73) and higher methoxy content (8.36 ± 0.28 %) in contrast to CP. However, a similar degree of esterification of 71.57 ± 0.79 and 70.55 ± 0.59 %, was observed in AP and CP respectively. Apple pectin demonstrated slight lower galacturonic acid (GalA) content of 68.10 ± 3.94 % in comparison to 72.31 ± 4.62 % of CP, which was further corroborated by reduced intensity in FTIR fingerprint region (912-1025 cm-1). Morphology revealed a sheet-like cloudy appearance indicating a significant presence of associated sugars whereas X-ray diffraction highlighted the highly amorphous nature of AP. AP and CP solutions (3-9 %) displayed a shear-thinning flow and viscoelastic behavior where the loss (G') moduli dominated over the storage moduli (G"). Owing to high degree of esterification, galacturonic acid content (>65 %) that aligns with commercial standards and viscoelastic behavior, the extracted AP holds promise for potential utilization in commercial applications. This study underscores the potential for sustainable utilization of prematurely dropped apples through pectin extraction, contributing to valorization of the wasted bioresource.

Identification and validation of stable reference genes for expression profiling of target genes in diverse ovine tissues.

Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1, and B2M emerged as the most stable RGs, while RPS9, RPS15, and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1, and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1, and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues.