(1) Background: Overconsumption of processed meats, fats, and carbohydrates drives the obesity epidemic in the USA. Associated with this epidemic are increases in metabolic diseases, such as type 2 diabetes, cardiovascular disease, and cancer. In this study, protein levels of adipocytokines isolated from visceral fat in mice fed high-fat diets with proteins modified through ammonium supplementation were analyzed to determine changes that occur as a result of dietary protein source and its modification based on age or sex. (2) Methods: Male and female C3H/HeJ mice were randomized into six customized diets—Group 1: CCN = Control Chow (CC) + Ammonium Hydroxide Enhancement (AHE); Group 2: CC = Control Chow; Group 3: HFBN = High Fat (HF) AHE Dietary Beef; Group 4: HFB = HF Beef; Group 5: HFCN = HF AHE Dietary Casein; Group 6: HFC = HF Dietary Casein. Mice were censored at six-month intervals, and visceral fat was collected for analysis. This study highlights sex- and age-related changes in cellular adipocytokine protein expression from 12 to 18 months. (3) Results: When compared to dietary casein, dietary-beef-fed mice showed increased expression of adiponectin, leptin, and MCP-1. In dietary casein protein diets, high fat content was correlated with the expression of pro-inflammatory adipocytokines leptin, MCP-1, resistin, VEGF-A, and TIMP-1. Sex-related differences were observed in adiponectin, leptin, and MCP-1 expression levels. AHE of dietary protein decreased the expression of adiponectin, leptin, MCP-1, and TIMP-1. Age-related changes in expression were observed in leptin, MCP-1, and VEGF-A. (4) Conclusions: Our results indicate that the source of dietary protein plays a critical role in determining adipocytokine expression in WAT. Furthermore, this study shows that in addition to dietary protein type (beef or casein), AHE and fat content also impact the relative expression of both pro-inflammatory and anti-inflammatory adipocytokines based on sex over time, with leptin and MCP-1 identified as the most frequently affected.

The excess consumption of a high-fat diet has been identified as one of the factors contributing to obesity. Women are at higher risk of adult obesity than men, predisposing them to a different set of detrimental disease conditions. Furthermore, aging studies show that physiological decline also has a serious impact on changes in the endocrine properties of white adipose tissue. However, there is still relatively little known about the factors associated with obesity and aging and their compounding impacts on women’s health. To investigate changes in adipocytokine secretion profiles, obesity was induced in female C3H/HeJ mice through the long-term consumption of a high-fat diet. Weight gain measurements and the Echo MRI analysis of fat composition showed that increases were due solely to the high fat content in the diet. Adipocytokine secretions were analyzed in media conditioned from harvested visceral fat tissue that was organ-cultured ex vivo. Adipocytokine analysis performed across diets and ages showed that there were significant increases in Adiponectin and Leptin secretion in high-fat diets, accelerating increases in Resistin secretion in high-fat diets. Aging induced the increased secretion of Lipcalin-2, Pentraxin-3, Serpin E1, MCP-1, and ICAM-1, regardless of diet. Furthermore, the comparisons of organoid-cultured protein secretions and flash-frozen tissue samples differed greatly, suggesting the WAT organoid cultures may yield information that is more reflective of in situ conditions. Taken together, our results show that high-fat diets and aging in C3H/HeJ female mice significantly impact secretions from adipose tissue, which may contribute to women’s health issues.

Excess consumption of a high-fat diet has been identified as one of the contributing factors for obesity. Women are at higher risk for adult obesity than men, predisposing them to a different set of detrimental disease conditions. Furthermore, aging studies show that physiological decline also has a serious impact on changes in the endocrine properties of white adipose tissue. Howev-er, there is still relatively little known about the factors associated with obesity and aging and their compounding impact on women’s health. To investigate adipocytokine secretion profile changes, female C3H/HeJ mice were induced to obesity through long-term consumption of a high-fat diet. Weight gain measurements and Echo MRI analysis of fat composition showed in-creases were a due solely to the high fat content in the diet. Adipocytokine secretions were ana-lyzed in media conditioned from harvested visceral fat tissue that was organ cultured ex-vivo. Adipocytokine analysis across diets and age showed significant increases in Adiponectin and Leptin secretion in high-fat diets, accelerated increases in Resistin secretion with high-fat diets. Aging induced increased secretion of Lipcalin-2, Pentraxin-3 and Serpin E1, MCP-1 and ICAM-1, regardless of diet. Furthermore, comparisons of organoid cultured protein secretions and flash-frozen tissue samples differed greatly, suggesting the WAT organoid cultures may yield information more reflective of in situ conditions. Taken together, our results show that diet and aging significantly impact secretions from adipose tissue, that may contribute to women’s health issues.

CRISPR-Cas9 systems have enabled unprecedented advances in genome engineering, particularly in developing treatments for human diseases, like cancer. Despite potential applications, limitations of Cas9 include its relatively large size and strict targeting requirements. Cas12j2, a variant ofCasΦ-2, shows promise for overcoming these limitations. However, its effectiveness in mammalian cells remains relatively unexplored. This study sought to develop an optimized CRISPR-Cas12j2 system for targeted knockout of the E6 oncogene in HPV-associated cancers. A combination of computational tools (ColabFold, CCTop, Cas-OFFinder, HADDOCK2.4, and Amber for Molecular Dynamics) was utilized to investigate the impact of engineered modifications on structural integrity and gRNA binding of Cas12j2 fusion constructs, in potential intracellular conditions. Cas12j2_F2, a Cas12j2 variant designed and evaluated in this study, behaves similarly to the wild-type Cas12j2 structure in terms of RMSD/RMSF profiles, compact Rg values, and minimal electrostatic perturbation. The computationally validated Cas12j2 variant was incorporated into a custom expression vector, co-expressing the engineered construct along with a dual gRNA for packaging into a viral vector for targeted knockout of HPV-associated cancers. This study provides a structural and computational foundation for the rational design of Cas12j2 fusion constructs with enhanced stability and functionality, supporting their potential application for precise genome editing in mammalian cells.