Autophagy is initiated at endoplasmic reticulum (ER)-associated omegasomes, yet how the ER contributes to mitochondrial cargo degradation during early autophagosome formation remains unclear. Gp78 is an ER-resident RING E3 ubiquitin ligase involved in ER-associated degradation (ERAD) that also promotes Parkin-independent mitophagy. However, the spatial relationship between Gp78 ubiquitin ligase activity, omegasome formation, and mitochondrial degradation has yet to be defined. Combining targeted mitochondria labeling and super-resolution STED microscopy, we show that the Gp78 ubiquitin ligase is localized to and regulates DFCP1-positive omegasome formation, where it drives mitochondrial protein breakdown. Using fluorescent mitophagy reporters and acute ivermectin-induced mitophagy, generation of mitophagy intermediates lacking mitochondrial outer membrane (TOMM20) was found to be dependent on the Gp78 RING finger as well as the BAG6-UBL4A-USP13 ubiquitin-ligase module, and independent of PINK1 and Parkin. Object-based analysis of 3D STED images further showed that both DFCP1- and LC3-positive structures associate primarily with intact mitochondrial fragments which show progressive degradation of mitochondrial content. Gp78 localizes to and promotes, via BAG6, the formation of DFCP1-positive omegasomes and mitochondrial protein degradation in these early autophagic structures. ER-derived omegasome biogenesis is therefore coupled to Gp78-dependent ubiquitin ligase activity and local degradation of mitochondrial cargo during mitophagy.

Originally thought to be a major endocytic portal, caveolae function as a membrane buffer, whereby caveolae flattening protects the plasma membrane from rupture under mechanical stress, such as hypotonic shock. However, the fate of the caveolae coat protein caveolin-1 upon caveolae flattening is not known. Here, extended hypotonic shock induces ubiquitin-independent, CLIC-dependent endocytosis of caveolin-1 to large, intracellular, CD44-positive, pH-neutral lysosomal vacuoles negative for multivesicular body markers. Caveolin-1 recycles from these vacuoles to the plasma membrane upon return to isotonic conditions. Caveolin-1 internalization occurs upon reduced cell volume due to extended hypotonic shock as well as in low-tension cells grown on reduced-stiffness hydrogels. Upon hypertonic shock, caveolin-1 internalization occurs in PC3 cells, lacking cavin-1 and caveolae, and is inhibited upon cavin-1 reintroduction. CLIC endocytosis of non-caveolar caveolin-1 to neutral pH lysosomal vacuoles identifies a non-caveolar endocytic and recycling pathway for caveolin-1 in response to reduced membrane tension.

There will be 66, 400 new cases of pancreatic cancer identified and 51, 750 will die by end of 2024 in the United States. KRAS is the most common oncogenic driver occurring in ≥85% of pancreatic cancer patients. Previously, we identified an overexpression of high affinity binding protein Interleukin-4 Receptor alpha (α) of Interleukin 4 (IL-4) in numerous solid human cancers including pancreatic cancer. Herein, we investigated if IL-4Rα and KRAS co-expression can act as a biomarker of disease severity in pancreatic cancer patients. One hundred and seven archival tumor specimens were examined by six investigators for IL-4Rα and KRAS expression by IHC and ISH techniques in a blinded manner. The data were stratified into pathological grades and stages of the patients. The results were analyzed statistically if individual/combined expression of IL-4Rα or KRAS can serve as biomarker/s of disease severity. Kaplan-Meier survival analysis was performed to investigate if these gene-expression had any influence on overall survival of the patients. Our IHC results demonstrate that ≥68% samples overexpressed IL-4Rα alone in moderately to poorly differentiated grade tumors and in samples from clinical stage II + III patients with ≥2+ immunostaining intensity. Interestingly, ≥80 % of the specimens with moderately to poorly differentiated grades showed a co-expression for KRAS and IL-4Rα (≥2+). In contrast, patients with well differentiated grade and clinical stage I revealed ≤ 73% co-expression of these genes. We observed a similar pattern of mRNA expression in these patients by ISH. Further, Kaplan-Meier analysis revealed a significantly shorter survival (P≤0.001) in patients with ≥2+ positive expression of IL-4Rα plus KRAS in moderately to poorly differentiated grades and advanced clinical stages. In contrast, the patients overexpressing either of these genes alone, regardless of grades and stages, survived relatively longer compared to those expressing both IL-4Rα and KRAS . Our data show that IL-4Rα and KRAS co-expression, correlate with disease severity in pancreatic cancer patients. Thus our study uncovers a novel role of combined expression of IL-4Rα and KRAS as a biomarker of prognosis and driver of oncogenesis in pancreatic cancer patients and may serve a unique druggable target/s for developing targeted cellular or gene therapy-based cancer immunotherapies. Ashley Lea, Toshio Fujisawa, Hiroyuki Isayama, Supreet Khanal, Santosh Panthi, Mona Mansouri, Samir Lababidi, Steven R. Bauer, Kyung Sung, Nirjal Bhattarai, Bharat H. Joshi. Co-expression of interleukin-4 receptor alpha and KRAS correlates with disease severity and prognosis in pancreatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2009.

The endoplasmic reticulum (ER) comprises smooth tubules, ribosome-studded sheets, and peripheral sheets that can present as tubular matrices. ER shaping proteins determine ER morphology, however, understanding their role in tubular matrix formation requires reconstructing the dynamic, convoluted ER network. Existing reconstruction methods are sensitive to parameters or require extensive annotation and training for deep learning. We introduce nERdy, an image processing based approach, and nERdy+, a D4-equivariant neural network, for accurate extraction and representation of ER networks and junction dynamics, outperforming previous methods. Comparison of stable and dynamic representations of the extracted ER structure reports on tripartite junction movement and distinguishes tubular matrices from peripheral ER networks. Analysis of live cell confocal and Stimulated emission depletion microscopy (STED) time series data shows that Atlastin and Reticulon 4 promote dynamic tubular matrix formation and enhance junction dynamics, identifying novel roles for these ER shaping proteins in regulating ER structure and dynamics.

IntroductionThe gut microbiome’s influence on weight management has gained significant interest for its potential to support better obesity therapeutics. Patient stratification leading to personalized nutritional intervention has shown benefits over one-size-fit-all diets. However, the efficacy and impact on the gut’s microbiome of personalizing weight loss diets based on individual factors remains under-investigated.MethodsThis study assessed the impact of Digbi Health’s personalized dietary and lifestyle program on weight loss and the gut microbiome end-points in 103 individuals. Participants’ weight loss patterns and gut microbiome profiles were analyzed from baseline to follow-up samples.ResultsSpecific microbial genera, functional pathways, and communities associated with BMI changes and the program’s effectiveness were identified. 80% of participants achieved weight loss. Analysis of the gut microbiome identified genera and functional pathways associated with a reduction in BMI, including Akkermansia, Christensenella, Oscillospiraceae, Alistipes, and Sutterella, short-chain fatty acid production, and degradation of simple sugars like arabinose, sucrose, and melibiose. Network analysis identified a microbiome community associated with BMI, which includes multiple taxa known for associations with BMI and obesity.DiscussionThe personalized dietary and lifestyle program positively impacted the gut microbiome and demonstrated significant associations between gut microbial changes and weight loss. These findings support the use of the gut microbiome as an endpoint in weight loss interventions, highlighting potential microbiome biomarkers for further research.

BACKGROUND:Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric antigen receptor construct using a high binding and codon optimised scFv-IL-13Rα2 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB. METHODS:We developed a scFv clone, designated 14-1, by biopanning the bound scFv phages using huIL-13Rα2Fc chimeric protein and compared its binding with our previously published clone 4-1. We performed bioinformatic analyses for complementary determining regions (CDR) framework and residue analyses of the light and heavy chains. This construct was packaged with helper plasmids to produce CAR-lentivirus and transduced human Jurkat T or activated T cells from peripheral blood mononuclear cells (PBMCs) to produce CAR-T cells and tested for their quality attributes in vitro and in vivo. Serum enzymes including body weight from non-tumour bearing mice were tested for assessing general toxicity of CAR-T cells. RESULTS:The binding of 14-1 clone is to IL-13Rα2Fc-chimeric protein is ∼5 times higher than our previous clone 4-1. The 14-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotype and biological attributes such as cell viability, potency, migration and T cell activation. Clone 14-1 migrated to IL-13Rα2Fc and cell free supernatants only from IL-13Rα2+ve confluent glioma tumour cells in a chemotaxis assay. scFv-IL-13Rα2-CAR-T cells specifically killed IL-13Rα2+ve but not IL-13Rα2-ve tumour cells in vitro and selectively caused significant release of IFN-γ only from IL-13Rα2+ve co-cultures. These CAR-T cells regressed IL-13Rα2+ve glioma xenografts in vivo without any general toxicity. In contrast, the IL-13Rα2 gene knocked-down U251 and U87 xenografts failed to respond to the CAR-T therapy. CONCLUSION:Taken together, we conclude that the novel scFv-IL-13Rα2 CAR-T cell therapy may offer an effective therapeutic option after designing a careful pre-clinical and clinical study.

Figure S5. Flow chart of the selection process of patients.

Figure S7. Histology of primary breast cancer tissue.

S1. Caspase induction in SKBR3 cells pre-incubated for 20 h in normoxia or hypoxia and treated with T-DM1 was measured using the Caspase-Glo 3/7 assay kit and the results are shown as mean luminescence (measured in relative light units [RLU]).

Caveolin-1 (CAV1), the main structural component of caveolae, is phosphorylated at tyrosine-14 (pCAV1), regulates signal transduction, mechanotransduction, and mitochondrial function, and plays contrasting roles in cancer progression. We report that CRISPR/Cas9 knockout (KO) of CAV1 increases mitochondrial oxidative phosphorylation, increases mitochondrial potential, and reduces ROS in MDA-MB-231 triple-negative breast cancer cells. Supporting a role for pCAV1, these effects are reversed upon expression of CAV1 phosphomimetic CAV1 Y14D but not non-phosphorylatable CAV1 Y14F. pCAV1 is a known effector of Rho-associated kinase (ROCK) signaling and ROCK1/2 signaling mediates CAV1 promotion of increased mitochondrial potential and decreased ROS production in MDA-MB-231 cells. CAV1/ROCK control of mitochondrial potential and ROS is caveolae-independent as similar results were observed in PC3 prostate cancer cells lacking caveolae. Increased mitochondrial health and reduced ROS in CAV1 KO MDA-MB-231 cells were reversed by knockdown of the autophagy protein ATG5, mitophagy regulator PINK1 or the mitochondrial fission protein Drp1 and therefore due to mitophagy. Use of the mitoKeima mitophagy probe confirmed that CAV1 signaling through ROCK inhibited basal mitophagic flux. Activation of AMPK, a major mitochondrial homeostasis protein inhibited by ROCK, is inhibited by CAV1-ROCK signaling and mediates the increased mitochondrial potential, decreased ROS, and decreased basal mitophagy flux observed in wild-type MDA-MB-231 cells. CAV1 regulation of mitochondrial health and ROS in cancer cells therefore occurs via ROCK-dependent inhibition of AMPK. This study therefore links pCAV1 signaling activity at the plasma membrane with its regulation of mitochondrial activity and cancer cell metabolism through control of mitophagy. Caveolin-1 (CAV1), the main structural component of caveolae, is phosphorylated at tyrosine-14 (pCAV1), and signals via Rho Kinase (ROCK) to regulate focal adhesion signaling and tumor cell migration and invasion. Here we show that pCAV1 also acts via ROCK signaling to prevent AMPK activation, limiting basal mitophagy resulting in accumulation of damaged mitochondria, reduced mitochondrial respiration, and increased ROS production. pCAV1 signaling activity therefore regulates mitochondrial activity and cancer cell metabolism through control of mitophagy.image

Supplementary Video 1 from Phosphorylated Caveolin-1 Regulates Rho/ROCK-Dependent Focal Adhesion Dynamics and Tumor Cell Migration and Invasion

Obesity is a significant health problem due to its profound health deteriorating effects and high costs for healthcare systems. There exist lifestyle and pharmacological interventions available to prevent and reverse obesity; however, at the population level, these have shown to be insufficient, and we continue to see a worldwide increase in obesity prevalence. The gut microbiome has been shown to influence the susceptibility to weight gain and difficulty in losing weight and to be associated with successful long-term weight loss. Therefore, multiple studies have suggested that obesity interventions should consider the gut microbiome as a primary target through an improved diet and a crucial endpoint to monitor. However, there is a paucity of evidence regarding how to tailor the diet for an individual’s microbiome and what changes are expected to occur due to successful weight management. Digital therapeutics solutions have emerged as an exciting alternative to increase population access, reduce costs, and have the potential to accompany individuals on their health-promoting journey closely. Digbi Health has developed a dietary and lifestyle program to achieve weight loss that effectively reduces weight and improves diverse health outcomes by prioritizing and personalizing food ingredients to match an individual’s genetic profile and nurture the gut microbiome. In this study, we analyze the weight loss pattern and microbiome profile of 103 individuals to identify the effects of the weight loss program on the gut microbiome between their baseline and follow-up samples. We found that 80% of individuals lost weight during the study. Analysis of their gut microbiome identified genera, functional pathways, and microbial communities associated with BMI changes and dietary and lifestyle program. The microbial genera and functional pathways associated with a reduction in BMI during the study include several previously reported in the literature, including Akkermansia, Christensenella , Oscillospiraceae, Alistipes , and Sutterella , short-chain fatty acid (SCFA) production and degradation of simple sugars like arabinose, sucrose, and melibiose. Network analysis identified two microbiome communities associated with BMI, one of which also significantly responded to the weight loss program, which includes multiple known associations with BMI and obesity. Our findings provide additional evidence for using the gut microbiome as an endpoint of weight loss program and highlight how it positively impacts the gut microbiome, with significant parallels in weight loss and health outcomes. These results provide additional evidence for known microbiome biomarkers of obesity and highlight new ones that warrant further research.

Supplementary Figure 1 from Human Adrenomedullin Up-regulates Interleukin-13 Receptor α2 Chain in Prostate Cancer In vitro and In vivo: A Novel Approach to Sensitize Prostate Cancer to Anticancer Therapy

Supplementary Figure Legends 1-5 from A Novel Role of Interleukin-13 Receptor α2 in Pancreatic Cancer Invasion and Metastasis

Background Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Methods To determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with 89 Zirconium-oxine ( 89 Zr-oxine), and characterized and compared their product attributes with non-labeled CAR-T cells. The 89 Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon-γ upon co-culture with IL-13Rα2 expressing glioma cells. Results We observed that radiolabeling of CAR-T cells with 89 Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells was similar to that of unlabeled cells as determined by TUNEL assay and caspase 3/7 enzyme activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN-γ) or T cell exhaustion(PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13Rα2Fc was similar to that of non-labeled cells. Conclusions Importantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with 89 Zr-oxine retain critical product attributes and suggest 89 Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.

Supplementary Figure 1 from Phosphorylated Caveolin-1 Regulates Rho/ROCK-Dependent Focal Adhesion Dynamics and Tumor Cell Migration and Invasion

Supplementary Data from Interleukin 13 Mediates Signal Transduction through Interleukin 13 Receptor α2 in Pancreatic Ductal Adenocarcinoma: Role of IL-13 Pseudomonas Exotoxin in Pancreatic Cancer Therapy

Supplementary Video 3 from Phosphorylated Caveolin-1 Regulates Rho/ROCK-Dependent Focal Adhesion Dynamics and Tumor Cell Migration and Invasion