Specific Membrane Research Articles

Abstract 3690: Monitoring prostate specific membrane antigen or androgen receptor expression on circulating stromal cells in advanced prostate cancer patients and their correlation with patient response

Abstract Background: Metastatic Prostate cancer (mPCa) mortality rates are high despite numerous treatment options against various mechanisms of action, including targeted therapies, i.e. prostate specific membrane antigen (PSMA)-targeted therapy (Pluvicto) or anti-androgen receptor (AR) (Enzulamide, Biculamide). Further, tumor biomarkers (i.e. PSMA and AR) can change over time and with resistance mechanisms that develop after treatments. This suggests that primary biopsies may not represent later PCa tumors necessitating a way to identify patients that may respond to later line therapies. Blood based biopsies can be used to monitor PCa treatment, which can include circulating tumor cells (CTCs) and the newly discovered cancer associated macrophage like cells (CAMLs), a cancer specific circulating phagocytic stromal cells. Interestingly, while PSMA & AR have been identified in CTCs, they have not been evaluated in CAMLs, nor have PSMA & AR expressions in CTCs & CAMLs been evaluated in anti-AR therapy settings. We measured CAMLs & CTCs in mPCa to evaluate their PSMA & AR expression, including in patients treated with anti-AR therapy. Methods: We evaluated CAMLs & CTCs in a multi-institutional prospective pilot study using n=30 mPCa patients with progressive disease, prior to starting a new line of therapy (T0). Whole peripheral blood (7.5mL) was filtered for CAMLs & CTCs and stained for PSMA (n=15) & AR (n=15). In addition, 15 patients were treated with anti-AR therapy as standard of care and responses by PET/CT were compared against AR expression. When possible, follow up samples (T1) were also procured. Results: CTCs were identified in 16% (n=5/30) of patients and CAMLs were identified in 96% (n=29/30), with 53% of patients having hyperengorged CAMLs ≥50 μm (n=16/30). CTCs in patients were not statistically significant for worse outcomes PFS HR= 0.1748, p=0.546 nor OS (HR= 1.303, p=0.865), nor were CAMLs prognostic for PFS or OS. However, patients with ≥50 μm CAMLs did have significantly worse PFS (HR= 5.9, CI95% 2.2-16.3, p=0.001) and worse OS (HR= 3.1, CI95% 1.3-7.1, p=0.016). Further, PSMA CAML/CTC expression was high in 60% (n=9/15) of patients and AR CAML/CTC expression was high in 53% (n=8/15). Of the n=15 patients who received anti-AR therapy after T0, high AR expressing CAMLs/CTCs (n=8/15) had a ~250% better mPFS of 10.3 months (mons) (progressing at 2.2-32.6 mons) vs low AR CAMLs/CTCs mPFS of 4 mons (progressing at 0.2-7.2 mons). Additional mOS analysis from T1 samples and 2-year survival is ongoing. Conclusion: We utilized liquid biopsies to monitor the expression of two common cell surface receptors (PSMA & AR) in CAMLs and CTCs in mPCa. Further, in a small subset of patients treated with AR therapy, high AR CTC/CAML expression appeared to correlate with better response rates, though larger prospective studies are needed. Citation Format: Dimpal M. Kasabwala, Raymond C. Bergan, R. Katherine Alpaugh, Daniel C. Danila, Tuan L. Chuang, Brenda Y. Hurtado, Daniel L. Adams. Monitoring prostate specific membrane antigen or androgen receptor expression on circulating stromal cells in advanced prostate cancer patients and their correlation with patient response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3690.

Abstract 1853: Prostate specific membrane antigen (PSMA) glycoforms and prostate cancer progression

Abstract Introduction: Prostate cancer (PCa) is the most common non-skin malignancy and the second leading cause of cancer related death in men. Prostate cancer is a heterogeneous disease with diverse clinical outcomes ranging from indolent asymptomatic cases to very aggressive and potentially lethal metastatic forms of the disease. Early detection of PCa is predominantly through determination of serum PSA levels and digital rectal examinations (DRE). Subsequently, biopsy based Gleason Grade Scores and TNM staging, tumor size or mpMRI are used to assess disease severity and progression. However, due to the low specificity of PSA concentration in diagnosis and prognosis, biopsy complications, and high mpMRI costs, there is still a critical need to identify and develop novel noninvasive biomarkers that discriminate indolent from aggressive PCa upon diagnosis. Our long-term objective is to discover biomarkers for aggressive PCa, develop and to implement them into robust clinical testing minimally invasive assays for PCa risk stratification at diagnosis. The current focus is on Prostate-Specific Membrane Antigen (PSMA), a membrane bound glycoprotein, that is significantly over-expressed in aggressive and metastatic prostate carcinomas and with poor patient prognosis. The role of its glycosylation status and composition in disease progression and its glycoforms on biomarker development and therapeutics is unknown. Aberrant glycosylation of glycoproteins occurs in many cancers, and it influences disease progression. We rationalize that differential glycosylation of PSMA correlate with progression to aggressive PCa and that PSMA glycopeptides and glycoforms can be used as diagnostics and prognosticators for aggressive disease. Methods/Results: PSMA was isolated from risk Grade Group stratified post-DRE urines, seminal plasma and prostate cancer cell lines with different metastatic phenotypes cell line lysates. Intact glycopeptides were isolated, purified and analyzed by qualitative and quantitative mass spectrometry to identify site specific glycoforms. Our data demonstrate differential expression of unique PSMA glycoforms that correlate with disease risk and severity. Conclusions: Differential glycosylation of PSMA correlates with PCa disease progression to aggressive phenotypes and may have utility in disease stratification. Ongoing studies focus on determining if these qualitative and quantitative changes have diagnostic/prognostic value and if these be integrated with other PCa biomarkers to improve their prognostic power in discriminating aggressive PCa. Acknowledgements: This work was supported by the DOD, USAMRAA, through the Prostate Cancer Research Program under Award No. W81XWH18-1-0228. Opinions, interpretations, and conclusions are those of the author and are not necessarily endorsed by the USAMRAA. Citation Format: Stephen Mackay, Ian O. Oduor, Tanya Burch, Dean A. Troyer, Oliver J. Semmes, Julius O. Nyalwidhe. Prostate specific membrane antigen (PSMA) glycoforms and prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1853.

Abstract 6023: 225Ac-FL-020 is a novel PSMA-targeting radionuclide drug conjugate (RDC) with superior in vivo anti-tumor activity

Abstract Despite significant developments over the last few decades, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Prostate specific membrane antigen (PSMA), a prostate cancer-specific membrane marker which directly correlates with androgen independence, metastasis, and disease progression, has been well-established as a radioligand target for the diagnosis and treatment of mCRPC. Lutetium-177 (177Lu)-PSMA-617 (PLUVICTOTM) was approved in 2022 for the treatment of progressive PSMA-positive mCRPC. However, only 30% of patients showed a radiological response in the registrational trial. Meanwhile, recent clinical experience has shown very limited efficacy in low PSMA expressing patients. These data collectively request for further improvement in the clinical benefit of PSMA-targeted radiotherapy. Actinium-225 (225Ac), an alpha emitter, is significantly more potent on cancer cell killing and has a shorter range in tissue penetration when compared to the beta emitter 177Lu. Such a profile supports the development of 225Ac based radiotherapies. Using our proprietary Clear-XTM technology platform, we developed 225Ac-FL-020, a novel 225Ac-based PSMA radioligand therapy candidate which has demonstrated prominent pre-clinical properties. We have assessed the binding affinity against PSMA of the non-labeled vector FL-020 in vitro. The in vivo biodistribution profile of FL-020 was characterized by SPECT/CT imaging and biodistribution using Indium-111 (111In)-FL-020 in PSMA high LNCaP tumor-bearing nude mice. In addition, anti-tumor activities of 225Ac-FL-020 were evaluated in the LNCaP xenograft model and directly compared to 225Ac-PSMA-617. FL-020 was found bound to LNCaP cells with an IC50 value of 51.55 nM. Meanwhile, the off-target screening showed that less than 50% inhibition of binding or activity was observed by FL-020 at 10 µM against 85 targets including receptors, ion channels, enzymes, and transporters, indicating the high selectivity of FL-020. Moreover, 111In-FL-020 displayed a very promising in vivo distribution profile with high and sustained tumor uptake and fast systemic clearance. Furthermore, 225Ac-FL-020 exhibited superior anti-tumor activity compared to 225Ac-PSMA-617 at the same dose level (10 KBq/mouse) in the LNCaP xenograft model with a favorable safety profile as indicated by body weight and hematological parameters. A mechanistic study was also conducted in 225Ac-FL-020-treated LNCaP tumor samples where DNA double-strand breaks and tumor cell apoptosis were observed, confirming the MOA of alpha emitters. Taken together, these results collectively demonstrate that 225Ac-FL-020 is a potent and selective PSMA-targeting radioligand therapy candidate with superior anti-tumor activity and a favorable safety profile warranting further clinical development. Citation Format: Fa Liu, Jiangwei Zhang, Junyu Yang, Kirstine T. Thrane, Mathias W. Hallund, Rikke V. Grønlund, Nicholas C. Wong. 225Ac-FL-020 is a novel PSMA-targeting radionuclide drug conjugate (RDC) with superior in vivo anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6023.

Abstract 6037: Copper-67 as a next-generation radioconjugate warhead for cancer therapy

Abstract Background and Purpose: Antibody drug conjugates (ADCs) have shown promise as cancer treatments. However, the efficacy of ADCs has been limited in solid tumors and can come with significant toxicities. Radioconjugates utilize various radionuclides that are chelated and can be conjugated to small molecules, peptides and antibodies. We propose Targeted Copper Theranostics (TCTs) which use copper-64 to image tumors and beta-emitting copper-67 to irradiate and kill cancer cells using various molecular targets of oncological relevance. We have evaluated the efficacy of copper-67 based therapy in three formats; peptides, antibodies, and pre-targeting of antibody conjugates in relevant tumor mouse models. Benefits of copper-67 [half-life, 61.8 h; 100% β- energy in the range of 0.18-0.56 MeV] include high purity, high specific activity, manufacturing in a centralized distribution model at high yields on electron accelerators, and half-life suitable to various targeting formats. Copper-64 and copper-67 provide multiple logistical benefits relative to other radionuclides including Ga-68, F-18 and Lu-177. Methods: We evaluated three tumor-targeting moieties conjugated to a sarcophagine bifunctional chelator (SAR), including: a peptide dimer targeting prostate specific membrane antigen (SAR-bisPSMA); an antibody 5B1, targeting the PDAC-specific biomarker carbohydrate antigen-19-9 (CA19-9); and a Tz-TCO IDEEA huA33 antibody/sarcophagine tetrazine conjugate targeting transmembrane glycoprotein A33. Various doses were administered of [67Cu]Cu-SAR-bisPSMA in LNCAP xenografts; [67Cu]Cu-SAR-5B1 (~10 μg) in FC1245 Ft2Ab PDAC tumor-bearing C57BL/6 mice; and [67Cu]Cu-MeCOSAR-Tz in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts 72 hours after administration of huA33-TCO. Results: Administration of either 7.5, 15 or 30 MBq of [67Cu]Cu-SAR-bisPSMA increased overall survival relative to the control group. Similarly, after administration of 4, 11, 18 MBq of [67Cu]Cu-5B1, treated groups enhanced overall survival to 32 days compared to control (14 days). Pre-targeting using ~18.5, 37.0 or 55.5 MBq or fractionated dosing of two 27.8 MBq doses of [67Cu]Cu-MeCOSAR-Tz showed efficient tumor growth reduction with enhanced overall survival for treated mice pre-administered huA33-TCO, from 68 days to >200 days (dose dependent) compared to control (21 days). Conclusion: We report the inhibition of tumor growth with minimal radiotoxicity and each format demonstrated significantly improved overall survival compared to controls. Collectively our results demonstrated that TCTs using the sarcophagine chelator with copper-67 are effective radioconjugates across multiple formats with an acceptable safety profile. Copper-67-based TCTs are being translated in multiple theranostic clinical trials in the USA and Australia. Citation Format: Matt Harris, Kurt Gehlsen, Lachlan McInnes, Jason Lewis. Copper-67 as a next-generation radioconjugate warhead for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6037.

Abstract 439: Tackling oxidative phosphorylation of glioblastoma stem cells through the specific impairment of transmembrane chloride intracellular channel 1

Abstract Solid cancer cells show higher energy and biomolecules requirements compared with healthy cells, making the targeting of cancer metabolic dysfunctions an appealing therapeutic approach. Tackling the tumor metabolism is a daunting task, due to the ability of cancer cells to re-organize their metabolism switching between glycolysis and oxidative phosphorylation. Cancer metabolic plasticity and re-programming is partially supported by altered ionic channels expression and activity. Our laboratory has described the chronic presence transmembrane form of Chloride Intracellular Channel 1 (tmCLIC1) as a marker of malignancy in tumor cells and a possible therapeutic target since it is mainly absent in healthy cells. CLIC1 is described as a metamorphic protein able to shuttle between the cytoplasmic form to a transmembrane conformation. In the present investigation, we show that tmCLIC1 acts as a privileged metformin target in glioblastoma stem-like cells. Metformin is the first-line drug for type 2 diabetes and has been proposed as an antineoplastic drug, but no direct evidence of its’ membrane permeability nor of a specific membrane receptor in cancer cells has been found yet. Here we demonstrate that metformin impairs tmCLIC1 activity by direct and specific binding coordinated by arginine 29. This residue mutation prevents metformin from tmCLIC1 binding and abolishes the drug's effect on glioblastoma cells in 2D and 3D models and its actions on mitochondrial respiration. In vivo experiments in zebrafish embryos and mice orthotopically engrafted with glioblastoma cells and treated with metformin have shown that metformin binding to tmCLIC1 is crucial for its anti-neoplastic effect. Citation Format: Francesca Cianci, Ivan Verduci, Riccardo Cazzoli, Saverio Minucci, Michele Mazzanti. Tackling oxidative phosphorylation of glioblastoma stem cells through the specific impairment of transmembrane chloride intracellular channel 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 439.

Abstract 5750: Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate cancer

Abstract Introduction: Treatment choices for early-stage prostate cancer include mostly watchful waiting, radical prostatectomy, or radiation therapy, whereas for advanced cases hormone therapy followed by chemo/radiotherapy are the preferred options, despite their notable side effects. There is a need for more effective therapy with lower side effects, especially for advanced scenarios. Unlike normal epithelial prostate cells, cancerous ones show reduced zinc (Zn) levels due to ZIP1 zinc transporter downregulation. Zn deficiency allows m-aconitase to remain active, making prostate cancer cells energy efficient and grow. High levels of Zn are needed to inhibit m-aconitase, the enzyme responsible for the first reaction in the Krebs cycle. This study aims to induce energy starvation and cell death of prostate cancer cells by inhibiting m-aconitase using the prostate-specific membrane antigen (PSMA)-targeting liposomal zinc. Methods: Liposomes were prepared by standard reverse phase evaporation method with DSPC, Cholesterol and DSPE-mPEG(2000) lipids. Zinc chloride was used to load Zn+2 into liposome following passive loading method. PSMA-expressing (PC3-PIP) and PSMA-null (PC3-LUC) human prostate cancer cells were used for in vitro studies. In the cell uptake study, cells were incubated with Dil-dye labeled liposomes for 2 hours before analyzing them by microscopy and flow cytometry. For the uptake inhibition study, cells were pre-incubated with PSMA-specific peptide for 2 hours before incubation with liposomes. Cytotoxicity studies were performed with PSMA-targeting Zn-loaded liposomes in PC3-PIP cells. Results: We conjugated the PSMA-specific peptide and control peptide (ASP3) with DSPE-PEG(3400)-NHS, and used them at various amounts (1 to 4 mol% of total lipid) to prepare Zn-loaded PSMA-targeting liposomes (TL) and control liposomes (CL), respectively. The average hydrodynamic diameter of all the liposomes was 120 to 130 nm, and zeta potential ranged from -16 to -12 mV. The encapsulation efficiency and loading capacity of Zn+2 were 0.6% and 8.5%, respectively. Cellular uptake studies showed a higher accumulation of PSMA-targeting liposome (TL) compared to non-targeting liposome (CL) in PSMA-expressing PC3 cells, whereas both liposomes showed low uptake in PSMA non-expressing cell. Furthermore, pre-treatment of PSMA-expressing cells with PSMA-specific peptide reduced the uptake of TL, indicating that the uptake of TL liposome is PSMA dependent. Finally, cytotoxicity studies showed that Zn-loaded TL, induces cell death in the PSMA-expressing cells. Overall, we developed PSMA-targeting Zn-containing liposomes that selectively target and kill PSMA-expressing prostate cancer cells. Future work includes testing the effect of Zn-loaded TL on m-aconitase activity and testing of in vivo efficacy of the formulation in prostate cancer models. Citation Format: Sujan K. Mondal, Alexander L. Klibanov, Anna Moore. Development of prostate specific membrane antigen-targeted liposomal zinc for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5750.

Abstract 7200: Non-cytotoxic inhibition of nuclear encoded mitochondrial protein MAGMAS resensitize treatment resistant prostate cancer cells to taxane therapy

Abstract Metastatic prostate cancer (PCa) is the leading cause of PCa-associated deaths and one of the leading causes of cancer mortality in men. Novel therapeutics, such as Prostate Specific Membrane Antigen (PSMA) ligand therapy, are emerging as effective treatment strategies for metastatic prostate cancer. However, most men succumb to Metastatic Castration Resistant Prostate Cancer (mCRPC) due to treatment resistance. MAGMAS, a 13.8 kDa mitochondrial protein, imports nuclear-encoded mitochondrial proteins into the mitochondrial matrix. MAGMAS is overexpressed in several aggressive tumors, such as breast cancer, glioblastoma, and prostate cancer. When overexpressed, MAGMAS plays a role as a cytoprotective protein by acting as a reactive oxygen species (ROS) scavenger that maintains sufficient ROS levels to promote cell proliferation but below the threshold levels that could lead to apoptosis. This study was designed to investigate the pro-survival and therapy-resistance functions of MAGMAS in treatment-resistant PCa cells. Predictively, we observed that the docetaxel (DTX)-resistant PC3-DR and DU145-DR PCa cell lines expressed significantly higher levels of MAGMAS compared to their drug-sensitive parental cell lines. We hypothesized that inhibiting Magmas reduces the aggressive properties of highly proliferative DTX-resistant metastatic PCa cell lines and re-sensitizes them to DTX. MAGMAS knockdown in DTX-resistant PC3 and DU145 cells in the presence of increasing DTX treatments enhanced sensitivity to DTX. Consistent with these results, pharmacological inhibition of MAGMAS with the novel BT#9 inhibitor in combination with increasing DTX concentrations also enhanced sensitivity to DTX in these cell lines. Additional studies using cell migration/invasion assays and tumor-sphere formation assays are underway to further establish MAGMAS as a potential therapeutic target to attenuate the aggressive properties of chemo-resistant PCa cells. Lastly, we will investigate the diagnostic potential of MAGMAS as a biomarker for advanced PCa and BT#9 as an imaging/therapeutic-specific agent for treatment-resistant tumors. Citation Format: Alfonso Duran, Kristen Whitley, Krystal Santiago, Adil Mohammed, Carlos Casiano, Bhaskar Das, Frankis Almaguel. Non-cytotoxic inhibition of nuclear encoded mitochondrial protein MAGMAS resensitize treatment resistant prostate cancer cells to taxane therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7200.

Modulation of Phospholipase A2 Membrane Activity by Anti-inflammatory Drugs.

The phospholipase A2 (PLA2) superfamily consists of lipolytic enzymes that hydrolyze specific cell membrane phospholipids and have long been considered a central hub of biosynthetic pathways, where their lipid metabolites exert a variety of physiological roles. A misregulated PLA2 activity is associated with mainly inflammatory-derived pathologies and thus has shown relevant therapeutic potential. Many natural and synthetic anti-inflammatory drugs (AIDs) have been proposed as direct modulators of PLA2 activity. However, despite the specific chemical properties that these drugs share in common, little is known about the indirect modulation able to finely tune membrane structural changes at the precise lipid-binding site. Here, we use a novel experimental strategy based on differential scanning calorimetry to systematically study the structural properties of lipid membrane systems during PLA2 cleavage and under the influence of several AIDs. For a better understanding of the AIDs-membrane interaction, we present a comprehensive and comparative set of molecular dynamics (MD) simulations. Our thermodynamic results clearly demonstrate that PLA2 cleavage is hindered by those AIDs that significantly reduce the lipid membrane cooperativity, while the rest of the AIDs oppositely tend to catalyze PLA2 activity to different extents. On the other hand, our MD simulations support experimental results by providing atomistic details on the binding, insertion, and dynamics of each AID on a pure lipid system; the drug efficacy to impact membrane cooperativity is related to the lipid order perturbation. This work suggests a membrane-based mechanism of action for diverse AIDs against PLA2 activity and provides relevant clues that must be considered in its modulation.

The small yeast GTPase Rho5 requires specific mitochondrial outer membrane proteins for translocation under oxidative stress and interacts with the VDAC Por1

Yeast Rho5 is a small GTPase which mediates the response to nutrient and oxidative stress, and triggers mitophagy and apoptosis. We here studied the rapid translocation of a GFP-tagged Rho5 to mitochondria under such stress conditions by live-cell fluorescence microscopy in the background of strains lacking different mitochondrial outer membrane proteins (MOMP). Fun14, Msp1 and Alo1 were found to be required for efficient recruitment of the GTPase, whereas translocation of Dck1 and Lmo1, the subunits of its dimeric GDP/GTP exchange factor (GEF), remained unaffected. An influence of the voltage-dependent anion channel (VDAC) Por1 on the association of GFP-Rho5 with mitochondria under oxidative stress conditions appeared to be strain-dependent. However, epistasis analyses and bimolecular fluorescence complementation (BiFC) studies indicate a genetic and physical interaction. All four strains lacking a single MOMP were investigated for their effect on mitophagy.

Domain-Targeted Membrane Partitioning of Specific Proteins with DNA Nanodevices.

The cell membrane exhibits a remarkable complexity of lipids and proteins that dynamically segregate into distinct domains to coordinate various cellular functions. The ability to manipulate the partitioning of specific membrane proteins without involving genetic modification is essential for decoding various cellular processes but highly challenging. In this work, by conjugating cholesterols or tocopherols at the three bottom vertices of the DNA tetrahedron, we develop two sets of nanodevices for the selective targeting of lipid-order (Lo) and lipid-disorder (Ld) domains on the live cell membrane. By incorporation of protein-recognition ligands, such as aptamers or antibodies, through toehold-mediated strand displacement, these DNA nanodevices enable dynamic translocation of target proteins between these two domains. We first used PTK7 as a protein model and demonstrated, for the first time, that the accumulation of PTK7 to the Lo domains could promote tumor cell migration, while sequestering it in the Ld domains would inhibit the movement of the cells. Next, based on their modular nature, these DNA nanodevices were extended to regulate the process of T cell activation through manipulating the translocation of CD45 between the Lo and the Ld domains. Thus, our work is expected to provide deep insight into the study of membrane structure and molecular interactions within diverse cell signaling processes.

Iridium Oxide Coordinatively Unsaturated Active Sites Govern the Electrocatalytic Oxidation of Water

AbstractA special membrane electrode assembly to measure operando X‐ray absorption spectra and resonant photoemission spectra of mesoporous templated iridium oxide films is used. These films are calcined to different temperatures to mediate the catalyst activity. By combining operando resonant photoemission measurements of different films with ab initio simulations these are able to unambiguously distinguish µ2‐O (bridging oxygen) and µ1‐O (terminal oxygen) in the near‐surface regions of the catalysts. The intrinsic activity of iridium oxide scales with the formation of µ1‐O (terminal oxygen) is found. Importantly, it is shown that the peroxo species do not accumulate under reaction conditions. Rather, the formation of µ1‐O species, which are active in O−O bond formation during the OER, is the most oxidized oxygen species observed, which is consistent with an O−O rate‐limiting step. Thus, the oxygen species taking part in the electrochemical oxidation of water on iridium electrodes are more involved and complex than previously stated. This result highlights the importance of employing theory together with true and complementary operando measurements capable of probing different aspects of catalysts surfaces during operation.

New Insights into Interactions between Mushroom Aegerolysins and Membrane Lipids.

Aegerolysins are a family of proteins that recognize and bind to specific membrane lipids or lipid domains; hence they can be used as membrane lipid sensors. Although aegerolysins are distributed throughout the tree of life, the most studied are those produced by the fungal genus Pleurotus. Most of the aegerolysin-producing mushrooms code also for proteins containing the membrane attack complex/perforin (MACPF)-domain. The combinations of lipid-sensing aegerolysins and MACPF protein partners are lytic for cells harboring the aegerolysin membrane lipid receptor and can be used as ecologically friendly bioinsecticides. In this work, we have recombinantly expressed four novel aegerolysin/MACPF protein pairs from the mushrooms Heterobasidion irregulare, Trametes versicolor, Mucidula mucida, and Lepista nuda, and compared these proteins with the already studied aegerolysin/MACPF protein pair ostreolysin A6-pleurotolysin B from P. ostreatus. We show here that most of these new mushroom proteins can form active aegerolysin/MACPF cytolytic complexes upon aegerolysin binding to membrane sphingolipids. We further disclose that these mushroom aegerolysins bind also to selected glycerophospholipids, in particular to phosphatidic acid and cardiolipin; however, these interactions with glycerophospholipids do not lead to pore formation. Our results indicate that selected mushroom aegerolysins show potential as new molecular biosensors for labelling phosphatidic acid.

Aryl Hydrocarbon Receptor Involvement in the Sodium-Dependent Glutamate/Aspartate Transporter Regulation in Cerebellar Bergmann Glia Cells.

Glutamate, the major excitatory neurotransmitter in the vertebrate brain, exerts its functions through the activation of specific plasma membrane receptors and transporters. Overstimulation of glutamate receptors results in neuronal cell death through a process known as excitotoxicity. A family of sodium-dependent glutamate plasma membrane transporters is responsible for the removal of glutamate from the synaptic cleft, preventing an excitotoxic insult. Glial glutamate transporters carry out more than 90% of the brain glutamate uptake activity and are responsible for glutamate recycling through the GABA/Glutamate/Glutamine shuttle. The aryl hydrocarbon receptor is a ligand-dependent transcription factor that integrates environmental clues through its ability to heterodimerize with different transcription factors. Taking into consideration the fundamental role of glial glutamate transporters in glutamatergic synapses and that these transporters are regulated at the transcriptional, translational, and localization levels in an activity-dependent fashion, in this contribution, we explored the involvement of the aryl hydrocarbon receptor, as a model of environmental integrator, in the regulation of the glial sodium-dependent glutamate/aspartate transporter. Using the model of chick cerebellar Bergmann glia cells, we report herein that the aryl hydrocarbon receptors exert a time-dependent decrease in the transporter mRNA levels and a diminution of its uptake activity. The nuclear factor kappa light chain enhancer of the activated B cell signaling pathway is involved in this regulation. Our results favor the notion of an environmentally dependent regulation of glutamate removal in glial cells and therefore strengthen the notion of the involvement of glial cells in xenobiotic neurotoxic effects.

Transfer of membrane(s) matter(s)-non-genetic inheritance of (metabolic) phenotypes?

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are anchored at the outer phospholipid layer of eukaryotic plasma membranes exclusively by a glycolipid. GPI-APs are not only released into extracellular compartments by lipolytic cleavage. In addition, certain GPI-APs with the glycosylphosphatidylinositol anchor including their fatty acids remaining coupled to the carboxy-terminus of their protein components are also detectable in body fluids, in response to certain stimuli, such as oxidative stress, radicals or high-fat diet. As a consequence, the fatty acid moieties of GPI-APs must be shielded from access of the aqueous environment by incorporation into membranes of extracellular vesicles or into micelle-like complexes together with (lyso)phospholipids and cholesterol. The GPI-APs released from somatic cells and tissues are transferred via those complexes or EVs to somatic as well as pluripotent stem cells with metabolic consequences, such as upregulation of glycogen and lipid synthesis. From these and additional findings, the following hypotheses are developed: i) Transfer of GPI-APs via EVs or micelle-like complexes leads to the induction of new phenotypes in the daughter cells or zygotes, which are presumably not restricted to metabolism. ii) The membrane topographies transferred by the concerted action of GPI-APs and interacting components are replicated by self-organization and self-templation and remain accessible to structural changes by environmental factors. iii) Transfer from mother cells and gametes to their daughter cells and zygotes, respectively, is not restricted to DNA and genes, but also encompasses non-genetic matter, such as GPI-APs and specific membrane constituents. iv) The intergenerational transfer of membrane matter between mammalian organisms is understood as an epigenetic mechanism for phenotypic plasticity, which does not rely on modifications of DNA and histones, but is regarded as molecular mechanism for the inheritance of acquired traits, such as complex metabolic diseases. v) The missing interest in research of non-genetic matter of inheritance, which may be interpreted in the sense of Darwin's "Gemmules" or Galton's "Stirps", should be addressed in future investigations of the philosophy of science and sociology of media.

Self-supervised representation learning of filtration barrier in kidney

While the advance of deep learning has allowed to automate many tasks in bioimage analysis, quantifying key visual features of biological objects in an image, such as cells, organs, or tissues, is still a multi-step and laborious task. It requires image segmentation and definition of features of interest, which often might be image- and problem-specific. This approach requires image labeling and training of the segmentation method as well as manual feature design and implementation of dedicated procedures for their quantification. Here we propose a self-supervised learning (SSL) approach to encoding in microscopy images morphological features of molecular structures that play role in disease phenotype and patient clinical diagnosis. We encode super-resolution images of slit diaphragm (SD)—a specialized membrane between podocyte cells in kidney—in a high-dimensional embedding space in an unsupervised manner, without the need of image segmentation and feature quantification. We inspect the embedding space and demonstrate its relationship to the morphometric parameters of the SD estimated with a previously published method. The SSL-derived image representations additionally reflect the level of albuminuria—a key marker of advancement of kidney disease in a cohort of chronic kidney disease patients. Finally, the embeddings allow for distinguishing mouse model of kidney disease from the healthy subjects with a comparable accuracy to classification based on SD morphometric features. In a one step and label-free manner the SSL approach offers possibility to encode meaningful details in biomedical images and allow for their exploratory, unsupervised analysis as well as further fine-tuning for specialized supervised tasks.

Comparative light and scanning electron microscopic studies of the lenses in the insectivorous bat (Pipistrellus kuhlii) and Egyptian fruit bat (Rousettus aegyptiacus).

Bats have the ability to fly without eye application in the darkness. In this study, we aimed to characterize the functional and structural acclimations of the lenses of two common bats with a various lifestyle in the Egyptian environment: the insectivorous bat (IB) (Pipistrellus kuhlii) and Egyptian fruit bat (FB) (Rousettus aegyptiacus). From each species, seven lenses were extracted from adult eyes. The scanning electron microscopic (SEM) and light microscopic examination of the lens were carried out. FB lenses were made up primarily of fiber cells and sheets, which were encapsulated by a thin collagenous capsule and covered by single epithelial layer anteriorly. On the other hand, the IB lens had two poles and was visibly oval shaped. Both lenses had epithelial cells of the same cuboidal form that were subjected to continuous division and differentiation into new fiber cells at the center. SEM revealed that the normal FB lens had regularly organized shells of fiber cells of intact lens fibers which were connected by membrane interdigitations with different shapes mainly ball-and-socket junctions through the superficial cortical fiber cells. The IB lens was composedof parallel, evenly spaced fibers with various types of interdigitations between fibers that can be seen and increased close to the middle region revealing tiny bumps along the scrubby portions and sockets and balls in the center of the wide portions. Near the center of both lenses, there were large interlocking paddles with little and lengthy protrusions along their short sides. In conclusion, our study discovered several ultrastructural and structural variations among the investigated species. The detection of specialized membrane interdigitations with different shapes protruding from the lens fiber sheets is considered the most characteristic of the FB lens. RESEARCH HIGHLIGHTS: FB lens has more organized sheets of fibers parallel to each other than IB lens. Different shapes of interdigitations protruded from the FB lens have been detected. Interlocking paddles, balls, and sockets with tongue-like fiber flabs are characteristic to FB lens.

Optimization and scale up of production of the PSMA imaging agent [18F]AlF-P16-093 on a custom automated radiosynthesis platform

BackgroundRecent advancements in positron emission tomograph (PET) using prostate specific membrane antigen (PSMA)-targeted radiopharmaceuticals have changed the standard of care for prostate cancer patients by providing more accurate information during staging of primary and recurrent disease. [68Ga]Ga-P16-093 is a new PSMA-PET radiopharmaceutical that demonstrated superior imaging performance in recent head-to-head studies with [68Ga]Ga-PSMA-11. To improve the availability of this new PSMA PET imaging agent, [18F]AlF-P16-093 was developed. The 18F-analog [18F]AlF-P16-093 has been synthesized manually at low activity levels using [18F]AlF2+ and validated in pre-clinical models. This work reports the optimization of the production of > 15 GBq of [18F]AlF-P16-093 using a custom automated synthesis platform.ResultsThe sensitivity of the radiochemical yield of [18F]AlF-P16-093 to reaction parameters of time, temperature and reagent amounts was investigated using a custom automated system. The automated system is a low-cost, cassette-based system designed for 1-pot syntheses with flow-controlled solid phase extraction (SPE) workup and is based on the Raspberry Pi Zero 2 microcomputer/Python3 ecosystem. The optimized none-decay-corrected yield was 52 ± 4% (N = 3; 17.5 ± 2.2 GBq) with a molar activity of 109 ± 14 GBq/µmole and a radiochemical purity of 98.6 ± 0.6%. Run time was 30 min. A two-step sequence was used: SPE-purified [18F]F− was reacted with 80 nmoles of freeze-dried AlCl3·6H2O at 65 °C for 5 min followed by reaction with 160 nmoles of P16-093 ligand at 40 °C for 4 min in a 1:1 mixture of ethanol:0.5 M pH 4.5 NaOAc buffer. The mixture was purified by SPE (> 97% recovery). The final product formulation (5 mM pH 7 phosphate buffer with saline) exhibited a rate of decline in radiochemical purity of ~ 1.4%/h which was slowed to ~ 0.4%/h when stored at 4 °C.ConclusionThe optimized method using a custom automated system enabled the efficient (> 50% none-decay-corrected yield) production of [18F]AlF-P16-093 with high radiochemical purity (> 95%). The method and automation system are simple and robust, facilitating further clinical studies with [18F]AlF-P16-093.

Membrane mimetic-dependence of GPCR energy landscapes

We leveraged variable-temperature 19F-NMR spectroscopy to compare the conformational equilibria of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), across a range of temperatures ranging from lower temperatures typically employed in 19F-NMR experiments to physiological temperature. A2AAR complexes with partial agonists and full agonists showed large increases in the population of a fully active conformation with increasing temperature. NMR data measured at physiological temperature were more in line with functional data. This was pronounced for complexes with partial agonists, where the population of active A2AAR was nearly undetectable at lower temperature but became evident at physiological temperature. Temperature-dependent behavior of complexes with either full or partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. Cellular signaling experiments correlated with the temperature-dependent conformational equilibria of A2AAR in lipid nanodiscs but not in some detergents, underscoring the importance of the membrane environment in studies of GPCR function.

Biochemically recurrent prostate cancer in the era of EMBARK and PSMA PET imaging: everything has changed, except the patients.

Patients with biochemically recurrent prostate cancer (BCR) after unsuccessful curative therapies frequently have an indolent and asymptomatic disease course for years. There are no prospective data showing that treating BCR improves overall survival despite new imaging strategies and emerging therapeutic data. Managing BCR requires a unique perspective in oncology that balances toxicities and disease kinetics. Prostate specific membrane antigen (PSMA) imaging is now widely available and can define subclinical disease in patients with BCR who otherwise have negative CT and bone scans, but limited data exists showing that treating PSMA-positive disease has long term impact. A phase 3 trial demonstrated that the androgen receptor pathway inhibitor enzalutamide either alone or with androgen deprivation therapy (ADT) was superior in delaying metastasis, relative to ADT alone. Survival benefits from this study remain unknown. BCR is a heterogeneous population where overtreatment may present greater risk to patients than a disease course that is often indolent. Management of BCR should be individualized based on disease kinetics. Given the unique biology of BCR, future therapeutic research should emphasize an approach that alters disease trajectory without accompanying side effects and should explore options beyond ADT-based strategies.

The phosphatidylinositol-5' phosphatase synaptojanin1 limits integrin-mediated invasion of Staphylococcus aureus.

The gram-positive bacterium Staphylococcus aureus can invade non-professional phagocytic cells by associating with the plasma protein fibronectin to exploit host cell integrins. Integrin-mediated internalization of these pathogens is facilitated by the local production of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) via an integrin-associated isoform of phosphatidylinositol-5' kinase. In this study, we addressed the role of PI-4,5-P2-directed phosphatases on internalization of S. aureus. ShRNA-mediated knockdown of individual phosphoinositide 5-phosphatases revealed that synaptojanin1 (SYNJ1) is counteracting invasion of S. aureus into mammalian cells. Indeed, shRNA-mediated depletion as well as genetic deletion of synaptojanin1 via CRISPR/Cas9 resulted in a gain-of-function phenotype with regard to integrin-mediated uptake. Surprisingly, the surface level of integrins was slightly downregulated in Synj1-KO cells. Nevertheless, these cells showed enhanced local accumulation of PI-4,5-P2 and exhibited increased internalization of S. aureus. While the phosphorylation level of the integrin-associated protein tyrosine kinase FAK was unaltered, the integrin-binding and -activating protein talin was enriched in the vicinity of S. aureus in synaptojanin1 knockout cells. Scanning electron microscopy revealed enlarged membrane invaginations in the absence of synaptojanin1 explaining the increased capability of these cells to internalize integrin-bound microorganisms. Importantly, the enhanced uptake by Synj1-KO cells and the exaggerated morphological features were rescued by the re-expression of the wild-type enzyme but not phosphatase inactive mutants. Accordingly, synaptojanin1 activity limits integrin-mediated invasion of S. aureus, corroborating the important role of PI-4,5-P2 during this process.IMPORTANCEStaphylococcus aureus, an important bacterial pathogen, can invade non-professional phagocytes by capturing host fibronectin and engaging integrin α5β1. Understanding how S. aureus exploits this cell adhesion receptor for efficient cell entry can also shed light on the physiological regulation of integrins by endocytosis. Previous studies have found that a specific membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), supports the internalization process. Here, we extend these findings and report that the local levels of PIP2 are controlled by the activity of the PIP2-directed lipid phosphatase Synaptojanin1. By dephosphorylating PIP2 at bacteria-host cell attachment sites, Synaptojanin1 counteracts the integrin-mediated uptake of the microorganisms. Therefore, our study not only generates new insight into subversion of cellular receptors by pathogenic bacteria but also highlights the role of host cell proteins acting as restriction factors for bacterial invasion at the plasma membrane.