Development and In Vivo Evaluation of Small-Molecule Ligands for Positron Emission Tomography of Immune Checkpoint Modulation Targeting Programmed Cell Death 1 Ligand 1.

A substantial portion of patients do not benefit from programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) checkpoint inhibition therapies, necessitating a deeper understanding of predictive biomarkers. Immunohistochemistry (IHC) has played a pivotal role in assessing PD-L1 expression, but small-molecule positron emission tomography (PET) tracers could offer a promising avenue to address IHC-associated limitations, i.e., invasiveness and PD-L1 expression heterogeneity. PET tracers would allow for improved quantification of PD-L1 through noninvasive whole-body imaging, thereby enhancing patient stratification. Here, a large series of PD-L1 targeting small molecules were synthesized, leveraging advantageous substructures to achieve exceptionally low nanomolar affinities. Compound 5c emerged as a promising candidate (IC50 = 10.2 nM) and underwent successful carbon-11 radiolabeling. However, a lack of in vivo tracer uptake in xenografts and notable accumulation in excretory organs was observed, underscoring the challenges encountered in small-molecule PD-L1 PET tracer development. The findings, including structure-activity relationships and in vivo biodistribution data, stand to illuminate the path forward for refining small-molecule PD-L1 PET tracers.

Epiberberine induced p53/p21-dependent G2/M cell cycle arrest and cell apoptosis in gastric cancer cells by activating γ-aminobutyric acid receptor- ß3.

BACKGROUND AND PURPOSE: Epiberberine (EPI) is one of the most important bioalkaloid found in the rhizome of Coptis chinensis, which has been observed to exhibit pharmaceutical effects against gastric cancer (GC). Nevertheless, the potential mechanism of EPI against GC cells still remains unclear. This study aimed to identify the core receptor on GC cells through which EPI inhibited the growth of GC cells and to explore the underlying inhibitory mechanisms. METHODS: To identify hub receptor targets that respond to EPI treatment, RNA sequencing (RNA-Seq) data from a tumor-bearing mouse model were analyzed using bioinformatics method and molecular docking. The binding interaction between EPI and GABRB3 was validated through western blotting based-cellular thermal shift assay (WB-CETSA). To further verify the binding region between EPI and GABRB3 through circular dichroism (CD) chromatography, fragments of the extracellular and transmembrane domains of the GABRB3 protein were expressed and purified in vitro. Stable cell lines with the overexpression or knockdown of GABRB3 were established using the recombinant lentivirus system. MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)) assay, colony formation assay, invasion and migration experiments, and flow cytometry were conducted to validate the inhibitory effect of EPI on the GC cells via GABRB3. Additionally, western blotting was utilized to explore the potential inhibitory mechanisms. RESULTS: Through the combination of multiple bioinformatics methods and molecular docking, we found that the γ-aminobutyric acid type A receptor subunit -ß3 (GABRB3) might be the critical receptor target in response to EPI treatment. The results of WB-CETSA analysis indicated that EPI significantly promoted the thermostability of the GABRB3 protein. Importantly, EPI could directly bind to GABRB3 and alter the secondary structure of GABRB3 fragments similar to the natural agonist, γ-aminobutyric acid (GABA). The EPI-induced suppression of the malignant phenotype of GC cells was dependent on the presence of GABRB3. GABRB3 expression was positively correlated with TP53 in patients with GC. The binding of EPI to GABRB3 stimulated p53 accumulation in GC cells. This activated the p21/CDK1/cyclinB1 pathway, resulting in G2/M cell cycle arrest, and induced the Bcl-2/BAX/Caspase axis-dependent cell apoptosis. CONCLUSION: This study revealed the target receptor for EPI in GC cells and provided new insights into its anticancer mechanisms.

KMT2C methyltransferase domain regulated INK4A expression suppresses prostate cancer metastasis.

BACKGROUND: Frequent truncation mutations of the histone lysine N-methyltransferase KMT2C have been detected by whole exome sequencing studies in various cancers, including malignancies of the prostate. However, the biological consequences of these alterations in prostate cancer have not yet been elucidated. METHODS: To investigate the functional effects of these mutations, we deleted the C-terminal catalytic core motif of Kmt2c specifically in mouse prostate epithelium. We analysed the effect of Kmt2c SET domain deletion in a Pten-deficient PCa mouse model in vivo and of truncation mutations of KMT2C in a large number of prostate cancer patients. RESULTS: We show here for the first time that impaired KMT2C methyltransferase activity drives proliferation and PIN formation and, when combined with loss of the tumour suppressor PTEN, triggers loss of senescence, metastatic dissemination and dramatically reduces life expectancy. In Kmt2c-mutated tumours we show enrichment of proliferative MYC gene signatures and loss of expression of the cell cycle repressor p16INK4A. In addition, we observe a striking reduction in disease-free survival of patients with KMT2C-mutated prostate cancer. CONCLUSIONS: We identified truncating events of KMT2C as drivers of proliferation and PIN formation. Loss of PTEN and KMT2C in prostate cancer results in loss of senescence, metastatic dissemination and reduced life expectancy. Our data demonstrate the prognostic significance of KMT2C mutation status in prostate cancer patients. Inhibition of the MYC signalling axis may be a viable treatment option for patients with KMT2C truncations and therefore poor prognosis.

Carbon Microtube Textile with MoS2 Nanosheets Grown on Both Outer and Inner Walls as Multifunctional Interlayer for Lithium-Sulfur Batteries.

The shuttle effect of soluble lithium polysulfides during the charge/discharge process is the key bottleneck hindering the practical application of lithium-sulfur batteries. Herein, a multifunctional interlayer is developed by growing metallic molybdenum disulfide nanosheets on both outer and inner walls of cotton cloth derived carbon microtube textile (MoS2@CMT). The hollow structure of CMT provides channels to favor electrolyte penetration, Li+ diffusion and restrains polysulfides via physical confinement. The hydrophilic and conductive 1T-MoS2 nanosheets facilitate chemisorption and kinetic behavior of polysulfides. The synergic effect of 1T-MoS2 nanosheets and CMT affords the MoS2@CMT interlayer with an efficient trapping-diffusion-conversion ability toward polysulfides. Therefore, the cell with the MoS2@CMT interlayer exhibits enhanced cycling life (765 mAh g-1 after 500 cycles at 0.5 C) and rate performance (974 mAh g-1 at 2 C and 740 mAh g-1 at 5 C). This study presents a pathway to develop low-cost multifunctional interlayers for advanced lithium-sulfur batteries.

Lmo3 deficiency in the mouse is associated with alterations in mood-related behaviors and a depression-biased amygdala transcriptome.

The highly conserved transcription factor LIM-only 3 (Lmo3) is involved in important neurodevelopmental processes in several brain areas including the amygdala, a central hub for the generation and regulation of emotions. Accordingly, a role for Lmo3 in the behavioral responses to ethanol and in the display of anxiety-like behavior in mice has been demonstrated while the potential involvement of Lmo3 in the control of mood-related behavior has not yet been explored. Using a mouse model of Lmo3 depletion (Lmo3z), we here report that genetic Lmo3 deficiency is associated with altered performance in behavioral paradigms assessing anxiety-like and depression-like traits and additionally accompanied by impairments in learned fear. Importantly, long-term potentiation (LTP) in the basolateral amygdala (BLA), a proposed cellular correlate of fear learning, is impaired in Lmo3z mice. RNA-Seq analysis of BLA tissue and gene set enrichment analysis (GSEA) of differentially expressed genes in Lmo3z mice reveals a significant overlap between genes overexpressed in Lmo3z mice and those enriched in the amygdala of a cohort of patients suffering from major depressive disorder. Consequently, we propose that Lmo3 may play a role in the regulation of gene networks that are relevant to the regulation of emotions. Future work may aid to further explore the role of Lmo3 in the pathophysiology of affective disorders and its genetic foundations.

Waste cotton cloth derived carbon microtube textile: a robust and scalable interlayer for lithium-sulfur batteries.

A robust carbonized cotton cloth interlayer composed of numerous knitted hollow carbon microtubes is simply derived from waste cotton cloth by scalable carbonization. The interlayer acts as an upper current collector and a lithium polysulfide barrier simultaneously, thus greatly improving the electrochemical performances of the lithium-sulfur batteries.

A role for miR-132 in learned safety.

Learned safety is a fear inhibitory mechanism, which regulates fear responses, promotes episodes of safety and generates positive affective states. Despite its potential as experimental model for several psychiatric illnesses, including post-traumatic stress disorder and depression, the molecular mechanisms of learned safety remain poorly understood, We here investigated the molecular mediators of learned safety, focusing on the characterization of miRNA expression in the basolateral amygdala (BLA). Comparing levels of 22 miRNAs in learned safety and learned fear trained mice, six safety-related miRNAs, including three members of the miR-132/-212 family, were identified. A gain-of-function approach based upon in-vivo transfection of a specific miRNA mimic, and miR-132/212 knock-out mice as loss-of-function tool were used in order to determine the relevance of miR-132 for learned safety at the behavioral and the neuronal functional levels. Using a designated bioinformatic approach, PTEN and GAT1 were identified as potential novel miR-132 target genes and further experimentally validated. We here firstly provide evidence for a regulation of amygdala miRNA expression in learned safety and propose miR-132 as signature molecule to be considered in future preclinical and translational approaches testing the transdiagnostic relevance of learned safety as intermediate phenotype in fear and stress-related disorders.

Enhanced synaptic plasticity and spatial memory in female but not male FLRT2-haplodeficient mice.

The Fibronectin Leucine-Rich Transmembrane protein 2 (FLRT2) has been implicated in several hormone -and sex-dependent physiological and pathological processes (including chondrogenesis, menarche and breast cancer); is known to regulate developmental synapses formation, and is expressed in the hippocampus, a brain structure central for learning and memory. However, the role of FLRT2 in the adult hippocampus and its relevance in sex-dependent brain functions remains unknown. We here used adult single-allele FLRT2 knockout (FLRT2+/-) mice and behavioral, electrophysiological, and molecular/biological assays to examine the effects of FLRT2 haplodeficiency on synaptic plasticity and hippocampus-dependent learning and memory. Female and male FLRT2+/- mice presented morphological features (including body masses, brain shapes/weights, and brain macroscopic cytoarchitectonic organization), indistinguishable from their wild type counterparts. However, in vivo examinations unveiled enhanced hippocampus-dependent spatial memory recall in female FLRT2+/- animals, concomitant with augmented hippocampal synaptic plasticity and decreased levels of the glutamate transporter EAAT2 and beta estrogen receptors. In contrast, male FLRT2+/- animals exhibited deficient memory recall and decreased alpha estrogen receptor levels. These observations propose that FLRT2 can regulate memory functions in the adulthood in a sex-specific manner and might thus contribute to further research on the mechanisms linking sexual dimorphism and cognition.

Sustained consumption of cocoa-based dark chocolate enhances seizure-like events in the mouse hippocampus.

While the consumption of caffeine and cocoa has been associated with a variety of health benefits to humans, some authors have proposed that excessive caffeine intake may increase the frequency of epileptic seizures in humans and reduce the efficiency of antiepileptic drugs. Little is known, however, about the proconvulsant potential of the sustained, excessive intake of cocoa on hippocampal neural circuits. Using the mouse as an experimental model, we examined the effects of the chronic consumption of food enriched in cocoa-based dark chocolate on motor and mood-related behaviours as well as on the excitability properties of hippocampal neurons. Cocoa food enrichment did not affect body weights or mood-related behaviours but rather promoted general locomotion and improved motor coordination. However, ex vivo electrophysiological analysis revealed a significant enhancement in seizure-like population spike bursting at the neurogenic dentate gyrus, which was paralleled by a significant reduction in the levels of GABA-α1 receptors thus suggesting that an excessive dietary intake of cocoa-enriched food might alter some of the synaptic elements involved in epileptogenesis. These data invite further multidisciplinary research aiming to elucidate the potential deleterious effects of chocolate abuse on behaviour and brain hyperexcitability.

The brain-tumor related protein podoplanin regulates synaptic plasticity and hippocampus-dependent learning and memory.

INTRODUCTION: Podoplanin is a cell-surface glycoprotein constitutively expressed in the brain and implicated in human brain tumorigenesis. The intrinsic function of podoplanin in brain neurons remains however uncharacterized. MATERIALS AND METHODS: Using an established podoplanin-knockout mouse model and electrophysiological, biochemical, and behavioral approaches, we investigated the brain neuronal role of podoplanin. RESULTS: Ex-vivo electrophysiology showed that podoplanin deletion impairs dentate gyrus synaptic strengthening. In vivo, podoplanin deletion selectively impaired hippocampus-dependent spatial learning and memory without affecting amygdala-dependent cued fear conditioning. In vitro, neuronal overexpression of podoplanin promoted synaptic activity and neuritic outgrowth whereas podoplanin-deficient neurons exhibited stunted outgrowth and lower levels of p-Ezrin, TrkA, and CREB in response to nerve growth factor (NGF). Surface Plasmon Resonance data further indicated a physical interaction between podoplanin and NGF. DISCUSSION: This work proposes podoplanin as a novel component of the neuronal machinery underlying neuritogenesis, synaptic plasticity, and hippocampus-dependent memory functions. The existence of a relevant cross-talk between podoplanin and the NGF/TrkA signaling pathway is also for the first time proposed here, thus providing a novel molecular complex as a target for future multidisciplinary studies of the brain function in the physiology and the pathology. Key messages Podoplanin, a protein linked to the promotion of human brain tumors, is required in vivo for proper hippocampus-dependent learning and memory functions. Deletion of podoplanin selectively impairs activity-dependent synaptic strengthening at the neurogenic dentate-gyrus and hampers neuritogenesis and phospho Ezrin, TrkA and CREB protein levels upon NGF stimulation. Surface plasmon resonance data indicates a physical interaction between podoplanin and NGF. On these grounds, a relevant cross-talk between podoplanin and NGF as well as a role for podoplanin in plasticity-related brain neuronal functions is here proposed.

Sorting nexin 12 interacts with BACE1 and regulates BACE1-mediated APP processing.

BACKGROUND: ß-site APP cleaving enzyme 1 (BACE1) cleaves ß-amyloid precursor protein (APP) to initiate the production of ß-amyloid (Aß), the prime culprit in Alzheimer's disease (AD). Dysregulation of the intracellular trafficking of BACE1 may affect Aß generation, contributing to AD pathology. In this study, we investigated whether BACE1 trafficking and BACE1-mediated APP processing/Aß generation are affected by sorting nexin 12 (SNX12), a member of the sorting nexin (SNX) family that is involved in protein trafficking regulation. RESULTS: Herein, we find that SNX12 is widely expressed in brain tissues and is mainly localized in the early endosomes. Overexpression of SNX12 does not affect the steady-state levels of APP, BACE1 or γ-secretase components, but dramatically reduces the levels of Aß, soluble APPß and APP ß-carboxyl terminal fragments. Downregulation of SNX12 has the opposite effects. Modulation of SNX12 levels does not affect γ-secretase activity or in vitro ß-secretase activity. Further studies reveal that SNX12 interacts with BACE1 and downregulation of SNX12 accelerates BACE1 endocytosis and decreases steady-state level of cell surface BACE1. Finally, we find that the SNX12 protein level is dramatically decreased in the brain of AD patients as compared to that of controls. CONCLUSION: This study demonstrates that SNX12 can regulate the endocytosis of BACE1 through their interaction, thereby affecting ß-processing of APP for Aß production. The reduced level of SNX12 in AD brains suggests that an alteration of SNX12 may contribute to AD pathology. Therefore, inhibition of BACE1-mediated ß-processing of APP by regulating SNX12 might serve as an alternative strategy in developing an AD intervention.