Characterisation of LPS+ bacterial extracellular vesicles along the gut-hepatic portal vein-liver axis.

Gut microbiome dysbiosis is a major contributing factor to several pathological conditions. However, the mechanistic understanding of the communication between gut microbiota and extra-intestinal organs remains largely elusive. Extracellular vesicles (EVs), secreted by almost every form of life, including bacteria, could play a critical role in this inter-kingdom crosstalk and are the focus of present study. Here, we present a novel approach for isolating lipopolysaccharide (LPS)+ bacterial extracellular vesicles (bEVLPS) from complex biological samples, including faeces, plasma and the liver from lean and diet-induced obese (DIO) mice. bEVLPS were extensively characterised using nanoparticle tracking analyses, immunogold labelling coupled with transmission electron microscopy, flow cytometry, super-resolution microscopy and 16S sequencing. In liver tissues, the protein expressions of TLR4 and a few macrophage-specific biomarkers were assessed by immunohistochemistry, and the gene expressions of inflammation-related cytokines and their receptors (n = 89 genes) were measured using a PCR array. Faecal samples from DIO mice revealed a remarkably lower concentration of total EVs but a significantly higher percentage of LPS+ EVs. Interestingly, DIO faecal bEVLPS showed a higher abundance of Proteobacteria by 16S sequencing. Importantly, in DIO mice, a higher number of total EVs and bEVLPS consistently entered the hepatic portal vein and subsequently reached the liver, associated with increased expression of TLR4, macrophage markers (F4/80, CD86 and CD206), cytokines and receptors (Il1rn, Ccr1, Cxcl10, Il2rg and Ccr2). Furthermore, a portion of bEVLPS escaped liver and entered the peripheral circulation. In conclusion, bEV could be the key mediator orchestrating various well-established biological effects induced by gut bacteria on distant organs.

Three water-soluble copper(II) N-heterocyclic carbene complexes: toward copper-catalyzed ketone reduction under sustainable conditions.

A series of tridentate copper(II) N-heterocyclic carbene (NHC) complexes with imidazole, benzimidazole, and 5,6-dimethylbenzimidazole azole rings were synthesized and comprehensively characterized via X-ray crystallography, ESI-MS, cyclic voltammetry, and UV-Vis and EPR spectroscopic studies. These complexes were then utilized for the optimization of ketone reduction under sustainable conditions using 2-acetylpyridine and phenylsilane. The relationships between product formation, temperature, reaction time, and catalyst loading for the hydrogenation reactions are covered in detail. Reduction of eighteen different aliphatic, cyclic, and aromatic ketones were demonstrated, which were compatible to produce the corresponding products in moderate to good yields. These systems were used to develop related DNA-hybrid catalytic systems, but only supported weak enantioselectivity. Further thermodynamic experiments showed Cu-NHC complexes did not demonstrate specific binding to DNA, which is consistent with their limited selectivity.

A Liquid Biopsy-Based Approach to Isolate and Characterize Adipose Tissue-Derived Extracellular Vesicles from Blood.

Obesity is a major risk factor for multiple chronic diseases. Anthropometric and imaging approaches are primarily used to assess adiposity, and there is a dearth of techniques to determine the changes in adipose tissue (AT) at the molecular level. Extracellular vesicles (EVs) have emerged as a novel and less invasive source of biomarkers for various pathologies. Furthermore, the possibility of enriching cell or tissue-specific EVs from the biofluids based on their unique surface markers has led to classifying these vesicles as "liquid biopsies", offering valuable molecular information on hard-to-access tissues. Here, we isolated small EVs from AT (sEVAT) of lean and diet-induced obese (DIO) mice, identified unique surface proteins on sEVAT by surface shaving followed by mass spectrometry, and developed a signature of five unique proteins. Using this signature, we pulled out sEVAT from the blood of mice and validated the specificity of isolated sEVAT by measuring the expression of adiponectin, 38 adipokines on an array, and several adipose tissue-related miRNAs. Furthermore, we provided evidence of sEV applicability in disease prediction by characterizing sEVAT from the blood of lean and DIO mice. Interestingly, sEVAT-DIO cargo showed a stronger pro-inflammatory effect on THP1 monocytes compared to sEVAT-Lean and a significant increase in obesity-associated miRNA expression. Equally important, sEVAT cargo revealed an obesity-associated aberrant amino acid metabolism that was subsequently validated in the corresponding AT. Lastly, we show a significant increase in inflammation-related molecules in sEVAT isolated from the blood of nondiabetic obese (>30 kg/m2) individuals. Overall, the present study offers a less-invasive approach to characterize AT.

MicroRNA expression in extracellular vesicles as a novel blood-based biomarker for Alzheimer's disease.

INTRODUCTION: Brain cell-derived small extracellular vesicles (sEVs) in blood offer unique cellular and molecular information related to the onset and progression of Alzheimer's disease (AD). We simultaneously enriched six specific sEV subtypes from the plasma and analyzed a selected panel of microRNAs (miRNAs) in older adults with/without cognitive impairment. METHODS: Total sEVs were isolated from the plasma of participants with normal cognition (CN; n = 11), mild cognitive impairment (MCI; n = 11), MCI conversion to AD dementia (MCI-AD; n = 6), and AD dementia (n = 11). Various brain cell-derived sEVs (from neurons, astrocytes, microglia, oligodendrocytes, pericytes, and endothelial cells) were enriched and analyzed for specific miRNAs. RESULTS: miRNAs in sEV subtypes differentially expressed in MCI, MCI-AD, and AD dementia compared to the CN group clearly distinguished dementia status, with an area under the curve (AUC) > 0.90 and correlated with the temporal cortical region thickness on magnetic resonance imaging (MRI). DISCUSSION: miRNA analyses in specific sEVs could serve as a novel blood-based molecular biomarker for AD. HIGHLIGHTS: Multiple brain cell-derived small extracellular vesicles (sEVs) could be isolated simultaneously from blood. MicroRNA (miRNA) expression in sEVs could detect Alzheimer's disease (AD) with high specificity and sensitivity. miRNA expression in sEVs correlated with cortical region thickness on magnetic resonance imaging (MRI). Altered expression of miRNAs in sEVCD31 and sEVPDGFRß suggested vascular dysfunction. miRNA expression in sEVs could predict the activation state of specific brain cell types.

The ß-Secretase 1 Enzyme as a Novel Therapeutic Target for Prostate Cancer.

Recent studies have demonstrated the association of APP and Aß with cancer, suggesting that BACE1 may play an important role in carcinogenesis. In the present study, we assessed BACE1's usefulness as a therapeutic target in prostate cancer (PCa). BACE1 expression was observed in human PCa tissue samples, patient-derived xenografts (PDX), human PCa xenograft tissue in nude mice, and transgenic adenocarcinoma of the mouse prostate (TRAMP) tissues by immunohistochemistry (IHC) analysis. Additionally, the downstream product of BACE1 activity, i.e., Aß1-42 expression, was also observed in these PCa tissues by IHC as well as by PET imaging in TRAMP mice. Furthermore, BACE1 gene expression and activity was confirmed in several established PCa cell lines (LNCaP, C4-2B-enzalutamide sensitive [S], C4-2B-enzalutamide resistant [R], 22Rv1-S, 22Rv1-R, PC3, DU145, and TRAMP-C1) by real-time PCR and fluorometric assay, respectively. Treatment with a pharmacological inhibitor of BACE1 (MK-8931) strongly reduced the proliferation of PCa cells in in vitro and in vivo models, analyzed by multiple assays (MTT, clonogenic, and trypan blue exclusion assays and IHC). Cell cycle analyses revealed an increase in the sub-G1 population and a significant modulation in other cell cycle stages (G1/S/G2/M) following MK-8931 treatment. Most importantly, in vivo administration of MK-8931 intraperitoneal (30 mg/kg) strongly inhibited TRAMP-C1 allograft growth in immunocompetent C57BL/6 mice (approximately 81% decrease, p = 0.019). Furthermore, analysis of tumor tissue using the prostate cancer-specific pathway array revealed the alteration of several genes involved in PCa growth and progression including Forkhead O1 (FOXO1). All together, these findings suggest BACE1 as a novel therapeutic target in advanced PCa.

Small extracellular vesicles in plasma reveal molecular effects of modified Mediterranean-ketogenic diet in participants with mild cognitive impairment.

Extracellular vesicles have emerged as a less-invasive nano-tool for discovering biomarkers of Alzheimer's disease and related dementia. Here, we analysed different neuron-enriched extracellular vesicles from plasma to predict response and molecular mechanisms of ketogenic diet's efficacy in mild cognitive impairment participants. The study was a randomized crossover design in which cognitively normal and mild cognitive impairment participants consumed a modified Mediterranean-ketogenic diet or American Heart Association diet for 6 weeks, followed by other diet after washout. L1 cell adhesion molecule, synaptophysin and neural cell adhesion molecule surface markers were used to enrich for neuron-secreted small extracellular vesicles (sEVL1CAM, sEVSYP and sEVNCAM). For the first time, we have presented multiple evidences, including immunogold labelling/transmission electron microscopy, clusters of differentiation 63-ELISA-based assay, confocal microscopy fluorescent images and flow cytometry data confirming the presence of L1 cell adhesion molecule on the surface of sEVL1CAM, validating purity and relative abundance of sEVL1CAM in the plasma. Cargo analysis of sEVL1CAM showed that modified Mediterranean-ketogenic diet intervention reduces amyloid beta 1-42 (50.3%, P = 0.011), p181-tau (34.9%, P = 0.033) and neurofilament light (54.2%, P = 0.020) in mild cognitive impairment participants. Moreover, sEVL1CAMshowed better sensitivity compared with CSF in analysing increased glutamate (6-folds, P < 0.0001) from mild cognitive impairment participants following modified Mediterranean-ketogenic diet intervention. sEVL1CAM characterization also suggested that modified Mediterranean-ketogenic diet differentially targets the expression of various glutamate receptors-glutamate receptor ionotropic NMDA1, glutamate receptor ionotropic NMDA2A, glutamate receptor ionotropic NMDA2B and glutamate receptor ionotropic AMPA type subunit 1. Importantly, these sEVL1CAM measures strongly correlated with corresponding clinical CSF biomarkers (neurogranin, amyloid beta 1-42, neurofilament light and tau). Furthermore, sEVL1CAM were loaded with less advanced glycation endproducts and exhibited anti-inflammatory activity following modified Mediterranean-ketogenic diet intervention. Most importantly, the expression of monocarboxylate transporter 2 on the surface of sEVL1CAM predicted the amyloid beta 1-42 response to modified Mediterranean-ketogenic diet intervention (area under the curve = 0.87, P = 0.0044) and offered a novel screening tool to identify participants responsive to this dietary intervention. Finally, sEVL1CAM, sEVSYP and sEVNCAM showed significantly high concordance in analysing amyloid beta 1-42 (Pearson correlation coefficient ≥ 0.63, P < 0.01) and neurofilament light (Pearson correlation coefficient ≥ 0.49, P < 0.05). Together, small extracellular vesicles in plasma offers promise in assessing the efficacy of dietary/therapeutic intervention against mild cognitive impairment/Alzheimer's disease.

Role of extracellular vesicles secretion in paclitaxel resistance of prostate cancer cells.

Aim: The development of chemotherapy resistance is the major obstacle in the treatment of advanced prostate cancer (PCa). Extracellular vesicles (EVs) secretion plays a significant role among different mechanisms contributing to chemoresistance. Hence, inhibition of EVs release may increase the efficacy of chemotherapeutic drugs against PCa. Methods: Paclitaxel (PTX) resistant PCa cells (PC3-R and DU145-R) were treated with GW4869, a known exosome biogenesis inhibitor. EVs were isolated from the conditioned media by ExoQuick-based precipitation method and characterized for concentration and size distribution by nanoparticle tracking analysis. The effect of GW4869 treatment on the survival and growth of PCa cells was assessed by MTT, and colony formation assays in vitro, and ectopic PC3-R xenografts in male athymic nude mice in vivo. The effect of other EV biogenesis inhibitors, imipramine and dimethyl amiloride (DMA), treatment was also analyzed on the survival of PC3-R cells. Results: GW4869 (10-20 µM) treatment of PTX resistant PCa cells significantly reduced the release of small EVs (50-100 nm size range) while increasing the release of larger EVs (> 150 nm in size), and inhibited their clonogenicity. Moreover, GW4869 (5-20 µM) treatment (24-72h) significantly inhibited the survival of PC3-R cells in a dose-dependent manner. We observed a similar growth inhibition with both imipramine (5-20 µg/mL) and DMA (5-20 µg/mL) treatment in PC3-R cells. Furthermore, GW4869 treatment (IP) in mice bearing PC3-R xenografts significantly reduced the tumor weight (65% reduction, P = 0.017) compared to the vehicle-treated control mice without causing any noticeable toxicity. Conclusion: Inhibiting the release of EVs could sensitize the resistant PCa cells to chemotherapy.

Leptin modulated microRNA-628-5p targets Jagged-1 and inhibits prostate cancer hallmarks.

MicroRNAs (miRNAs) are single-stranded non-coding RNA molecules that play a regulatory role in gene expression and cancer cell signaling. We previously identified miR-628-5p (miR-628) as a potential biomarker in serum samples from men with prostate cancer (PCa) (Srivastava et al. in Tumour Biol 35:4867-4873, 10.1007/s13277-014-1638-1, 2014). This study examined the detailed cellular phenotypes and pathways regulated by miR-628 in PCa cells. Since obesity is a significant risk factor for PCa, and there is a correlation between levels of the obesity-associated hormone leptin and PCa development, here we investigated the functional relationship between leptin and miR-628 regulation in PCa. We demonstrated that exposure to leptin downregulated the expression of miR-628 and increased cell proliferation/migration in PCa cells. We next studied the effects on cancer-related phenotypes in PCa cells after altering miR-628 expression levels. Enforced expression of miR-628 in PCa cells inhibited cell proliferation, reduced PCa cell survival/migration/invasion/spheroid formation, and decreased markers of cell stemness. Mechanistically, miR-628 binds with the JAG1-3'UTR and inhibits the expression of Jagged-1 (JAG1). JAG1 inhibition by miR-628 downregulated Notch signaling, decreased the expression of Snail/Slug, and modulated epithelial-mesenchymal transition and invasiveness in PC3 cells. Furthermore, expression of miR-628 in PCa cells increased sensitivity towards the drugs enzalutamide and docetaxel by induction of cell apoptosis. Collectively our data suggest that miR-628 is a key regulator of PCa carcinogenesis and is modulated by leptin, offering a novel therapeutic opportunity to inhibit the growth of advanced PCa.

Copper(II) NHC Catalyst for the Formation of Phenol from Arylboronic Acid.

Arylboronic acids are commonly used in modern organic chemistry to form new C-C and C-heteroatom bonds. These activated organic synthons show reactivity with heteroatoms in a range of substrates under ambient oxidative conditions. This broad reactivity has limited their use in protic, renewable solvents like water, ethanol, and methanol. Here, we report our efforts to study and optimize the activation of arylboronic acids by a copper(II) N-heterocyclic carbene (NHC) complex in aqueous solution and in a range of alcohols to generate phenol and aryl ethers, respectively. The optimized reactivity showcases the ability to make targeted C-O bonds, but also identifies conditions where water and alcohol activation could be limiting for C-C and C-heteroatom bond-forming reactions. This copper(II) complex shows strong reactivity toward arylboronic acid activation in aqueous medium at ambient temperature. The relationship between product formation and temperature and catalyst loading are described. Additionally, the effects of buffer, pH, base, and co-solvent are explored with respect to phenol and ether generation reactions. Characterization of the new copper(II) NCN-pincer complex by X-ray crystallography, HR-MS, cyclic voltammetry, FT-IR and UV-Vis spectral studies is reported.

Carnitine Palmitoyltransferase 1 Regulates Prostate Cancer Growth under Hypoxia.

Hypoxia and hypoxia-related biomarkers are the major determinants of prostate cancer (PCa) aggressiveness. Therefore, a better understanding of molecular players involved in PCa cell survival under hypoxia could offer novel therapeutic targets. We previously reported a central role of mitochondrial protein carnitine palmitoyltransferase (CPT1A) in PCa progression, but its role in regulating PCa survival under hypoxia remains unknown. Here, we employed PCa cells (22Rv1 and MDA-PCa-2b) with knockdown or overexpression of CPT1A and assessed their survival under hypoxia, both in cell culture and in vivo models. The results showed that CPT1A knockdown in PCa cells significantly reduced their viability, clonogenicity, and sphere formation under hypoxia, while its overexpression increased their proliferation, clonogenicity, and sphere formation. In nude mice, 22Rv1 xenografts with CPT1A knockdown grew significantly slower compared to vector control cells (~59% reduction in tumor volume at day 29). On the contrary, CPT1A-overexpressing 22Rv1 xenografts showed higher tumor growth compared to vector control cells (~58% higher tumor volume at day 40). Pathological analyses revealed lesser necrotic areas in CPT1A knockdown tumors and higher necrotic areas in CPT1A overexpressing tumors. Immunofluorescence analysis of tumors showed that CPT1A knockdown strongly compromised the hypoxic areas (pimonidazole+), while CPT1A overexpression resulted in more hypoxia areas with strong expression of proliferation biomarkers (Ki67 and cyclin D1). Finally, IHC analysis of tumors revealed a significant decrease in VEGF or VEGF-D expression but without significant changes in biomarkers associated with microvessel density. These results suggest that CPT1A regulates PCa survival in hypoxic conditions and might contribute to their aggressiveness.