Airway management of postburn neck contracture in pediatric patient - A challenge for anesthesiologist!

Pediatric difficult airway management is more challenging for an anesthesiologist due to anatomical and physiological differences as compared to adults. Moreover, the familiarity with the use of difficult airway equipment in adults does not equate to proficiency for the same in children. So, here we are presenting the management of a unique case of a difficult airway due to postburn neck contracture in a 4-year-old child, which was managed successfully with the help of a video laryngoscope after the failure attempt with a flexible fiberoptic bronchoscope.

Emergence of Extracellular Vesicles as "Liquid Biopsy" for Neurological Disorders: Boom or Bust.

Extracellular vesicles (EVs) have emerged as an attractive liquid biopsy approach in the diagnosis and prognosis of multiple diseases and disorders. The feasibility of enriching specific subpopulations of EVs from biofluids based on their unique surface markers has opened novel opportunities to gain molecular insight from various tissues and organs, including the brain. Over the past decade, EVs in bodily fluids have been extensively studied for biomarkers associated with various neurological disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, bipolar disorder, major depressive disorders, substance use disorders, human immunodeficiency virus-associated neurocognitive disorder, and cancer/treatment-induced neurodegeneration. These studies have focused on the isolation and cargo characterization of either total EVs or brain cells, such as neuron-, astrocyte-, microglia-, oligodendrocyte-, pericyte-, and endothelial-derived EVs from biofluids to achieve early diagnosis and molecular characterization and to predict the treatment and intervention outcomes. The findings of these studies have demonstrated that EVs could serve as a repetitive and less invasive source of valuable molecular information for these neurological disorders, supplementing existing costly neuroimaging techniques and relatively invasive measures, like lumbar puncture. However, the initial excitement surrounding blood-based biomarkers for brain-related diseases has been tempered by challenges, such as lack of central nervous system specificity in EV markers, lengthy protocols, and the absence of standardized procedures for biological sample collection, EV isolation, and characterization. Nevertheless, with rapid advancements in the EV field, supported by improved isolation methods and sensitive assays for cargo characterization, brain cell-derived EVs continue to offer unparallel opportunities with significant translational implications for various neurological disorders. SIGNIFICANCE STATEMENT: Extracellular vesicles present a less invasive liquid biopsy approach in the diagnosis and prognosis of various neurological disorders. Characterizing these vesicles in biofluids holds the potential to yield valuable molecular information, thereby significantly impacting the development of novel biomarkers for various neurological disorders. This paper has reviewed the methodology employed to isolate extracellular vesicles derived from various brain cells in biofluids, their utility in enhancing the molecular understanding of neurodegeneration, and the potential challenges in this research field.

Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EVISF ) reveals sex-dependent changes in microglial EV proteome in response to Aß pathology.

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimer's disease (AD), relaying important physiological information about their host tissues. The internal cargo of EVs is protected from degradation, making EVs attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EVISF ) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labelling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EVISF cargo. EVISF were 40-150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g., APPswe, PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with Aß deposition. Genotype, age, and Aß deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EVISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse- EVISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EVISF and offers a unique opportunity to explore the role of EVs in AD.

Chymase in Plasma and Urine Extracellular Vesicles: Novel Biomarkers for Primary Hypertension.

BACKGROUND: Extracellular vesicles (EVs) have emerged as a promising liquid biopsy for various diseases. For the first time, using plasma and urinary EVs, we assessed the activity of renin-angiotensin system (RAS), a central regulator of renal, cardiac, and vascular physiology, in patients with control (Group I) or uncontrolled (Group II) primary hypertension. METHODS: EVs were isolated from 34 patients with history of hypertension, and characterized for size and concentration by nanoparticle tracking analyses, exosomal biomarkers by immunogold labeling coupled with transmission electron microscopy, flow cytometry and immunoblotting. EVs were analyzed for the hydrolytic activity of chymase, angiotensin converting enzyme (ACE), ACE2, and neprilysin (NEP) by HPLC. RESULTS: Plasma and urinary EVs were enriched for small EVs and expressed exosomal markers (CD63, CD9, and CD81). The size of urinary EVs (but not plasma EVs) was significantly larger in Group II compared to Group I. Differential activity of RAS enzymes was observed, with significantly higher chymase activity compared to ACE, ACE2, and NEP in plasma EVs. Similarly, urinary EVs exhibited higher chymase and NEP activity compared to ACE and ACE2 activity. Importantly, compared to Group I, significantly higher chymase activity was observed in urinary EVs (p = 0.03) from Group II, while no significant difference in activity was observed for other RAS enzymes. CONCLUSIONS: Bioactive RAS enzymes are present in plasma and urinary EVs. Detecting chymase in plasma and urinary EVs uncovers a novel mechanism of angiotensin II-forming enzyme and could also mediate cell-cell communication and modulate signaling pathways in recipient cells.

Therapeutic efficacy and promise of stem cell-derived extracellular vesicles in Alzheimer's disease and other aging-related disorders.

The term extracellular vesicles (EVs) refers to a variety of heterogeneous nanovesicles secreted by almost all cell types, primarily for intercellular communication and maintaining cellular homeostasis. The role of EVs has been widely reported in the genesis and progression of multiple pathological conditions, and these vesicles are suggested to serve as 'liquid biopsies'. In addition to their use as biomarkers, EVs secreted by specific cell types, especially with stem cell properties, have shown promise as cell-free nanotherapeutics. Stem cell-derived EVs (SC-EVs) have been increasingly used as an attractive alternative to stem cell therapies and have been reported to promote regeneration of aging-associated tissue loss and function. SC-EVs treatment ameliorates brain and peripheral aging, reproductive dysfunctions and inhibits cellular senescence, thereby reversing several aging-related disorders and dysfunctions. The anti-aging therapeutic potential of SC-EVs depends on multiple factors, including the type of stem cells, the age of the source stem cells, and their physiological state. In this review, we briefly describe studies related to the promising effects of SC-EVs against various aging-related pathologies, and then we focus in-depth on the therapeutic benefits of SC-EVs against Alzheimer's disease, one of the most devastating neurodegenerative diseases in elderly individuals. Numerous studies in transgenic mouse models have reported the usefulness of SC-EVs in targeting the pathological hallmarks of Alzheimer's disease, including amyloid plaques, neurofibrillary tangles, and neuroinflammation, leading to improved neuronal protection, synaptic plasticity, and cognitive measures. Cell culture studies have further identified the underlying molecular mechanisms through which SC-EVs reduce amyloid beta (Aß) levels or shift microglia phenotype from pro-inflammatory to anti-inflammatory state. Interestingly, multiple routes of administration, including nasal delivery, have confirmed that SC-EVs could cross the blood-brain barrier. Due to this, SC-EVs have also been tested to deliver specific therapeutic cargo molecule/s (e.g., neprilysin) to the brain. Despite these promises, several challenges related to quality control, scalability, and biodistribution remain, hindering the realization of the vast clinical promise of SC-EVs.

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.

Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EV-ISF) reveals sex-dependent changes in microglial EV proteome in response to Aß pathology.

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimer's disease (AD), relaying important physiological information about their host tissues. Circulating EVs are protected from degradation, making them attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EVISF) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labeling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EVISF cargo. EVISF were 40-150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g. APPswe,PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with A deposition. Genotype, age, and Aß deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EVISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse- EVISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EVISF and offers a unique opportunity to explore the role of EVs in AD.

PET imaging of kappa opioid receptors and receptor expression quantified in neuron-derived extracellular vesicles in socially housed female and male cynomolgus macaques.

Recent positron emission tomography (PET) studies of kappa opioid receptors (KOR) in humans reported significant relationships between KOR availability and social status, as well as cocaine choice. In monkey models, social status influences physiology, receptor pharmacology and behavior; these variables have been associated vulnerability to cocaine abuse. The present study utilized PET imaging to examine KOR availability in socially housed, cocaine-naïve female and male monkeys, and peripheral measures of KORs with neuron-derived extracellular vesicles (NDE). KOR availability was assessed in dominant and subordinate female and male cynomolgus macaques (N = 4/rank/sex), using PET imaging with the KOR selective agonist [11C]EKAP. In addition, NDE from the plasma of socially housed monkeys (N = 13/sex; N = 6-7/rank) were isolated by immunocapture method and analyzed for OPRK1 protein expression by ELISA. We found significant interactions between sex and social rank in KOR availability across 12 of 15 brain regions. This was driven by female data, in which KOR availability was significantly higher in subordinate monkeys compared with dominant monkeys; the opposite relationship was observed among males, but not statistically significant. No sex or rank differences were observed for NDE OPRK1 concentrations. In summary, the relationship between brain KOR availability and social rank was different in female and male monkeys. This was particularly true in female monkeys. We hypothesize that lower [11C]EKAP binding potentials were due to higher concentrations of circulating dynorphin, which is consistent with greater vulnerability in dominant compared with subordinate females. These findings suggest that the KOR is an important target for understanding the neurobiology associated with vulnerability to abused drugs and sex differences, and detectable in peripheral circulation.

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.

[18F]KS1, a novel ascorbate-based ligand images ROS in tumor models of rodents and nonhuman primates.

Over production of reactive oxygen species (ROS) caused by altered redox regulation of signaling pathways is common in many types of cancers. While PET imaging is recognized as the standard tool for cancer imaging, there are no clinically-approved PET radiotracers for ROS-imaging in cancer diagnosis and treatment. An ascorbate-based radio ligand promises to meet this urgent need. Our laboratory recently synthesized [18F] KS1, a fluoroethoxy furanose ring-containing ascorbate derivative, to track ROS in prostate tumor-bearing mice. Here we report cell uptake assays of [18F]KS1 with different ROS-regulating agents, PET imaging in head and neck squamous cell carcinoma (HNSCC) mice, and doxorubicin-induced rats; PET imaging in healthy and irradiated hepatic tumor-bearing rhesus to demonstrate its translational potential. Our preliminary evaluations demonstrated that KS1 do not generate ROS in tumor cells at tracer-level concentrations and tumor-killing properties at pharmacologic doses. [18F]KS1 uptake was low in HNSCC pretreated with ROS blockers, and high with ROS inducers. Tumors in high ROS-expressing SCC-61 took up significantly more [18F]KS1 than rSCC-61 (low-ROS expressing HNSCC); high uptake in doxorubicin-treated rats compared to saline-treated controls. Rodent biodistribution and PET imaging of [18F]KS1 in healthy rhesus monkeys demonstrated its favorable safety, pharmacokinetic properties with excellent washout profile, within 3.0 h of radiotracer administration. High uptake of [18F]KS1 in liver tumor tissues of the irradiated hepatic tumor-bearing monkey showed target selectivity. Our strong data in vitro, in vivo, and ex vivo here supports the high translational utility of [18F]KS1 to image ROS.

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.

The impacts of exosomes on bone metastatic progression and their potential clinical utility.

Bone is one of the most common sites of cancer metastasis. Once cancer metastasizes to the bone, the mortality rate of cancer patients dramatically increases. Although the exact mechanisms for this observation remain elusive, recent studies have revealed that the complex crosstalk between bone marrow microenvironment and bone metastatic cancer cells is responsible for the induction of treatment resistance. Consequently, bone metastasis is currently considered incurable. Bone metastasis not only impairs the patients' survival, but also negatively affects their quality of life by causing painful complications. It has recently been implicated the regulatory role of exosomes in cancer development and/or progression as a delivery biomaterial between cancer cells and tumor microenvironment. However, little is known as to how exosomes contribute to the progression of bone metastasis by impaction on the crosstalk between bone metastatic cancer cells and bone marrow microenvironment. Here, we highlighted the emerging roles of cancer-derived exosomes in (i) the process of dissemination and bone colonization of bone metastatic cancer cells, (ii) the enhancement of crosstalk between bone marrow microenvironment and bone metastatic cancer cells, (iii) the development of its resultant painful complications, and (iv) the clinical applications of exosomes in the bone metastatic setting.

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.

Exosomal Metabolic Signatures Are Associated with Differential Response to Neoadjuvant Chemotherapy in Patients with Breast Cancer.

Neoadjuvant chemotherapy (NAC) is commonly used in breast cancer (BC) patients to increase eligibility for breast-conserving surgery. Only 30% of patients with BC show pathologic complete response (pCR) after NAC, and residual disease (RD) is associated with poor long-term prognosis. A critical barrier to improving NAC outcomes in patients with BC is the limited understanding of the mechanisms underlying differential treatment outcomes. In this study, we evaluated the ability of exosomal metabolic profiles to predict NAC response in patients with BC. Exosomes isolated from the plasma of patients after NAC were used for metabolomic analyses to identify exosomal metabolic signatures associated with the NAC response. Among the 16 BC patients who received NAC, eight had a pCR, and eight had RD. Patients with RD had 2.52-fold higher exosome concentration in their plasma than those with pCR and showed significant enrichment of various metabolic pathways, including citrate cycle, urea cycle, porphyrin metabolism, glycolysis, and gluconeogenesis. Additionally, the relative exosomal levels of succinate and lactate were significantly higher in patients with RD than in those with pCR. These data suggest that plasma exosomal metabolic signatures could be associated with differential NAC outcomes in BC patients and provide insight into the metabolic determinants of NAC response in patients with BC.

Intraligand Excited States Turn a Ruthenium Oligothiophene Complex into a Light-Triggered Ubertoxin with Anticancer Effects in Extreme Hypoxia.

Ru(II) complexes that undergo photosubstitution reactions from triplet metal-centered (3MC) excited states are of interest in photochemotherapy (PCT) due to their potential to produce cytotoxic effects in hypoxia. Dual-action systems that incorporate this stoichiometric mode to complement the oxygen-dependent photosensitization pathways that define photodynamic therapy (PDT) are poised to maintain antitumor activity regardless of the oxygenation status. Herein, we examine the way in which these two pathways influence photocytotoxicity in normoxia and in hypoxia using the [Ru(dmp)2(IP-nT)]2+ series (where dmp = 2,9-dimethyl-1,10-phenanthroline and IP-nT = imidazo[4,5-f][1,10]phenanthroline tethered to n = 0-4 thiophene rings) to switch the dominant excited state from the metal-based 3MC state in the case of Ru-phen-Ru-1T to the ligand-based 3ILCT state for Ru-3T and Ru-4T. Ru-phen-Ru-1T, having dominant 3MC states and the largest photosubstitution quantum yields, are inactive in both normoxia and hypoxia. Ru-3T and Ru-4T, with dominant 3IL/3ILCT states and long triplet lifetimes (τTA = 20-25 µs), have the poorest photosubstitution quantum yields, yet are extremely active. In the best instances, Ru-4T exhibit attomolar phototoxicity toward SKMEL28 cells in normoxia and picomolar in hypoxia, with phototherapeutic index values in normoxia of 105-1012 and 103-106 in hypoxia. While maximizing excited-state deactivation through photodissociative 3MC states did not result in bonafide dual-action PDT/PCT agents, the study has produced the most potent photosensitizer we know of to date. The extraordinary photosensitizing capacity of Ru-3T and Ru-4T may stem from a combination of very efficient 1O2 production and possibly complementary type I pathways via 3ILCT excited states.

Mass Spectrometry-Based Proteome Profiling of Extracellular Vesicles Derived from the Cerebrospinal Fluid of Adult Rhesus Monkeys Exposed to Cocaine throughout Gestation.

Cocaine use disorder has been reported to cause transgenerational effects. However, due to the lack of standardized biomarkers, the effects of cocaine use during pregnancy on postnatal development and long-term neurobiological and behavioral outcomes have not been investigated thoroughly. Therefore, in this study, we examined extracellular vesicles (EVs) in adult (~12 years old) female and male rhesus monkeys prenatally exposed to cocaine (n = 11) and controls (n = 9). EVs were isolated from the cerebrospinal fluid (CSF) and characterized for the surface expression of specific tetraspanins, concentration (particles/mL), size distribution, and cargo proteins by mass spectrometry (MS). Transmission electron microscopy following immunogold labeling for tetraspanins (CD63, CD9, and CD81) confirmed the successful isolation of EVs. Nanoparticle tracking analyses showed that the majority of the particles were <200 nm in size, suggesting an enrichment for small EVs (sEV). Interestingly, the prenatally cocaine-exposed group showed ~54% less EV concentration in CSF compared to the control group. For each group, MS analyses identified a number of proteins loaded in CSF-EVs, many of which are commonly listed in the ExoCarta database. Ingenuity pathway analysis (IPA) demonstrated the association of cargo EV proteins with canonical pathways, diseases and disorders, upstream regulators, and top enriched network. Lastly, significantly altered proteins between groups were similarly characterized by IPA, suggesting that prenatal cocaine exposure could be potentially associated with long-term neuroinflammation and risk for neurodegenerative diseases. Overall, these results indicate that CSF-EVs could potentially serve as biomarkers to assess the transgenerational adverse effects due to prenatal cocaine exposure.