Extracellular vesicles as a potential delivery platform for CRISPR-Cas based therapy in epithelial ovarian cancer.

Ovarian Cancer (OC) is the most common gynecological malignancy and the eighth most diagnosed cancer in females worldwide. Presently, it ranks as the fifth leading cause of cancer-related mortality among patients globally. Major factors contributing to the lethality of OC worldwide include delayed diagnosis, chemotherapy resistance, high metastatic rates, and the heterogeneity of subtypes. Despite continuous efforts to develop novel targeted therapies and chemotherapeutic agents, challenges persist in the form of OC resistance and recurrence. In the last decade, CRISPR-Cas-based genome editing has emerged as a powerful tool for modifying genetic and epigenetic mechanisms, holding potential for treating numerous diseases. However, a significant challenge for therapeutic applications of CRISPR-Cas technology is the absence of an optimal vehicle for delivering CRISPR molecular machinery into targeted cells or tissues. Recently, extracellular vesicles (EVs) have gained traction as potential delivery vehicles for various therapeutic agents. These heterogeneous, membrane-derived vesicles are released by nearly all cells into extracellular spaces. They carry a molecular cargo of proteins and nucleic acids within their intraluminal space, encased by a cholesterol-rich phospholipid bilayer membrane. EVs actively engage in cell-to-cell communication by delivering cargo to both neighboring and distant cells. Their inherent ability to shield molecular cargo from degradation and cross biological barriers positions them ideally for delivering CRISPR-Cas ribonucleoproteins (RNP) to target cells. Furthermore, they exhibit higher biocompatibility, lower immunogenicity, and reduced toxicity compared to classical delivery platforms such as adeno-associated virus, lentiviruses, and synthetic nanoparticles. This review explores the potential of employing different CRISPR-Cas systems to target specific genes in OC, while also discussing various methods for engineering EVs to load CRISPR components and enhance their targeting capabilities.

Enhancing precision targeting of ovarian cancer tumor cells in vivo through extracellular vesicle engineering.

Extracellular vesicles (EVs) function as natural mediators of intercellular communication, secreted by cells to facilitate cell-cell signaling. Due to their low toxicity, immunogenicity, biodegradability, and potential to encapsulate therapeutic drugs, EVs hold significant therapeutic promise. Nevertheless, their limited targeting ability often diminishes their therapeutic impact. Therefore, enhancing EVs by incorporating targeting units onto their membranes could bolster their targeting capabilities, enabling them to accumulate in specific cells and tissues. In this study, we engineered EVs to fuse ephrin-B2 with the EV membrane protein LAMP2b. This modification aimed to direct the engineered EVs toward the ephrin-B4 receptor expressed on the surface of ovarian cancer cells. The engineered EVs retained their inherent properties, including size, expression of EV membrane proteins, and morphology, upon isolation. In vitro experiments using real-time imaging revealed that EVs engineered with the ephrin-B2 ligand exhibited substantial internalization and uptake by ovarian cancer cells, in stark contrast to native EVs. In vivo, the engineered EVs carrying the ephrin-B2 ligand effectively targeted ovarian cancer cells, surpassing the targeting efficiency of control EVs. This innovative approach establishes a novel targeting system, enhancing the uptake of EVs by ovarian cancer cells. Our findings underscore the potential of using EVs to target cancer cells, thereby enhancing the effectiveness of anti-cancer therapies while minimizing off-target effects and toxicity in normal cells and organs.

Chemically-Induced Lipoprotein Breakdown for Improved Extracellular Vesicle Purification.

Extracellular vesicles (EVs) are nanosized biomolecular packages involved in intercellular communication. EVs are released by all cells, making them broadly applicable as therapeutic, diagnostic, and mechanistic components in (patho)physiology. Sample purity is critical for correctly attributing observed effects to EVs and for maximizing therapeutic and diagnostic performance. Lipoprotein contaminants represent a major challenge for sample purity. Lipoproteins are approximately six orders of magnitude more abundant in the blood circulation and overlap in size, shape, and density with EVs. This study represents the first example of an EV purification method based on the chemically-induced breakdown of lipoproteins. Specifically, a styrene-maleic acid (SMA) copolymer is used to selectively breakdown lipoproteins, enabling subsequent size-based separation of the breakdown products from plasma EVs. The use of the polymer followed by tangential flow filtration or size-exclusion chromatography results in improved EV yield, preservation of EV morphology, increased EV markers, and reduced contaminant markers. SMA-based EV purification enables improved fluorescent labeling, reduces interactions with macrophages, and enhances accuracy, sensitivity, and specificity to detect EV biomarkers, indicating benefits for various downstream applications. In conclusion, SMA is a simple and effective method to improve the purity and yield of plasma-derived EVs, which favorably impacts downstream applications.

Genomic communication via circulating extracellular vesicles and long-term health consequences of COVID-19.

COVID-19 continues to affect an unprecedented number of people with the emergence of new variants posing a serious challenge to global health. There is an expansion of knowledge in understanding the pathogenesis of Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the impact of the acute disease on multiple organs. In addition, growing evidence reports that the impact of COVID-19 on different organs persists long after the recovery phase of the disease, leading to long-term consequences of COVID-19. These long-term consequences involve pulmonary as well as extra-pulmonary sequelae of the disease. Noteably, recent research has shown a potential association between COVID-19 and change in the molecular cargo of extracellular vesicles (EVs). EVs are vesicles released by cells and play an important role in cell communication by transfer of bioactive molecules between cells. Emerging evidence shows a strong link between EVs and their molecular cargo, and regulation of metabolism in health and disease. This review focuses on current knowledge about EVs and their potential role in COVID-19 pathogenesis, their current and future implications as tools for biomarker and therapeutic development and their possible effects on long-term impact of COVID-19.

Extracellular vesicle-mediated targeting strategies for long-term health benefits in gestational diabetes.

Extracellular vesicles (EVs) are critical mediators of cell communication, playing important roles in regulating molecular cross-talk between different metabolic tissues and influencing insulin sensitivity in both healthy and gestational diabetes mellitus (GDM) pregnancies. The ability of EVs to transfer molecular cargo between cells imbues them with potential as therapeutic agents. During pregnancy, the placenta assumes a vital role in metabolic regulation, with multiple mechanisms of placenta-mediated EV cross-talk serving as central components in GDM pathophysiology. This review focuses on the role of the placenta in the pathophysiology of GDM and explores the possibilities and prospects of targeting the placenta to address insulin resistance and placental dysfunction in GDM. Additionally, we propose the use of EVs as a novel method for targeted therapeutics in treating the dysfunctional placenta. The primary aim of this review is to comprehend the current status of EV targeting approaches and assess the potential application of these strategies in placental therapeutics, thereby delivering molecular cargo and improving maternal and fetal outcomes in GDM. We propose that EVs have the potential to revolutionize GDM management, offering hope for enhanced maternal-fetal health outcomes and more effective treatments.

Effects of periodontal cells-derived extracellular vesicles on mesenchymal stromal cell function.

OBJECTIVE: To enrich and compare three extracellular vesicles-EV subtypes (apoptotic bodies, microvesicles and small EV) from three periodontal cells (periodontal ligament cells-PDLCs, alveolar bone-derived osteoblasts-OBs and gingival fibroblasts-GFs), and assess uptake and cell function changes in buccal fat pad-derived mesenchymal stromal cells (BFP-MSCs). BACKGROUND: Periodontal cells such as PDLCs, OBs and GFs have the potential to enhance bone and periodontal regeneration, but face significant challenges, such as the regulatory and cost implications of in vitro cell culture and storage. To address these challenges, it is important to explore alternative 'cell-free' strategies, such as extracellular vesicles which have emerged as promising tools in regenerative medicine, to facilitate osteogenic differentiation and bone regeneration. METHODS AND MATERIALS: Serial centrifuges at 2600 and 16 000 g were used to isolate apoptotic bodies and microvesicles respectively. Small EV-sEV was enriched by our in-house size exclusion chromatography (SEC). The cellular uptake, proliferation, migration and osteogenic/adipogenic differentiation genes were analysed after EVs uptake in BFP-MSCs. RESULTS: Three EV subtypes were enriched and characterised by morphology, particle size and EV-associated protein expression-CD9. Cellular uptake of the three EVs subtypes was observed in BFP-MSCs for up to 7 days. sEV from the three periodontal cells promoted proliferation, migration and osteogenic gene expression. hOBs-sEV showed superior levels of osteogenesis markers compared to that hPDLCs-sEV and hGFs-sEV, while hOBs-16k EV promoted adipogenic gene expression compared to that from hPDLCs and hGFs. CONCLUSIONS: Our proof-of-concept data demonstrate that hOBs-sEV might be an alternative cell-free therapeutic for bone tissue engineering.

Explainable discovery of disease biomarkers: The case of ovarian cancer to illustrate the best practice in machine learning and Shapley analysis.

OBJECTIVE: Ovarian cancer is a significant health issue with lasting impacts on the community. Despite recent advances in surgical, chemotherapeutic and radiotherapeutic interventions, they have had only marginal impacts due to an inability to identify biomarkers at an early stage. Biomarker discovery is challenging, yet essential for improving drug discovery and clinical care. Machine learning (ML) techniques are invaluable for recognising complex patterns in biomarkers compared to conventional methods, yet they can lack physical insights into diagnosis. eXplainable Artificial Intelligence (XAI) is capable of providing deeper insights into the decision-making of complex ML algorithms increasing their applicability. We aim to introduce best practice for combining ML and XAI techniques for biomarker validation tasks. METHODS: We focused on classification tasks and a game theoretic approach based on Shapley values to build and evaluate models and visualise results. We described the workflow and apply the pipeline in a case study using the CDAS PLCO Ovarian Biomarkers dataset to demonstrate the potential for accuracy and utility. RESULTS: The case study results demonstrate the efficacy of the ML pipeline, its consistency, and advantages compared to conventional statistical approaches. CONCLUSION: The resulting guidelines provide a general framework for practical application of XAI in medical research that can inform clinicians and validate and explain cancer biomarkers.

The impact of obesity and uncontrolled asthma during pregnancy on metabolic and inflammatory pathways.

OBJECTIVE: Asthma and obesity are both inflammatory complications of pregnancy and when combined contribute to an increased risk of uncontrolled asthma during pregnancy and poor perinatal outcomes. Our previous work has identified the presence of maternal asthma is associated with a proinflammatory milieu in the placenta and reduced fetal growth. The current study was designed to determine the relationships between immunomodulatory metabolic pathways and inflammation and establish whether these pathways are associated with uncontrolled asthma in obese pregnant women. METHODS: Fifty-three obese (BMI >30) pregnant women were recruited prospectively. Participants were classified as having no asthma, controlled asthma, and uncontrolled asthma based on a doctor diagnosis and assessment using the Asthma Control Questionnaire (ACQ). Circulating plasma concentrations of metabolic hormones leptin, adiponectin, insulin, glucose, and extracellular vesicle (EVs) associated cytokines were measured at 18- and 36-weeks gestation. RESULTS: Concentrations of metabolic and inflammatory markers among obese participants with or without asthma were not significantly different throughout gestation. However total adiponectin concentrations increased as gestation progressed in obese, non-asthmatic women but did not increase in women with asthma. Plasma adiponectin and leptin levels in women with uncontrolled asthma were positively correlated with EV inflammatory markers including GM-CSF, IL-6, TNFα and IFNγ protein. CONCLUSIONS: This study demonstrated that most metabolic markers remain unchanged with the presence and severity of asthma in obese pregnant women. However, differences in the associations between metabolic and inflammatory pathways were observed in women with asthma and may be one of the mechanisms contributing to uncontrolled asthma in obese pregnant women.

Peptide-based LDH5 inhibitors enter cancer cells and impair proliferation.

Lactate dehydrogenase 5 (LDH5) is overexpressed in many cancers and is a potential target for anticancer therapy due to its role in aerobic glycolysis. Small-molecule drugs have been developed as competitive inhibitors to bind substrate/cofactor sites of LDH5, but none reached the clinic to date. Recently, we designed the first LDH5 non-competitive inhibitor, cGmC9, a peptide that inhibits protein-protein interactions required for LDH5 enzymatic activity. Peptides are gaining a large interest as anticancer agents to modulate intracellular protein-protein interactions not targetable by small molecules; however, delivery of these peptides to the cytosol, where LDH5 and other anticancer targets are located, remains a challenge for this class of therapeutics. In this study, we focused on the cellular internalisation of cGmC9 to achieve LDH5 inhibition in the cytosol. We designed cGmC9 analogues and compared them for LDH5 inhibition, cellular uptake, toxicity, and antiproliferation against a panel of cancer cell lines. The lead analogue, [R/r]cGmC9, specifically impairs proliferation of cancer cell lines with high glycolytic profiles. Proteomics analysis showed expected metabolic changes in response to decreased glycolysis. This is the first report of a peptide-based LDH5 inhibitor able to modulate cancer metabolism and kill cancer cells that are glycolytic. The current study demonstrates the potential of using peptides as inhibitors of intracellular protein-protein interactions relevant for cancer pathways and shows that active peptides can be rationally designed to improve their cell permeation.

Comprehensive Strategy for Identifying Extracellular Vesicle Surface Proteins as Biomarkers for Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a liver disorder that has become a global health concern due to its increasing prevalence. There is a need for reliable biomarkers to aid in the diagnosis and prognosis of NAFLD. Extracellular vesicles (EVs) are promising candidates in biomarker discovery, as they carry proteins that reflect the pathophysiological state of the liver. In this review, we developed a list of EV proteins that could be used as diagnostic biomarkers for NAFLD. We employed a multi-step strategy that involved reviewing and comparing various sources of information. Firstly, we reviewed papers that have studied EVs proteins as biomarkers in NAFLD and papers that have studied circulating proteins as biomarkers in NAFLD. To further identify potential candidates, we utilized the EV database Vesiclepedia.org to qualify each protein. Finally, we consulted the Human Protein Atlas to search for candidates' localization, focusing on membrane proteins. By integrating these sources of information, we developed a comprehensive list of potential EVs membrane protein biomarkers that could aid in diagnosing and monitoring NAFLD. In conclusion, our multi-step strategy for identifying EV-based protein biomarkers for NAFLD provides a comprehensive approach that can also be applied to other diseases. The protein candidates identified through this approach could have significant implications for the development of non-invasive diagnostic tests for NAFLD and improve the management and treatment of this prevalent liver disorder.

Hydrogel Nanoarchitectonics: An Evolving Paradigm for Ultrasensitive Biosensing.

The integration of nanoarchitectonics and hydrogel into conventional biosensing platforms offers the opportunities to design physically and chemically controlled and optimized soft structures with superior biocompatibility, better immobilization of biomolecules, and specific and sensitive biosensor design. The physical and chemical properties of 3D hydrogel structures can be modified by integrating with nanostructures. Such modifications can enhance their responsiveness to mechanical, optical, thermal, magnetic, and electric stimuli, which in turn can enhance the practicality of biosensors in clinical settings. This review describes the synthesis and kinetics of gel networks and exploitation of nanostructure-integrated hydrogels in biosensing. With an emphasis on different integration strategies of hydrogel with nanostructures, this review highlights the importance of hydrogel nanostructures as one of the most favorable candidates for developing ultrasensitive biosensors. Moreover, hydrogel nanoarchitectonics are also portrayed as a promising candidate for fabricating next-generation robust biosensors.

The link between gestational diabetes and cardiovascular diseases: potential role of extracellular vesicles.

Extracellular vesicles are critical mediators of cell communication. They encapsulate a variety of molecular cargo such as proteins, lipids, and nucleic acids including miRNAs, lncRNAs, circular RNAs, and mRNAs, and through transfer of these molecular signals can alter the metabolic phenotype in recipient cells. Emerging studies show the important role of extracellular vesicle signaling in the development and progression of cardiovascular diseases and associated risk factors such as type 2 diabetes and obesity. Gestational diabetes mellitus (GDM) is hyperglycemia that develops during pregnancy and increases the future risk of developing obesity, impaired glucose metabolism, and cardiovascular disease in both the mother and infant. Available evidence shows that changes in maternal metabolism and exposure to the hyperglycemic intrauterine environment can reprogram the fetal genome, leaving metabolic imprints that define life-long health and disease susceptibility. Understanding the factors that contribute to the increased susceptibility to metabolic disorders of children born to GDM mothers is critical for implementation of preventive strategies in GDM. In this review, we discuss the current literature on the fetal programming of cardiovascular diseases in GDM and the impact of extracellular vesicle (EV) signaling in epigenetic programming in cardiovascular disease, to determine the potential link between EV signaling in GDM and the development of cardiovascular disease in infants.

A novel technique using chronic infusion of small extracellular vesicles from gestational diabetes mellitus causes glucose intolerance in pregnant mice.

Small extracellular vesicles (sEVs) play a central role in cell-to-cell communication in normal physiology and in disease, including gestational diabetes mellitus (GDM). The goal of the present study was to test the hypothesis that chronic administration of sEVs isolated from GDM causes glucose intolerance in healthy pregnant mice. Small EVs were isolated from plasma between 24 and 28 weeks gestation from healthy pregnant women (controls) and GDM, and infused intravenously for 4 days in late pregnant mice using a mini-osmotic pump. Subsequently in vivo glucose tolerance was assessed, and muscle and adipose tissue insulin sensitivity and islet glucose stimulated insulin secretion (GSIS) were determined in vitro. Mice infused with sEVs from GDM developed glucose intolerance. Administration of sEVs from controls, but not sEVs from GDM women, stimulated islet GSIS and increased fasting insulin levels in pregnant mice. Neither infusion of sEVs from controls nor from GDM women affected muscle insulin sensitivity, placental insulin or mTOR signaling, placental and fetal weight. Moreover, these results were not associated with immunomodulatory effects as human sEVs did not activate mouse T cells in vitro. We suggest that circulating sEVs regulate maternal glucose homeostasis in pregnancy and may contribute to the attenuated islet insulin secretion and more pronounced glucose intolerance in GDM as compared with healthy pregnancy.

Extracellular Vesicles and Preeclampsia: Current Knowledge and Future Research Directions.

Preeclampsia (PE) is associated with long-term morbidity in mothers and lifelong morbidities for their children, ranging from cerebral palsy and cognitive delay in preterm infants, to hypertension, diabetes and obesity in adolescents and young adults. There are several processes that are critical for development of materno-fetal exchange, including establishing adequate perfusion of the placenta by maternal blood, and the formation of the placental villous vascular tree. Recent studies provide persuasive evidence that placenta-derived extracellular vesicles (EVs) represent a significant intercellular communication pathway, and that they may play an important role in placental and endothelial cell (both fetal and maternal) function. These functions are known to be altered in PE. EVs can carry and transport a wide range of bioactive molescules that have potential to be used as biomarkers and therapeutic delivery tools for PE. EV content is often parent cell specific, thus providing an insight or "thumbprint" of the intracellular environment of the originating cell (e.g., human placenta). EV have been identified in plasma under both normal and pathological conditions, including PE. The concentration of EVs and their content in plasma has been reported to increase in association with disease severity and/or progression. Placenta-derived EVs have been identified in maternal plasma during normal pregnancy and PE pregnancies. They contain placenta-specific proteins and miRNAs and, as such, may be differentiated from maternally-derived EVs. The aim of this review, thus, is to describe the potential roles of EVs in preecmpatic pregnancies, focussing on EVs secreted from placental cells. The biogenesis, specificity of placental EVs, and methods used to characterise EVs in the context of PE pregnancies will be also discussed.

Extracellular Vesicle Nanoarchitectonics for Novel Drug Delivery Applications.

Extracellular vesicles (EVs) can transfer intercellular messages in various (patho)physiological processes and transport biomolecules to recipient cells. EVs possess the capacity to evade the immune system and remain stable over long periods, identifying them as natural carriers for drugs and biologics. However, the challenges associated with EVs isolation, heterogeneity, coexistence with homologous biomolecules, and lack of site-specific delivery, have impeded their potential. In recent years, the amalgamation of EVs with rationally engineered nanostructures has been proposed for achieving effective drug loading and site-specific delivery. With the advancement of nanotechnology and nanoarchitectonics, different nanostructures with tunable size, shapes, and surface properties can be integrated with EVs for drug loading, target binding, efficient delivery, and therapeutics. Such integration may enable improved cellular targeting and the protection of encapsulated drugs for enhanced and specific delivery to target cells. This review summarizes the recent development of nanostructure amalgamated EVs for drug delivery, therapeutics, and real-time monitoring of disease progression. With a specific focus on the exosomal cargo, diverse drug delivery system, and biomimetic nanostructures based on EVs for selective drug delivery, this review also chronicles the needs and challenges of EV-based biomimetic nanostructures and provides a future outlook on the strategies posed.

Extracellular vesicle-associated miRNAs are an adaptive response to gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is a serious public health issue affecting 9-15% of all pregnancies worldwide. Recently, it has been suggested that extracellular vesicles (EVs) play a role throughout gestation, including mediating a placental response to hyperglycaemia. Here, we investigated the EV-associated miRNA profile across gestation in GDM, assessed their utility in developing accurate, multivariate classification models, and determined the signaling pathways in skeletal muscle proteome associated with the changes in the EV miRNA profile. METHODS: Discovery: A retrospective, case-control study design was used to identify EV-associated miRNAs that vary across pregnancy and clinical status (i.e. GDM or Normal Glucose Tolerance, NGT). EVs were isolated from maternal plasma obtained at early, mid and late gestation (n = 29) and small RNA sequencing was performed. Validation: A longitudinal study design was used to quantify expression of selected miRNAs. EV miRNAs were quantified by real-time PCR (cases = 8, control = 14, samples at three times during pregnancy) and their individual and combined classification efficiencies were evaluated. Quantitative, data-independent acquisition mass spectrometry was use to establish the protein profile in skeletal muscle biopsies from normal and GDM. RESULTS: A total of 2822 miRNAs were analyzed using a small RNA library, and a total of 563 miRNAs that significantly changed (p < 0.05) across gestation and 101 miRNAs were significantly changed between NGT and GDM. Analysis of the miRNA changes in NGT and GDM separately identified a total of 256 (NGT-group), and 302 (GDM-group) miRNAs that change across gestation. A multivariate classification model was developed, based on the quantitative expression of EV-associated miRNAs, and the accuracy to correctly assign samples was > 90%. We identified a set of proteins in skeletal muscle biopsies from women with GDM associated with JAK-STAT signaling which could be targeted by the miRNA-92a-3p within circulating EVs. Interestingly, overexpression of miRNA-92a-3p in primary skeletal muscle cells increase insulin-stimulated glucose uptake. CONCLUSIONS: During early pregnancy, differently-expressed, EV-associated miRNAs may be of clinical utility in identifying presymptomatic women who will subsequently develop GDM later in gestation. We suggest that miRNA-92a-3p within EVs might be a protected mechanism to increase skeletal muscle insulin sensitivity in GDM.

Regulation of glucose homeostasis by small extracellular vesicles in normal pregnancy and in gestational diabetes.

The mechanisms underpinning maternal metabolic adaptations to a healthy pregnancy and in gestational diabetes mellitus (GDM) remain poorly understood. We hypothesized that small extracellular vesicles (sEVs) isolated from healthy pregnant women promote islet glucose-stimulated insulin secretion (GSIS) and peripheral insulin resistance in nonpregnant mice and that sEVs from GDM women fail to stimulate insulin secretion and cause exacerbated insulin resistance. Small EVs were isolated from plasma of nonpregnant, healthy pregnant, and GDM women at 24-28 weeks of gestation. We developed a novel approach in nonpregnant mice involving a mini-osmotic pump for continuous 4-day jugular venous infusion of sEVs and determined their effects on glucose tolerance in vivo and islets and skeletal muscle in vitro. Fasting insulin was elevated in mice infused with pregnant sEVs as compared to sEVs from nonpregnant and GDM women. Mice infused with sEVs from GDM women developed glucose intolerance. GSIS was increased in mice infused with healthy pregnancy sEVs compared to mice receiving nonpregnant sEVs. GSIS and muscle basal insulin signaling, and insulin responsiveness were attenuated in mice infused with GDM sEVs. sEVs represent a novel mechanism regulating maternal glucose homeostasis in pregnancy and we speculate that altered sEV content contributes to the development of GDM.

A novel DNA binding protein-based platform for electrochemical detection of miRNA.

We present a novel amplification-free sandwich type platform assay for electrochemical detection of miRNA. The assay is based on T4 DNA polymerase mediated synthesis of the p53 binding DNA sequence at the 3' end of target miRNA. The resulting miRNA-DNA chimera is detected via an electrochemical sandwich hybridization assay where HRP-labelled p53 binds to its recognition sequence and an amperometric signal is generated by hydroquinone-mediated enzymatic reduction of H2O2. The limit of detection of our assay was estimated to be 22 fM with a linear dynamic range of 100 fM-1 nM. This new platform method of detecting miRNA shows superior performance to conventional electrochemical miRNA biosensors and has the potential for amplification-free analysis of miRNA with high specificity and sensitivity.

A phase III randomized clinical trial comparing sentinel node biopsy with no retroperitoneal node dissection in apparent early-stage endometrial cancer - ENDO-3: ANZGOG trial 1911/2020.

BACKGROUND: Sentinel node biopsy is a surgical technique to explore lymph nodes for surgical staging of endometrial cancer, which has replaced full retroperitoneal lymph node dissection. However, the effectiveness of sentinel node biopsy, its value to patients, and potential harms compared with no-node dissection have never been shown in a randomized trial. PRIMARY OBJECTIVES: Stage 1 will test recovery from surgery. Stage 2 will compare disease-free survival at 4.5 years between patients randomized to sentinel node biopsy versus no retroperitoneal node dissection. STUDY HYPOTHESIS: The primary hypothesis for stage 1 is that treatment with sentinel node biopsy will not cause detriment to patient outcomes (lymphedema, morbidity, loss of quality of life) and will not increase treatment-related morbidity or health services costs compared with patients treated without a retroperitoneal node dissection at 12 months after surgery. The primary hypothesis for stage 2 is that disease-free survival at 4.5 years after surgery in patients without retroperitoneal node dissection is not inferior to those receiving sentinel node biopsy. TRIAL DESIGN: This phase III, open-label, two-arm, multistage, randomized non-inferiority trial (ENDO-3) will determine the value of sentinel node biopsy for surgical management of endometrial cancer. Patients with endometrial cancer are randomized to receive: (1) laparoscopic/robotic hysterectomy, bilateral salpingo-oophorectomy with sentinel node biopsy or (2) laparoscopic/robotic hysterectomy, bilateral salpingo-oophorectomy without retroperitoneal node dissection. In stage 1, 444 patients will be enrolled to demonstrate feasibility and quality of life. If this is demonstrated, we will enroll another 316 patients in stage 2. MAJOR INCLUSION AND EXCLUSION CRITERIA: Inclusion criteria include women aged 18 years or older with histologically confirmed endometrial cancer; clinical stage 1, who meet the criteria for laparoscopic or robotic total hysterectomy and bilateral salpingo-oophorectomy. Patients with uterine mesenchymal tumors are excluded. PRIMARY ENDPOINTS: The endpoint for stage 1 is surgical recovery, with the proportion of patients returning to usual daily activities at 3 months post-surgery as measured with the EQ-5D. Stage 2 is disease-free survival at 4.5 years. SAMPLE SIZE: 760 participants (both stages). ESTIMATED DATES FOR COMPLETING ACCRUAL AND PRESENTING RESULTS: Stage 1 commenced in January 2021 and is planned to be completed in December 2024 when 444 participants have completed 12 months' follow-up. Stage 2 will enroll a further 316 participants for a total of 760 patients. TRIAL REGISTRATION: NCT04073706.

Salivary Outer Membrane Vesicles and DNA Methylation of Small Extracellular Vesicles as Biomarkers for Periodontal Status: A Pilot Study.

Periodontitis is an inflammatory disease, associated with a microbial dysbiosis. Early detection using salivary small extracellular vesicles (sEVs) biomarkers may facilitate timely prevention. sEVs derived from different species (i.e., humans, bacteria) are expected to circulate in saliva. This pilot study recruited 22 participants (seven periodontal healthy, seven gingivitis and eight periodontitis) and salivary sEVs were isolated using the size-exclusion chromatography (SEC) method. The healthy, gingivitis and periodontitis groups were compared in terms of salivary sEVs in the CD9+ sEV subpopulation, Gram-negative bacteria-enriched lipopolysaccharide (LPS+) outer membrane vesicles (OMVs) and global DNA methylation pattern of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and N6-Methyladenosine (m6dA). It was found that LPS+ OMVs, global 5mC methylation and four periodontal pathogens (T. denticola, E. corrodens, P. gingivalis and F. nucleatum) that secreted OMVs were significantly increased in periodontitis sEVs compared to those from healthy groups. These differences were more pronounced in sEVs than the whole saliva and were more superior in distinguishing periodontitis than gingivitis, in comparison to healthy patients. Of note, global 5mC hypermethylation in salivary sEVs can distinguish periodontitis patients from both healthy controls and gingivitis patients with high sensitivity and specificity (AUC = 1). The research findings suggest that assessing global sEV methylation may be a useful biomarker for periodontitis.