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Recent studies claim that immune checkpoint inhibitors are effective in defective mismatch repair (dMMR) cancers. This raises the question of whether similar therapies are effective in PanNETs (pancreatic neuroendocrine tumors); however, in general, assessment of MMR status in PanNETs has been inconsistent in previous studies. MGMT (O6-methylguanine-DNA methyltransferase) is potentially important for guiding temozolomide (TMZ) therapy in glioblastoma. The number of reports on MGMT expression and promoter methylation in PanNETs are limited.In this study we assessed the expression of MGMT and MMR proteins MSH2, MSH6, MLH1 and PMS2 in a series of PanNETs by IHC. The methylation status of MGMT and MMR genes in a subset of PanNETs was further assessed by MS-MLPA analysis. Survival curves were constructed using the Kaplan-Meier method, and differences were assessed using the log-rank test. Multivariate Cox proportional hazards regression models were used to determine the prognostic value of the variables.According to evaluation criteria for mismatch repair defects, none of PanNETs shown nuclear staining loss for MSH2, MSH6, MLH1, and PMS2. MGMT low-intensity PanNETs were more commonly found in higher grade, higher Ki67 index and non-functional tumors (P 0.05). In multivariate analysis, stage III-IV and low-intensity MGMT were shown to be independent risk factors for progression of PanNETs in the entire cohort, non-functioning subgroup and G2 subgroup (P 0.05 for all). MGMT promoter methylation tended to be higher in the group with low expression of MGMT, However, methylation of MGMT did not statistically correlate with low expression of MGMT (P = 0.153).In conclusion, our study suggests that decreased expression of MGMT but not MMR is associated with a higher risk of progression of pancreatic neuroendocrine tumors.

International audience; This paper reports the fabrication, testing and obtained performance of a plasmonic sensor employing a gold (Au) nanohole array chip coated with tungsten disulphide (WS2), which is then functionalized for the detection of protein–protein interactions. A key novelty is that the WS2 was deposited as a monoatomic layer using a wafer-scale synthesis method that successfully provided a film of both high quality and uniform thickness. The deposited WS2 film was transferred onto a Au nanohole array chip using a novel method and was subsequently functionalized with biotin. The final sensor was tested and it demonstrated efficient real-time and label-free plasmonic detection of biotin–streptavidin coupling. Specifically, compared to a standard (i.e. uncoated) Au nanohole-based sensor, our WS2-coated Au nanohole array boosted the spectral shift of the resonance wavelength by ∼190%, resulting in a 7.64-fold improvement of the limit of detection (LOD).

Introduction: Recent studies have suggested that alternative lengthening of telomeres (ALT) is associated with metastasis and poor survival in pancreatic neuroendocrine tumors (PanNETs). This study evaluated whether this association is applicable to Chinese patients as well as the potential somatic mutations associated with ALT. Methods: We assessed the prevalence of ALT by performing telomere-specific fluorescence in situ hybridization and analyzed DAXX/ATRX expression using immunohistochemistry in 112 Chinese patients with PanNETs to evaluate the association between ALT and clinical outcomes. A subset of the noninsulinoma samples (28/60) was subjected to Sanger sequencing and targeted sequencing. Results: The ALT-positive phenotype was identified in 23.2% (26/112) of the samples. The clinicopathologic factors significantly associated with progression in the noninsulinoma (n = 60) cohort were the female sex (p = 0.006), Ki-67 index (p < 0.001), World Health Organization grade (p = 0.031), and ALT positivity (p = 0.013). Patients with ALT-positive PanNETs had significantly shorter progression-free survival than those with ALT-negative PanNETs in the entire cohort (p < 0.001), noninsulinoma subgroup (p = 0.01), and G2 subgroup (p = 0.001). ALT-positive samples frequently harbored somatic mutations in DAXX, ATRX, MEN1, SETBP1, PRKDC, and GNAS. Conclusions: We confirmed that ALT positivity is an effective risk predictor, especially in the noninsulinoma and G2 subgroups. ALT is also related to somatic mutations in MEN1, SETBP1, PRKDC, and GNAS, in addition to DAXX and ATRX.

Huilin Shao, Yue Zhang, Jie Yan, Xinchao Ban, Xiaojie Fan, Xiaoyan Chang, Zhaohui Lu, Yan Wu, Liju Zong, Shengwei Mo, Shuangni Yu, Jie Chen Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, People’s Republic of ChinaCorrespondence: Shuangni Yu; Jie Chen Tel +86 10 69159375; +86 10 69159389Email yushn@pumch.cn; chenjie@pumch.cnBackground: There is an urgent need for the development of effective noninvasive biomarkers for early pancreatic cancer diagnosis. MicroRNAs (miRNAs) are promising candidates that can be identified in peripheral blood and can act as “liquid biopsy” biomarkers. miR-483-3p is overexpressed in the tumor tissue of pancreatic duct adenocarcinoma, but its potential as noninvasive biomarker remains unknown.Methods: We conducted locked nucleic acid in situ hybridization (LNA-ISH) for miR-483-3p in archival tissues of 107 patients with PDAC. We also used immunohistochemistry to evaluate SMAD4 expression, the putative miR-483-3p target gene. miR-483-3p expression level was also assessed using quantitative real-time PCR (qRT-PCR) in serum and serum exosome samples from 63 patients with PDAC and 22 healthy individuals.Results: LNA-ISH showed that miR-483-3p was overexpressed in PDAC and PanIN tissues compared to normal pancreatic duct cells. miR-483-3p expression levels correlated with increases in PanIN lesion grade. miR-483-3p expression negatively correlated with Smad4 expression (γ=− 0.770, p< 0.0001) in PDAC and PanIN tissues. Circulating miR-483-3p levels were significantly elevated in the serum and serum exosomes of PDAC patients compared to healthy controls (p< 0.0001 and p< 0.01, respectively). Specifically, serum miR-483-3p levels were able to distinguish patients with early stage (≤ 2cm) PDAC from healthy controls with an AUC of 0.83 [95% CI, 0.70– 0.96]. Higher serum exosomal miR-483-3p levels predicted worse survival in PDAC patients and serum exosomal miR-483-3p also proved to be an independent prognostic factor for PDAC (hazard ratio = 3.307; 95% CI=1.104 to 9.903; p=0.033). In vitro studies also showed that miR-483-3p promoted pancreatic cancer cell migration and invasion.Conclusion: miR-483-3p overexpression occurs early in PDAC development and is present in premalignant PanIN lesions. Serum miR-483-3p may act as an early PDAC diagnostic biomarker and serum exosomal miR-483-3p may be a PDAC prognostic biomarker.Keywords: pancreatic ductal adenocarcinoma, exosome, microRNA-483-3p, liquid biopsy, biomarker

Background and purpose: Various blood biomarkers reflecting brain amyloid-β (Aβ) load have recently been proposed with promising results. However, to date, no comparative study amongst blood biomarkers has been reported. Our objective was to examine the diagnostic performance and cost effectiveness of three blood biomarkers on the same cohort. Methods: Using the same cohort (n = 68), the performances of the single-molecule array (Simoa) Aβ40, Aβ42, Aβ42/Aβ40 and the amplified plasmonic exosome (APEX) Aβ42 blood biomarkers were compared using amyloid positron emission tomography (PET) as the reference standard. The extent to which these blood tests can reduce the recruitment cost of clinical trials was also determined by identifying amyloid positive (Aβ+) participants. Results: Compared to Simoa biomarkers, APEX-Aβ42 showed significantly higher correlations with amyloid PET retention values and excellent diagnostic performance (sensitivity 100%, specificity 93.3%, area under the curve 0.995). When utilized for clinical trial recruitment, our simulation showed that pre-screening with blood biomarkers followed by a confirmatory amyloid PET imaging would roughly half the cost (56.8% reduction for APEX-Aβ42 and 48.6% for Simoa-Aβ42/Aβ40) compared to the situation where only PET imaging is used. Moreover, with 100% sensitivity, APEX-Aβ42 pre-screening does not increase the required number of initial participants. Conclusions: With its high diagnostic performance, APEX is an ideal candidate for Aβ+ subject identification, monitoring and primary care screening, and could efficiently enrich clinical trials with Aβ+ participants whilst halving recruitment costs. (Less)

Summary Extracellular vesicles (EVs) harbor a rich glycome—diverse sugar modifications—that mediates and reflects disease progression. Despite its clinical potential, the analysis of EV glycans remains challenging due to limitations of current glycomics technologies. Here, we develop a dedicated analytical platform to directly profile EV glycans in native biofluids. Termed integrated magnetic analysis of glycans in extracellular vesicles (iMAGE), the platform utilizes rationally designed polycore magnetic nanoparticles to transduce EV-bound glycans, but not that of free-floating glycoproteins, into magnetic signals; the resultant signals are readily quantified through a built-in magnetoresistance sensor. The iMAGE assay is thus fast (

Massively parallel DNA sequencing is established, yet high-throughput protein profiling remains challenging. Here, we report a barcoding approach that leverages the combinatorial sequence content and the configurational programmability of DNA nanostructures for high-throughput multiplexed profiling of the subcellular expression and distribution of proteins in whole cells. The barcodes are formed by in situ hybridization of tetrahedral DNA nanostructures and short DNA sequences conjugated with protein-targeting antibodies, and by nanostructure-assisted ligation (either enzymatic or chemical) of the nanostructures and exogenous DNA sequences bound to nanoparticles of different sizes (which cause these localization sequences to differentially distribute across subcellular compartments). Compared with linear DNA barcoding, the nanostructured barcodes enhance the signal by more than 100-fold. By implementing the barcoding approach on a microfluidic device for the analysis of rare patient samples, we show that molecular subtypes of breast cancer can be accurately classified and that subcellular spatial markers of disease aggressiveness can be identified.

Pancreatic neuroendocrine neoplasms (PanNENs) have an unpredictable clinical course that varies from indolent to highly malignant. No immunohistochemical markers are available for reliable prediction of the biological behavior of early stage PanNENs. Minichromosome maintenance protein 7 (MCM7) is a putative powerful marker of cell proliferation. Whether the expression of MCM7 is related to the risk of PanNENs progression remains unclear. We assessed the clinical behavior of 156 PanNENs with respect to stage, grade, Ki-67 index, MCM7 index, and other pathologic features. A high MCM7 index was significantly associated with larger tumor size (P.001), nonfunctioning tumor (P.001), increased grade (P.0001), and later TNM stage (P.001). In multivariate analysis, G2/G3 (hazard ratio [HR], 2.21; 95% confidence interval [CI], 1.35-3.62; P.001), stage III/IV (HR, 2.11; 95% CI, 1.31-3.41; P.001), and MCM7 labeling index5% (HR, 3.81; 95% CI, 1.30-11.17; P = .02) were independent negative prognostic factors related to the risk of tumor progression in stage I-IV disease. MCM7 labeling index5% was associated with an increased risk of progression in stages I-V, I-III, and I-II. Our study confirms that MCM7 is a valuable marker for assessing the progression of PanNENs, especially in patients with early stage disease and without distant metastasis.

The controlled assembly of nanomaterials into desired architectures presents many opportunities; however, current preparations lack spatial precision and versatility in developing complex nano-architectures. Inspired by the amphiphilic nature of surfactants, we develop a facile approach to guide nanomaterial integration – spatial organization and distribution – in metal-organic frameworks (MOFs). Named surfactant tunable spatial architecture (STAR), the technology leverages the varied interactions of surfactants with nanoparticles and MOF constituents, respectively, to direct nanoparticle arrangement while molding the growing framework. By surfactant matching, the approach achieves not only tunable and precise integration of diverse nanomaterials in different MOF structures, but also fast and aqueous synthesis, in solution and on solid substrates. Employing the approach, we develop a dual-probe STAR that comprises peripheral working probes and central reference probes to achieve differential responsiveness to biomarkers. When applied for the direct profiling of clinical ascites, STAR reveals glycosylation signatures of extracellular vesicles and differentiates cancer patient prognosis., Current methods for controlled assembly of nanomaterials into desired architectures often lack the precision and versatility to develop complex architectures. Here the authors report STAR, surfactant tunable spatial architecture, to guide nanomaterial integration in metal-organic frameworks.

PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses.

Accessible and adaptable nucleic acid diagnostics remains a critical challenge in managing the evolving COVID‐19 pandemic. Here, an integrated molecular nanotechnology that enables direct and programmable detection of SARS‐CoV‐2 RNA targets in native patient specimens is reported. Termed synergistic coupling of responsive equilibrium in enzymatic network (SCREEN), the technology leverages tunable, catalytic molecular nanostructures to establish an interconnected, collaborative architecture. SCREEN mimics the extraordinary organization and functionality of cellular signaling cascades. Through programmable enzyme–DNA nanostructures, SCREEN activates upon interaction with different RNA targets to initiate multi‐enzyme catalysis; through system‐wide favorable equilibrium shifting, SCREEN directly transduces a single target binding into an amplified electrical signal. To establish collaborative equilibrium coupling in the architecture, a computational model that simulates all reactions to predict overall performance and optimize assay configuration is developed. The developed platform achieves direct and sensitive RNA detection (approaching single‐copy detection), fast response (assay reaction is completed within 30 min at room temperature), and robust programmability (across different genetic loci of SARS‐CoV‐2). When clinically evaluated, the technology demonstrates robust and direct detection in clinical swab lysates to accurately diagnose COVID‐19 patients., A molecular nanotechnology is developed utilizing interconnected enzyme–DNA nanostructures to achieve cooperative multi‐enzyme catalysis. Through programmable nanostructures and favorable equilibrium coupling in the system architecture, the technology achieves sensitive and versatile detection for the direct measurement of SARS‐CoV‐2 RNA targets in native patient specimens (30 min at room temperature).

Transition-state molecular switches enable hyper responsive detection of SARS-CoV-2 in native clinical samples., Despite the importance of nucleic acid testing in managing the COVID-19 pandemic, current detection approaches remain limited due to their high complexity and extensive processing. Here, we describe a molecular nanotechnology that enables direct and sensitive detection of viral RNA targets in native clinical samples. The technology, termed catalytic amplification by transition-state molecular switch (CATCH), leverages DNA-enzyme hybrid complexes to form a molecular switch. By ratiometric tuning of its constituents, the multicomponent molecular switch is prepared in a hyperresponsive state—the transition state—that can be readily activated upon the binding of sparse RNA targets to turn on substantial enzymatic activity. CATCH thus achieves superior performance (~8 RNA copies/μl), direct fluorescence detection that bypasses all steps of PCR (

Nature Biotechnology asks a selection of faculty about the most exciting frontier in their field and the most needed technologies for advancing knowledge and applications. What will be the most important areas of research in biotech over the coming years? Which technologies will be most important to advance knowledge and applications in these areas? Nature Biotechnology reached out to a set of faculty doing outstanding work in research areas representative of the journal’s remit and asked them to contribute their vision of where their fields are going.

The survival of patients with glioblastoma (GBM) remains poor despite radiotherapy and temozolomide (TMZ) chemotherapy after resection. The DNA repair enzymes O6-methylguanine-DNA methyltransferase (MGMT) and alkylpurine-DNA-N-glycosylase (APNG) play key roles in repairing TMZ-induced DNA damages, and resistance to TMZ is correlated to the levels of these enzymes in tissues. Despite the need to monitor treatment efficacy serially, repeat brain biopsies are invasive and complex, and can entail significant complications. Herein, we elaborate on the use of a microfluidic chip designed to measure mRNA levels of MGMT and APNG in enriched cancer exosomes from blood. We demonstrate that exosomal mRNA levels reflect these key enzyme levels in parent cells and that serial exosome analyses of MGMT and APNG levels for patients undergoing TMZ treatment can be correlated to treatment response. As such, this technology has the potential to monitor and predict chemotherapy efficacy in GBM patients in a minimally invasive manner.

The utility of mechanical metamaterials for biomedical applications has seldom been explored. Here we show that a metamaterial that is mechanically responsive to antibody-mediated biorecognition can serve as an optical interferometric mask to molecularly profile extracellular vesicles in ascites fluid from patients with cancer. The metamaterial consists of a hydrogel responsive to temperature and redox activity functionalized with antibodies to surface biomarkers on extracellular vesicles, and is patterned into micrometric squares on a gold-coated glass substrate. Through plasmonic heating, the metamaterial is maintained in a transition state between a relaxed form and a buckled state. Binding of extracellular vesicles from the patient samples to the antibodies on the hydrogel causes it to undergo crosslinking, induced by free radicals generated via the activity of horseradish peroxidase conjugated to the antibodies. Hydrogel crosslinking causes the metamaterial to undergo fast chiral re-organization, inducing amplified changes in its mechanical deformation and diffraction patterns, which are detectable by a smartphone camera. The mechanical metamaterial may find broad utility in the sensitive optical immunodetection of biomolecules.

Biologically templated plasmonics enables simultaneous biophysical and biomolecular analysis of exosome biomarkers., Exosomes are nanoscale vesicles distinguished by characteristic biophysical and biomolecular features; current analytical approaches, however, remain univariate. Here, we develop a dedicated platform for multiparametric exosome analysis—through simultaneous biophysical and biomolecular evaluation of the same vesicles—directly in clinical biofluids. Termed templated plasmonics for exosomes, the technology leverages in situ growth of gold nanoshells on vesicles to achieve multiselectivity. For biophysical selectivity, the nanoshell formation is templated by and tuned to distinguish exosome dimensions. For biomolecular selectivity, the nanoshell plasmonics locally quenches fluorescent probes only if they are target-bound on the same vesicle. The technology thus achieves multiplexed analysis of diverse exosomal biomarkers (e.g., proteins and microRNAs) but remains unresponsive to nonvesicle biomarkers. When implemented on a microfluidic, smartphone-based sensor, the platform is rapid, sensitive, and wash-free. It not only distinguished biomarker organizational states in native clinical samples but also showed that the exosomal subpopulation could more accurately differentiate patient prognosis.

Current technologies to measure drug–target interactions require complex processing and invasive tissue biopsies, limiting their clinical utility for cancer treatment monitoring. Here we develop an analytical platform that leverages circulating extracellular vesicles (EVs) for activity-based assessment of tumour-specific drug–target interactions in patient blood samples. The technology, termed extracellular vesicle monitoring of small-molecule chemical occupancy and protein expression (ExoSCOPE), utilizes bio-orthogonal probe amplification and spatial patterning of molecular reactions within matched plasmonic nanoring resonators to achieve in situ analysis of EV drug dynamics. It measures changes in drug occupancy and protein composition in molecular subpopulations of EVs. When used to monitor various targeted therapies, the ExoSCOPE revealed EV signatures that closely reflected cellular treatment efficacy. We further applied the technology for clinical cancer diagnostics and treatment monitoring. Using a small volume of blood, the ExoSCOPE accurately classified disease status and rapidly distinguished between targeted treatment outcomes, within 24 h after treatment initiation. In this work, the authors develop a platform that leverages extracellular vesicles to measure drug–target engagement and apply it to monitor the outcomes of targeted treatments in lung cancer patients.

Rapid, visual detection of pathogen nucleic acids has broad applications in infection management. Here we present a modular detection platform, termed enzyme-assisted nanocomplexes for visual identification of nucleic acids (enVision). The system consists of an integrated circuit of enzyme–DNA nanostructures, which function as independent recognition and signaling elements, for direct and versatile detection of pathogen nucleic acids from infected cells. The built-in enzymatic cascades produce a rapid color readout for the naked eye; the assay is thus fast (, Rapid, visual detection of pathogens is important for point-of-care diagnostics. Here the authors present enVision, which uses enzyme-DNA complexes to detect pathogen nucleic acids and provide a rapid, smartphone compatible readout.

Accurate and accessible nucleic acid diagnostics is critical to reducing the spread of COVID-19 and resuming socioeconomic activities. Here, we present an integrated platform for the direct detection of SARS-CoV-2 RNA targets near patients. Termed electrochemical system integrating reconfigurable enzyme-DNA nanostructures (eSIREN), the technology leverages responsive molecular nanostructures and automated microfluidics to seamlessly transduce target-induced molecular activation into an enhanced electrochemical signal. Through responsive enzyme-DNA nanostructures, the technology establishes a molecular circuitry that directly recognizes specific RNA targets and catalytically enhances signaling; only upon target hybridization, the molecular nanostructures activate to liberate strong enzymatic activity and initiate cascading reactions. Through automated microfluidics, the system coordinates and interfaces the molecular circuitry with embedded electronics; its pressure actuation and liquid-guiding structures improve not only analytical performance but also automated implementation. The developed platform establishes a detection limit of 7 copies of RNA target per μl, operates against the complex biological background of native patient samples, and is completed in

Extracellular vesicles (EVs) are diverse, nanoscale membrane vesicles actively released by cells. Similar-sized vesicles can be further classified (e.g., exosomes, microvesicles) based on their biogenesis, size, and biophysical properties. Although initially thought to be cellular debris, and thus under-appreciated, EVs are now increasingly recognized as important vehicles of intercellular communication and circulating biomarkers for disease diagnoses and prognosis. Despite their clinical potential, the lack of sensitive preparatory and analytical technologies for EVs poses a barrier to clinical translation. New analytical platforms including molecular ones are thus actively being developed to address these challenges. Recent advances in the field are expected to have far-reaching impact in both basic and translational studies. This article aims to present a comprehensive and critical overview of emerging analytical technologies for EV detection and their clinical applications.

Programmable Detection of SARS‐CoV‐2 In article number 2101155, Huilin Shao and co‐workers report the collaborative coupling of enzyme–DNA nanostructures to establish multi‐enzyme catalysis. Through programmable nanostructures and favorable equilibrium architecture, the technology achieves direct and sensitive detection of SARS‐CoV‐2 RNA in native patient samples, within 30 minutes at room temperature. [Image: see text]

DNA tetrahedron as the simplest 3D DNA nanostructure has been applied widely in biomedicine and biosensing. Herein, we design and fabricate a series of circular assemblies of DNA tetrahedron with high purity and decent yields. These circular nanostructures are confirmed by endonuclease digestion, gel electrophoresis and atomic force microscopy. Inspired by rotary protein motor, we demonstrate these circular architectures can serve as a stator for a rotary DNA motor to achieve the circular rotation. The DNA motor can rotate on the stators for several cycles, and the locomotion of the motor is monitored by the real-time fluorescent measurements. MOE (Min. of Education, S’pore) Published version

Extracellular vesicles (EVs) are diverse, nanoscale membrane vesicles released by cells into the circulation. As an emerging class of circulating biomarkers, EVs contain a trove of molecular information and play important roles in mediating intercellular communication. These EV molecular cargoes are differentially organized in the vesicles; they could be inherited from the parent cells or bound to the EV membrane through surface interactions. While the inherited constituents could serve as cell surrogate biomarkers, extravesicular association could reflect structural states of the bound molecules, revealing distinct subpopulations with different biophysical and/or biochemical properties. Despite the clinical potential of EVs and their diverse contents, conventional sensing technologies have limited compatibility to reveal nanoscale EV features. Complementary analytical platforms are being developed to address these technical challenges and expand the biomedical applications of EVs, to establish novel correlations and empower new diagnostics. This article provides a perspective on recent developments in sensor technologies to profile the diverse contents-different molecular types, quantities, and organizational states-in extracellular vesicles.

Objective: The identification of DNA polymerase epsilon (POLE) mutation subtypes in endometrial cancer is critical for molecular classification. The mutation of the POLE gene could only be detected by sequencing until now. We propose to validate and develop the feasibility of using BaseScope, an in situ hybridization (ISH) assay, for the detection of POLE mutations in high-grade endometrioid carcinomas (EC). Methods: Among 51 paraffin-embedded samples of high-grade EC, BaseScope-ISH assays were used to detect the RNA mutation status of the POLE gene, mainly focusing on two hotspot mutations of P286R and V411L. The number of positive signals in the cytoplasm was counted, setting the positive threshold and determining the in situ hybridization results. The sensitivity and specificity of BaseScope-ISH assay were compared with that of the Sanger sequencing results. Results: Based on the BaseScope assay, there were 19 positive samples and 32 negative samples in a total of 51 samples. Of the 19 positive samples, 10 samples showed P286R site mutations in the POLE gene, while the other nine samples were V411L site mutations. Only one sample with the V411L site mutation identified by Sanger sequencing showed negative signal value. The remaining 31 cases without the P286R site mutation or V411L site mutations all showed negative signal. This analysis result showed the sensitivity was 95% and the specificity was 100% for the BaseScope assay detecting POLE mutants in high-grade EC. Conclusion: In the case of high-grade EC, combined with morphological characteristics, the BaseScope assay can effectively and specifically identify POLE mutation cases, providing a reliable foundation for the application of clinical diagnosis and molecular classification.

Sweat is a readily accessible bodily fluid for detecting biomarkers such as pH, glucose etc., enabling continuous and non-invasive assessment of the well-being of individuals. Our proposed work aims at leveraging pulse oximeter chips in current-day fitness trackers for real-time continuous monitoring of pH in sweat. We achieve that by fabricating a highly responsive and long-term reusable pH sweat sensor on a flexible material to achieve skin conformity, targeting the sensor to work at the reflected infrared (880nm) and red (660nm) photoplethysmograph (PPG) signal intensities recorded by pulse oximeters. The sensor can be readily mounted atop any wearable with a pulse oximeter. We have successfully demonstrated a low-cost, low-power, highly-responsive and long-term reusable wrist-worn wearable prototype, pH Watch, for real-time continuous monitoring of pH value of sweat. We conducted on-body trials with 10 participants and pH Watch achieves an accuracy of $\approx$91%. We also showed that the integration of our sweat sensor does not hinder the pulse oximeter from measuring heart rate and SpO\textsubscript2, and users can continue with their daily activities with motion artifacts removed efficiently from PPG signals using the TROIKA framework, resulting in heart rate and SpO\textsubscript2 measurements with an accuracy of $\approx$95% and $\approx$96% respectively when validated against commercial finger pulse oximeter measurements. To the best of our knowledge, pH Watch is the first demonstration of a reusable sweat sensor that can be readily integrated into today's smart watches with pulse oximeters, paving the way for ubiquitous sensing of biomarkers.

Neurodegenerative diseases are heterogeneous disorders characterized by a progressive loss of function and/or death of nerve cells, leading to severe cognitive and functional decline. Due to the complex pathology, early detection and intervention are critical to the development of successful treatments; however, current diagnostic approaches are limited to subjective, late-stage clinical findings. Extracellular vesicles (EVs) have recently emerged as a promising circulating biomarker for neurodegenerative diseases. Actively released by diverse cells, EVs are nanoscale membrane vesicles. They abound in blood, readily cross the blood-brain barrier, and carry diverse molecular cargoes in different organizational states: these molecular cargoes are inherited from the parent cells or bound to the EV membrane through surface associations. Specifically, EVs have been found to be associated with several important pathogenic proteins of neurodegenerative diseases, and their involvement could alter disease progression. This article provides an overview of EVs as circulating biomarkers of neurodegenerative diseases and introduces new technological advances to characterize the biophysical properties of EV-associated biomarkers for accurate, blood-based detection of neurodegenerative diseases.

Motor neuron diversification and regionalization are important hallmarks of spinal cord development and rely on fine spatiotemporal release of molecular cues. Here, we present a dedicated platform to engineer complex molecular profiles for directed neuronal differentiation. Methods: The technology, termed microhexagon interlace for generation of versatile and fine gradients (microHIVE), leverages on an interlocking honeycomb lattice of microstructures to dynamically pattern molecular profiles at a high spatial resolution. By packing the microhexagons as a divergent, mirrored array, the platform not only enables maximal mixing efficiency but also maintains a small device footprint. Results: Employing the microHIVE platform, we developed optimized profiles of growth factors to induce rostral-caudal patterning of spinal motor neurons, and directed stem cell differentiation in situ into a spatial continuum of different motor neuron subtypes. Conclusions: The differentiated cells showed progressive RNA and protein signatures, consistent with that of representative brachial, thoracic and lumbar regions of the human spinal cord. The microHIVE platform can thus be utilized to develop advanced biomimetic systems for the study of diseases in vitro.

Massively parallel DNA sequencing is established, yet high-throughput protein profiling remains challenging. Here, we report a barcoding approach that leverages the combinatorial sequence content and the configurational programmability of DNA nanostructures for high-throughput multiplexed profiling of the subcellular expression and distribution of proteins in whole cells. The barcodes are formed by in situ hybridization of tetrahedral DNA nanostructures and short DNA sequences conjugated with protein-targeting antibodies, and by nanostructure-assisted ligation (either enzymatic or chemical) of the nanostructures and exogenous DNA sequences bound to nanoparticles of different sizes (which cause these localization sequences to differentially distribute across subcellular compartments). Compared with linear DNA barcoding, the nanostructured barcodes enhance the signal by more than 100-fold. By implementing the barcoding approach on a microfluidic device for the analysis of rare patient samples, we show that molecular subtypes of breast cancer can be accurately classified and that subcellular spatial markers of disease aggressiveness can be identified.

Exosomes have emerged as a promising biomarker. These vesicles abound in biofluids and harbor molecular constituents from their parent cells, thereby offering a minimally-invasive avenue for molecular analyses. Despite such clinical potential, routine exosomal analysis, particularly the protein assay, remains challenging, due to requirements for large sample volumes and extensive processing. We have been developing miniaturized systems to facilitate clinical exosome studies. These systems can be categorized into two components: microfluidics for sample preparation and analytical tools for protein analyses. In this report, we review a new assay platform, nano-plasmonic exosome (nPLEX), in which sensing is based on surface plasmon resonance to achieve label-free exosome detection. Looking forward, we also discuss some potential challenges and improvements in exosome studies.

Microvesicles (MVs) are an increasingly important source for biomarker discovery and clinical diagnostics. The small size of MVs and their presence in complex biological environment, however, pose technical challenges in sample preparation, particularly when sample volumes are small. We herein present an acoustic nanofilter system that size-specifically separates MVs in a continuous and contact-free manner. The separation uses ultrasound standing waves to exert differential acoustic force on MVs according to their size and density. By optimizing the design of the ultrasound transducers and underlying electronics, we were able to achieve a high separation yield and resolution. The "filter size-cutoff" can be controlled electronically in situ, which enables versatile MV-size selection. We applied the acoustic nanofilter to isolate nanoscale (200 nm) vesicles from cell culture media as well as MVs in stored red blood cell products. With the capacity for rapid and contact-free MV isolation, the developed system could become a versatile preparatory tool for MV analyses.

The growing emphasis on personalized medicine significantly increases the need to analyze key molecular markers. In comparison to tissue biopsies, circulating biomarkers (liquid biopsies) can be conveniently and repeatedly obtained from biofluids with minimal complications. In particular, exosomes have recently emerged as a promising circulating biomarker. Exosomes are nanometer-sized membrane vesicles actively shed off by cells and possess unique advantages: they abound in biofluids, readily cross the blood brain barrier and harbor diverse molecular contents. A sensor platform capable of sensitive and rapid detection of exosomes would thus be an invaluable tool in translating their clinical potential. In this talk, I will describe various systems that we have developed for quantitative analyses of circulating cancer exosomes. By enabling rapid, sensitive and cost-effective detection of circulating biomarkers, these platforms could significantly expand the reach of preclinical and clinical research, in informing therapy selection, rationally directing trials, and improving sequential monitoring to achieve better clinical outcomes.

Exosomes show potential for cancer diagnostics because they transport molecular contents of the cells from which they originate. Detection and molecular profiling of exosomes is technically challenging and often requires extensive sample purification and labeling. Here we describe a label-free, high-throughput approach for quantitative analysis of exosomes. Our nano-plasmonic exosome (nPLEX) assay is based on transmission surface plasmon resonance through periodic nanohole arrays. Each array is functionalized with antibodies to enable profiling of exosome surface proteins and proteins present in exosome lysates. We show that this approach offers improved sensitivity over previous methods, enables portable operation when integrated with miniaturized optics and allows retrieval of exosomes for further study. Using nPLEX to analyze ascites samples from ovarian cancer patients, we find that exosomes derived from ovarian cancer cells can be identified by their expression of CD24 and EpCAM, suggesting the potential of exosomes for diagnostics.