@article{nokey,

title = {Combating Antimicrobial Resistance via Single-Cell Diagnostic Technologies Powered by Droplet Microfluidics},

author = {K. Hsieh and KE. Mach and P. Zhang and JC. Liao and TH. Wang},

url = {https://pubmed.ncbi.nlm.nih.gov/34898173/},

doi = {DOI: 10.1021/acs.accounts.1c00462},

year  = {2022},

date = {2022-01-18},

journal = {Accounts of Chemical Research},

volume = {55},

pages = {123–133},

abstract = {Antimicrobial resistance is a global threat that if left unchecked could lead to 10 million annual mortalities by 2050. One factor contributing to the rise of multi-drug-resistant (MDR) pathogens is the reliance on traditional culture-based pathogen identification (ID) and antimicrobial susceptibility testing (AST) that typically takes several days. This delay of objective pathogen ID and AST information to inform clinical decision making results in clinicians treating patients empirically often using first-line, broad-spectrum antibiotics, contributing to the misuse/overuse of antibiotics. To combat the rise in MDR pathogens, there is a critical demand for rapid ID and AST technologies. Among the advances in ID and AST technologies in the past decade, single-cell diagnostic technologies powered by droplet microfluidics offer great promise due to their potential for high-sensitivity detection and rapid turnaround time. Our laboratory has been at the forefront of developing such technologies and applying them to diagnosing urinary tract infections (UTIs), one of the most common infections and a frequent reason for the prescription of antimicrobials. For pathogen ID, we first demonstrated the highly sensitive, amplification-free detection of single bacterial cells by confining them in picoliter-scale droplets and detection with fluorogenic peptide nucleic acid (PNA) probes that target their 16S rRNA (rRNA), a well-characterized marker for phylogenic classification. We subsequently improved the PNA probe design and enhanced detection sensitivity. For single-cell AST, we first employed a growth indicator dye and engineered an integrated device that allows us to detect growth from single bacterial cells under antibiotic exposure within 1 h, equivalent to two to three bacterial replications. To expand beyond testing a single antibiotic condition per device, a common limitation for droplet microfluidics, we developed an integrated programmable droplet microfluidic device for scalable single-cell AST. Using the scalable single-cell AST platform, we demonstrated the generation of up to 32 droplet groups in a single device with custom antibiotic titers and the capacity to scale up single-cell AST, and providing reliable pathogen categories beyond a binary call embodies a critical advance. Finally, we developed an integrated ID and AST platform. To this end, we developed a PNA probe panel that can identify nearly 90% of uropathogens and showed the quantitative detection of 16S rRNA from single bacterial cells in droplet-enabled AST after as little as 10 min of antibiotic exposure. This platform achieved both ID and AST from minimally processed urine samples in 30 min, representing one of the fastest turnaround times to date. In addition to tracing the development of our technologies, we compare them with contemporary research advances and offer our perspectives for future development, with the vision that single-cell ID and AST technologies powered by droplet microfluidics can indeed become a useful diagnostic tool for combating antimicrobial resistance},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Li2022b,

title = {Combinatorial nanodroplet platform for screening antibiotic combinations},

author = {H. Li and P. Zhang and K. Hsieh and T.H. Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2022/lc/d1lc00865j},

doi = {DOI	https://doi.org/10.1039/D1LC00865J},

year  = {2022},

date = {2022-01-11},

journal = {Lab on a Chip},

volume = {22},

pages = {621-631},

abstract = {The emergence and spread of multidrug resistant bacterial strains and concomitant dwindling of effective antibiotics pose worldwide healthcare challenges. To address these challenges, advanced engineering tools are developed to personalize antibiotic treatments by speeding up the diagnostics that is critical to prevent antibiotic misuse and overuse and make full use of existing antibiotics. Meanwhile, it is necessary to investigate novel antibiotic strategies. Recently, repurposing mono antibiotics into combinatorial antibiotic therapies has shown great potential for treatment of bacterial infections. However, widespread adoption of drug combinations has been hindered by the complexity of screening techniques and the cost of reagent consumptions in practice. In this study, we developed a combinatorial nanodroplet platform for automated and high-throughput screening of antibiotic combinations while consuming orders of magnitude lower reagents than the standard microtiter-based screening method. In particular, the proposed platform is capable of creating nanoliter droplets with multiple reagents in an automatic manner, tuning concentrations of each component, performing biochemical assays with high flexibility (e.g., temperature and duration), and achieving detection with high sensitivity. A biochemical assay, based on the reduction of resazurin by the metabolism of bacteria, has been characterized and employed to evaluate the combinatorial effects of the antibiotics of interest. In a pilot study, we successfully screened pairwise combinations between 4 antibiotics for a model Escherichia coli strain.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens},

author = {AY. Trick and FE. Chen and L. Chen and PW. Lee and AC. Hasnain and HH. Mostafa and KC. Carroll and TH. Wang},

url = {https://onlinelibrary.wiley.com/doi/full/10.1002/admt.202101013},

doi = { https://doi.org/10.1002/admt.202101013},

year  = {2022},

date = {2022-01-07},

journal = {Advanced Materials Technologies},

volume = {7},

number = {2101013},

issue = {6},

abstract = {The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kranenbarg2021,

title = {High performance methylated DNA markers for detection of colon adenocarcinoma},

author = {RAM. Klein Kranenbarg and AH. Vali and JNM. IJzermans and TR. Pisanic and TH. Wang and N. Azad and S. Sukumar and M. Fackler},

url = {https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-021-01206-2},

doi = {https://doi.org/10.1186/s13148-021-01206-2},

year  = {2021},

date = {2021-12-13},

journal = {Clinical Epigenetics},

volume = {13},

issue = {1},

pages = {1-15},

abstract = {Background

Colon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. In this study, we systematically selected and tested methylated markers that demonstrate high sensitivity and specificity for detection of CC in tissue and circulating cell-free DNA.



Methods

Hierarchical analysis of 22 candidate CpG loci was conducted using The Cancer Genome Atlas (TCGA) COAD 450K HumanMethylation database. Methylation of 13 loci was analyzed using quantitative multiplex methylation-specific PCR (QM-MSP) in a training set of fresh frozen colon tissues (N = 53). Hypermethylated markers were identified that were highest in cancer and lowest in normal colon tissue using the 75th percentile in Mann–Whitney analyses and the receiver operating characteristic (ROC) statistic. The cumulative methylation status of the marker panel was assayed in an independent test set of fresh frozen colon tissues (N = 52) using conditions defined and locked in the training set. A minimal marker panel of 6 genes was defined based on ROC area under the curve (AUC). Plasma samples (N = 20 colorectal cancers, stage IV and N = 20 normal) were tested by cMethDNA assay to evaluate marker performance in liquid biopsy.



Results

In the test set of samples, compared to normal tissue, a 6-gene panel showed 100% sensitivity and 90% specificity for detection of CC, and an AUC of 1.00 (95% CI 1.00, 1.00). In stage IV colorectal cancer plasma versus normal, an 8-gene panel showed 95% sensitivity, 100% specificity, and an AUC of 0.996 (95% CI 0.986, 1.00) while a 5-gene subset showed 100% sensitivity, 100% specificity, and an AUC of 1.00 (95% CI 1.00, 1.00), highly concordant with our observations in tissue.



Conclusions

We identified high performance methylated DNA marker panels for detection of CC. This knowledge has set the stage for development and implementation of novel, automated, self-contained CC detection assays in tissue and blood which can expeditiously and accurately detect colon cancer in both developed and underdeveloped regions of the world, enabling optimal use of limited resources in low- and middle-income countries.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhang2021,

title = {A Cascaded Droplet Microfluidic Platform Enables High-throughput Single Cell Antibiotic Susceptibility Testing at Scale},

author = {P. Zhang and A. Kaushik and K. Hsieh and S. Li and S. Lewis and K.E. Mach and .C. Liao and K.C. Carroll},

url = {https://onlinelibrary.wiley.com/doi/full/10.1002/smtd.202101254},

doi = {https://doi.org/10.1101/2021.06.25.21259551},

year  = {2021},

date = {2021-12-08},

urldate = {2021-07-02},

journal = {Small Methods},

volume = {6},

issue = {1},

abstract = {The global threat of antibiotic resistance underscores critical but unmet needs for rapid antibiotic susceptibility testing (AST) technologies. To this end, droplet microfluidic-based single-cell AST offers promise by achieving unprecedented rapidity, but its potential for clinical use is marred by the capacity of testing one to few antibiotic conditions per device, which falls short from the required scale in clinically relevant scenarios. To lift the scalability constraint in rapid single-cell AST technologies, a new cascaded droplet microfluidic platform that can streamline bacteria/antibiotic mixing, single-cell encapsulation within picoliter droplets, incubation, and detection in a continuous, assembly-line-like workflow is developed. The scalability of the platform is demonstrated by generating 32 groups of ≈10 000 droplets with custom antibiotic conditions within a single device, from which a new statistics-based method is used to analyze the single cell data and produce clinically useful antibiograms with minimum inhibitory concentrations in ≈90 min for the first antibiotic, plus 2 min for each subsequent antibiotic condition. Potential clinical utility of this platform is demonstrated by testing three clinical isolates and eight urine specimens against four frequently used antibiotics, and 100% and 93.8% categorical agreements are achieved compared to laboratory-based results that became available after 48 h.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Bridging the gap between development of point-of-care nucleic acid testing and patient care for sexually transmitted infections},

author = {K. Hsieh, and JH. Melendez and CA. Gaydos and TH. Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2022/lc/d1lc00665g},

doi = {DOI	https://doi.org/10.1039/D1LC00665G},

year  = {2021},

date = {2021-11-22},

journal = {Lab on a Chip},

volume = {22},

issue = {3},

pages = {476-511},

abstract = {The incidence rates of sexually transmitted infections (STIs), including the four major curable STIs – chlamydia, gonorrhea, trichomoniasis and, syphilis – continue to increase globally, causing medical cost burden and morbidity especially in low and middle-income countries (LMIC). There have seen significant advances in diagnostic testing, but commercial antigen-based point-of-care tests (POCTs) are often insufficiently sensitive and specific, while near-point-of-care (POC) instruments that can perform sensitive and specific nucleic acid amplification tests (NAATs) are technically complex and expensive, especially for LMIC. Thus, there remains a critical need for NAAT-based STI POCTs that can improve diagnosis and curb the ongoing epidemic. Unfortunately, the development of such POCTs has been challenging due to the gap between researchers developing new technologies and healthcare providers using these technologies. This review aims to bridge this gap. We first present a short introduction of the four major STIs, followed by a discussion on the current landscape of commercial near-POC instruments for the detection of these STIs. We present relevant research toward addressing the gaps in developing NAAT-based STI POCT technologies and supplement this discussion with technologies for HIV and other infectious diseases, which may be adapted for STIs. Additionally, as case studies, we highlight the developmental trajectory of two different POCT technologies, including one approved by the United States Food and Drug Administration (FDA). Finally, we offer our perspectives on future development of NAAT-based STI POCT technologies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{H.Melendez2021,

title = {Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae using a Phenotypic-Molecular Assay and Lyophilized Antimicrobials},

author = {JH.Melendez and FE. Chen and TH. Wang},

url = {https://www.sciencedirect.com/science/article/pii/S0732889321002819},

doi = {https://doi.org/10.1016/j.diagmicrobio.2021.115590},

year  = {2021},

date = {2021-10-24},

urldate = {2021-10-24},

journal = {Diagnostic Microbiology and Infectious Disease},

volume = {115590},

abstract = {Gonorrhea is an urgent global public health threat as Neisseria gonorrhoeae (Ng) has progressively developed resistance to all antibiotics commonly used for treatment. Surveillance of antimicrobial susceptibility trends is critical to monitor the emergence and spread of antimicrobial resistance. The gold standard methods for antimicrobial susceptibility testing (AST) of Ng are laborious and time-consuming. We evaluated a phenotypic molecular approach, involving a short cultivation step and quantitative PCR, with lyophilized antimicrobials to characterize antimicrobial susceptibility in Ng. There was excellent concordance between AST performed with liquid and lyophilized ciprofloxacin, penicillin, and tetracycline using the pheno-molecular assay, following a four-hour incubation step. The categorical agreement between the pheno-molecular assay and the gold standard AST results was 92.4% for characterization of antimicrobial susceptibility. Essential agreement between the two methods was 91.9%. Characterization of ceftriaxone susceptibility in Ng using the pheno-molecular assay required a six-hour incubation step.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ying2021,

title = {Methylation-based Cell-free DNA Signature for Early Detection of Pancreatic Cancer},

author = {L. Ying and A. Sharma and A. Chhoda and N. Ruzgar and N. Hasan and R. Kwak and CL. Wolfgang and TH. Wang and JW. Kunstman and RR. Salem and LD. Wood and C. Iacobuzio-Donahue and EB. Schneider and JJ. Farrell and N. Ahuja },

url = {https://pubmed.ncbi.nlm.nih.gov/34860810/},

doi = {DOI: 10.1097/MPA.0000000000001919},

year  = {2021},

date = {2021-10-01},

journal = {Pancreas },

volume = {50},

issue = {9},

pages = {1267-1273},

abstract = {Objectives: The potential of DNA methylation alterations in early pancreatic cancer (PC) detection among pancreatic tissue cell-free DNA seems promising. This study investigates the diagnostic capacity of the 4-gene methylation biomarker panel, which included ADAMTS1, BNC1, LRFN5, and PXDN genes, in a case-control study.



Methods: A genome-wide pharmacoepigenetic approach identified ADAMTS1, BNC1, LRFN5, and PXDN genes as putative targets. Tissue samples including stage I-IV PC (n = 44), pancreatic intraepithelial neoplasia (n = 15), intraductal papillary mucinous neoplasms (n = 24), and normal pancreas (n = 8), and cell-free DNA, which was acquired through methylation on beads technology from PC (n = 22) and control patients (n = 10), were included. The 2-∆ct was the outcome of interest and underwent receiver operating characteristic analysis to determine the diagnostic accuracy of the panel.



Results: Receiver operating characteristic analysis revealed an area under the curve of 0.93 among ADAMTS1, 0.76 among BNC1, 0.75 among PXDN, and 0.69 among LRFN5 gene. The combination gene methylation panel (ADAMTS1, BNC1, LRFN5, and PXDN) had an area under the curve of 0.94, with a sensitivity of 100% and specificity of 90%.



Conclusions: This methylation-based biomarker panel had promising accuracy for PC detection and warranted further validation in prospective PC surveillance trials.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {High resolution estimates of relative gene abundance with quantitative ratiometric regression PCR (qRR-PCR)},

author = {A.Y. Trick and F.E. Chen and J.A. Schares and B.E. Freml and P. Lor and Y. Yun and T.H. Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2021/an/d1an01397a},

year  = {2021},

date = {2021-09-30},

journal = {Analyst},

volume = {146},

pages = {6463-6469},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Magnetofluidic immuno-PCR for point-of-care COVID-19 serological testing},

author = {P. Zhang and L. Chen and J. Hu and A.Y. Trick and F.E. Chen and K. Hsieh and Y. Zhao and B. Coleman and K. Kruczynski and T.R Pisanic II and C. D Heaney and W.A, Clarke and Tza-Huei Wang},

url = {https://pubmed.ncbi.nlm.nih.gov/34600203/},

doi = {10.1016/j.bios.2021.113656},

year  = {2021},

date = {2021-09-23},

urldate = {2021-09-23},

journal = {Biosensors and Bioelectronic},

volume = {195},

number = {113656},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {A Rapid Single-Cell Antimicrobial Susceptibility Testing Workflow for Bloodstream Infections},

author = {B. Forsyth and P. Torab and J.H. Lee and T. Malcom and T.H. Wang and J.C. Liao and S.Yang and E. Kvam and C. Puleo and P.K. Wong},

doi = {https://doi.org/10.3390/bios11080288},

year  = {2021},

date = {2021-08-22},

urldate = {2021-08-22},

journal = {Biosensors},

volume = {11},

number = {8},

pages = {288},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2021,

title = {Portable Magnetofluidic Device for Point-of-Need Detection of African Swine Fever},

author = {L. Chen and K. Wen and F.E. Chen and A.Y. Trick and H. Liu and S. Shao and W. Yu and K. Hsieh and Z. Wang and J. Shen and T.H. Wang},

url = {https://pubs.acs.org/doi/full/10.1021/acs.analchem.1c01814},

year  = {2021},

date = {2021-07-28},

journal = {Analytical Chemistry},

volume = {93},

number = {31},

pages = {10940-10946},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2021b,

title = {Point-of-Care CRISPR-Cas-Assisted SARS-CoV-2 Detection in an Automated and Portable Droplet Magnetofluidic Device},

author = { F.E. Chen and P.W. Lee and A.Y. Trick and J.S. Park and L. Chen and K. Shah and H. Mostafa and K.C. Carroll and K. Hsieh and T.H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8170879/},

doi = {doi: 10.1016/j.bios.2021.113390},

year  = {2021},

date = {2021-06-02},

journal = {Biosensors and Bioelectronics},

volume = {113390},

abstract = {In the fight against COVID-19, there remains an unmet need for point-of-care (POC) diagnostic testing tools that can rapidly and sensitively detect the causative SARS-CoV-2 virus to control disease transmission and improve patient management. Emerging CRISPR-Cas-assisted SARS-CoV-2 detection assays are viewed as transformative solutions for POC diagnostic testing, but lack of streamlined sample preparation and full integration within an automated and portable device hamper their potential for POC use. We report herein POC-CRISPR – a single-step CRISPR-Cas-assisted assay that leverages facile magnetic concentration and transport of nucleic acid-binding magnetic beads to incorporate sample preparation with minimal manual operation. Moreover, POC-CRISPR has been adapted into a compact thermoplastic cartridge within a palm-sized yet fully-integrated and automated device. During analytical evaluation, POC-CRISPR was able detect 1 genome equivalent/μL SARS-CoV-2 RNA from a sample volume of 100 μL in < 30 min. Finally, when evaluated with 27 unprocessed clinical nasopharyngeal swab eluates that were pre-typed by standard RT-qPCR (Cq values ranged from 18.3 to 30.2 for the positive samples), POC-CRISPR achieved 27 out of 27 concordance and could detect positive samples with high SARS-CoV-2 loads (Cq < 25) in 20 min.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {A phase 2 trial of gemcitabine and docetaxel in patients with metastatic colorectal adenocarcinoma with methylated checkpoint with forkhead and ring finger domain promoter and/or microsatellite instability phenotype},

author = {M. Baretti and E. Karunasena and M. Zahurak and R. Walker and Y. Zhao and T.R. Pisanic 2nd and T.H. Wang and T.F. Greten and A.G. Duffy and E. Gootjes and G. Meijer and HMW Verheul and N. Ahuja and J.G. Herman and N.S. Azad},

url = {https://pubmed.ncbi.nlm.nih.gov/33811727/},

doi = {10.1111/cts.12960},

year  = {2021},

date = {2021-05-14},

journal = {Clinical and Translational Science},

volume = {14},

pages = {954-963},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Trick2021,

title = {A portable magnetofluidic platform for detecting sexually transmitted infections and antimicrobial susceptibility},

author = {Alexander Y. Trick and Johan H. Melendez and Fan-En Chen and Liben Chen and Annet Onzia and Aidah Zawedde and Edith Nakku-Joloba and Peter Kyambadde and Emmanuel Mande and Joshua Matovu and Maxine Atuheirwe and Richard Kwizera and Elizabeth A. Gilliams and Yu-Hsiang Hsieh and Charlotte A. Gaydos and Yukari C. Manabe and Matthew M. Hamill and Tza-Huei Wang},

url = {https://stm.sciencemag.org/content/13/593/eabf6356},

doi = {DOI: 10.1126/scitranslmed.abf6356},

year  = {2021},

date = {2021-05-12},

journal = {SCIENCE TRANSLATIONAL MEDICINE},

volume = {13},

number = {593},

pages = {eab6356},

abstract = {Abstract

Effective treatment of sexually transmitted infections (STIs) is limited by diagnostics that cannot deliver results rapidly while the patient is still in the clinic. The gold standard methods for identification of STIs are nucleic acid amplification tests (NAATs), which are too expensive for widespread use and have lengthy turnaround times. To address the need for fast and affordable diagnostics, we have developed a portable, rapid, on-cartridge magnetofluidic purification and testing (PROMPT) polymerase chain reaction (PCR) test. We show that it can detect Neisseria gonorrhoeae, the pathogen causing gonorrhea, with simultaneous genotyping of the pathogen for resistance to the antimicrobial drug ciprofloxacin in <15 min. The duplex test was integrated into a low-cost thermoplastic cartridge with automated processing of penile swab samples from patients using magnetic beads. A compact instrument conducted DNA extraction, PCR, and analysis of results while relaying data to the user via a smartphone app. This platform was tested on penile swab samples from sexual health clinics in Baltimore, MD, USA (n = 66) and Kampala, Uganda (n = 151) with an overall sensitivity and specificity of 97.7% (95% CI, 94.7 to 100%) and 97.6% (95% CI, 94.1 to 100%), respectively, for N. gonorrhoeae detection and 100% concordance with culture results for ciprofloxacin resistance. This study paves the way for delivering accessible PCR diagnostics for rapidly detecting STIs at the point of care, helping to guide treatment decisions and combat the rise of antimicrobial resistant pathogens.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhang2021b,

title = {Facile syringe filter-enabled bacteria separation, enrichment, and buffer exchange for clinical isolation-free digital detection and characterization of bacterial pathogens in urine},

author = {P. Zhang and A. Kaushik and K.E. Mach and K. Hsieh and J.C. Liao and T.H, Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2021/an/d1an00039j},

year  = {2021},

date = {2021-03-22},

journal = {Analyst},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kaushik2021,

title = {Droplet-based single-cell measurements of 16S rRNA enable integrated bacteria identification and pheno-molecular antimicrobial susceptibility testing from clinical samples in 30 min},

author = {A.M. Kaushik and K. Hsieh and K.E. Mach and S. Lewis and C.M. Puleo and K.C. Carroll and J.C. Liao and T.H. Wang},

url = {https://onlinelibrary.wiley.com/doi/full/10.1002/advs.202003419},

doi = {https://doi.org/10.1002/advs.202003419},

year  = {2021},

date = {2021-02-01},

journal = {Advanced Science},

volume = {8},

number = {6},

abstract = {Empiric broad‐spectrum antimicrobial treatments of urinary tract infections (UTIs) have contributed to widespread antimicrobial resistance. Clinical adoption of evidence‐based treatments necessitates rapid diagnostic methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) with minimal sample preparation. In response, a microfluidic droplet‐based platform is developed for achieving both ID and AST from urine samples within 30 min. In this platform, fluorogenic hybridization probes are utilized to detect 16S rRNA from single bacterial cells encapsulated in picoliter droplets, enabling molecular identification of uropathogenic bacteria directly from urine in as little as 16 min. Moreover, in‐droplet single‐bacterial measurements of 16S rRNA provide a surrogate for AST, shortening the exposure time to 10 min for gentamicin and ciprofloxacin. A fully integrated device and screening workflow were developed to test urine specimens for one of seven unique diagnostic outcomes including the presence/absence of Gram‐negative bacteria, molecular ID of the bacteriaas Escherichia coli, an Enterobacterales, or other organism, and assessment of bacterial susceptibility to ciprofloxacin. In a 50‐specimen clinical comparison study, the platform demonstrates excellent performance compared to clinical standard methods (areas‐under‐curves, AUCs >0.95), within a small fraction of the turnaround time, highlighting its clinical utility.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chamling2021,

title = {Single-Cell Transcriptomic Reveals Molecular Diversity and Developmental Heterogeneity of Human Stem Cell-derived Oligodendrocyte Lineage Cells},

author = {X. Chamling and A. Kallman and W. Fang and C. Berlinicke and J. Mertz and P. Devkota and I.M. Pantoja and M. Smith and Z. Ji and C. Chang and A. Kaushik and L. Chen and K. Whartenby and P. Calabresi and H.Q. Mao and H. Ji and T.H. Wang, and D. Zack},

url = {https://www.nature.com/articles/s41467-021-20892-3},

year  = {2021},

date = {2021-01-28},

journal = {Nature Communications},

volume = {12},

number = {652},

abstract = {Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of human oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, here we describe time-course single-cell-transcriptomic analysis of developing human stem cell-derived oligodendrocyte-lineage-cells (hOLLCs). The study includes hOLLCs derived from both genome engineered embryonic stem cell (ESC) reporter cells containing an Identification-and-Purification tag driven by the endogenous PDGFRα promoter and from unmodified induced pluripotent (iPS) cells. Our analysis uncovers substantial transcriptional heterogeneity of PDGFRα-lineage hOLLCs. We discover sub-populations of human oligodendrocyte progenitor cells (hOPCs) including a potential cytokine-responsive hOPC subset, and identify candidate regulatory genes/networks that define the identity of these sub-populations. Pseudotime trajectory analysis defines developmental pathways of oligodendrocytes vs astrocytes from PDGFRα-expressing hOPCs and predicts differentially expressed genes between the two lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of these pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2020,

title = {Toward Decentralizing Antibiotic Susceptibility Testing via Ready-to-Use Microwell Array and Resazurin-Aided Colorimetric Readout},

author = {F.E. Chen and A. Kaushik and K. Hsieh and E. Chang and L. Chen and P. Zhang and T.H. Wang},

url = {https://pubs.acs.org/doi/10.1021/acs.analchem.0c04095},

doi = { https://doi.org/10.1021/acs.analchem.0c04095},

year  = {2021},

date = {2021-01-26},

urldate = {2020-11-29},

journal = {Analytical Chemistry},

volume = {93},

number = {3},

pages = {1260–1265},

abstract = {In the face of the global threat from drug-resistant superbugs, there remains an unmet need for simple and accessible diagnostic tools that can perform important antibiotic susceptibility testing against pathogenic bacteria and guide antibiotic treatments outside of centralized clinical laboratories. As a potential solution to this important problem, we report herein the development of a microwell array-based resazurin-aided colorimetric antibiotic susceptibility test (marcAST). At the core of marcAST is a ready-to-use microwell array device that is preassembled with custom titers of various antibiotics and splits bacterial samples upon a simple syringe injection step to initiate AST against all antibiotics. We also employ resazurin, which changes from blue to pink in the presence of growing bacteria, to accelerate and enable colorimetric readout in our AST. Even with its simplicity, marcAST can accurately measure the minimum inhibitory concentrations of reference bacterial strains against common antibiotics and categorize the antibiotic susceptibilities of clinically isolated bacteria. With more characterization and refinement, we envision that marcAST can become a potentially useful tool for performing AST without trained personnel, laborious procedures, or bulky instruments, thereby decentralizing this important test for combating drug-resistant superbugs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Scherer2021,

title = {Digital electrical impedance analysis for single bacterium sensing and antimicrobial susceptibility testing},

author = { B. Scherer and C. Surrette and H. Li, and P. Torab, and E. Kvam and C. Galligan and S. Go and G. Grossmann and T. Hammond and T. Johnson and R. St-Pierre and J. Nelson and R.A. Potyrailo and T. Khire and K. Hsieh and T.H. Wang and P. Kin Wong and C. Puleo },

url = {https://pubs.rsc.org/en/content/articlelanding/2021/lc/d0lc00937g#!divAbstract},

doi = {https://doi.org/10.1039/D0LC00937G},

year  = {2021},

date = {2021-01-26},

journal = {Lab on a Chip},

abstract = {Single-molecule and single-cell analysis techniques have opened new opportunities for characterizing and analyzing heterogeneity within biological samples. These detection methods are often referred to as digital assays because the biological sample is partitioned into many small compartments and each compartment contains a discrete number of targets (e.g. cells). Using digital assays, researchers can precisely detect and quantify individual targets, and this capability has made digital techniques the basis for many modern bioanalytical tools (including digital PCR, single cell RNA sequencing, and digital ELISA). However, digital assays are dominated by optical analysis systems that typically utilize microscopy to analyze partitioned samples. The utility of digital assays may be dramatically enhanced by implementing cost-efficient and portable electrical detection capabilities. Herein, we describe a digital electrical impedance sensing platform that enables direct multiplexed measurement of single cell bacterial cells. We outline our solutions to the challenge of multiplexing impedance sensing across many culture compartments and demonstrate the potential for rapidly differentiating antimicrobial resistant versus susceptible strains of bacteria.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Park2020,

title = {Digital CRISPR/Cas‐Assisted Assay for Rapid and Sensitive Detection of SARS‐CoV‐2},

author = {J.S. Park and K. Hsieh and L. Chen and A. Kaushik and A.Y. Trick and T.H. Wang},

doi = {https://doi.org/10.1002/advs.202003564},

year  = {2021},

date = {2021-01-12},

urldate = {2020-11-20},

journal = {Advanced Science},

abstract = {The unprecedented demand for rapid diagnostics in response to the COVID‐19 pandemic has brought the spotlight onto clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated systems (Cas)‐assisted nucleic acid detection assays. Already benefitting from an elegant detection mechanism, fast assay time, and low reaction temperature, these assays can be further advanced via integration with powerful, digital‐based detection. Thus motivated, the first digital CRISPR/Cas‐assisted assay—coined digitization‐enhanced CRISPR/Cas‐assisted one‐pot virus detection (deCOViD)—is developed and applied toward SARS‐CoV‐2 detection. deCOViD is realized through tuning and discretizing a one‐step, fluorescence‐based, CRISPR/Cas12a‐assisted reverse transcription recombinase polymerase amplification assay into sub‐nanoliter reaction wells within commercially available microfluidic digital chips. The uniformly elevated digital concentrations enable deCOViD to achieve qualitative detection in <15 min and quantitative detection in 30 min with high signal‐to‐background ratio, broad dynamic range, and high sensitivity—down to 1 genome equivalent (GE) µL−1 of SARS‐CoV‐2 RNA and 20 GE µL−1 of heat‐inactivated SARS‐CoV‐2, which outstrips its benchtop‐based counterpart and represents one of the fastest and most sensitive CRISPR/Cas‐assisted SARS‐CoV‐2 detection to date. Moreover, deCOViD can detect RNA extracts from clinical samples. Taken together, deCOViD opens a new avenue for advancing CRISPR/Cas‐assisted assays and combating the COVID‐19 pandemic and beyond.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{park2021,

title = {Ligation-Enabled Fluorescence-Coding PCR for High-Dimensional Fluorescence-Based Nucleic Acid Detection},

author = { J.S. Park and T. Pisanic and Y. Zhang and T.H. Wang},

url = {https://pubmed.ncbi.nlm.nih.gov/33427441/},

doi = {https://doi.org/10.1021/acs.analchem.0c04221},

year  = {2021},

date = {2021-01-11},

journal = {Analytical Chemistry},

volume = {93},

number = {4},

pages = { 2351–2358},

abstract = {Polymerase chain reaction (PCR) is by far the most commonly used method of nucleic acid amplification and has likewise been employed for a plethora of diagnostic purposes. Nonetheless, multiplexed PCR-based detection schemes have hitherto been largely limited by technical challenges associated with nonspecific interactions and other limitations inherent to traditional fluorescence-based assays. Here, we describe a novel strategy for multiplexed PCR-based analysis called Ligation-eNabled fluorescence-Coding PCR (LiNC PCR) that exponentially enhances the multiplexing capability of standard fluorescence-based PCR assays. The technique relies upon a simple, preliminary ligation reaction in which target DNA sequences are converted to PCR template molecules with distinct endpoint fluorescence signatures. Universal TaqMan probes are used to create target-specific multicolor fluorescence signals that can be readily decoded to identify amplified targets of interest. We demonstrate the LiNC PCR technique by implementing a two-color-based assay for detection of 10 ovarian cancer epigenetic biomarkers at analytical sensitivities as low as 60 template molecules with no detectable target cross-talk. Overall, LiNC PCR provides a simple and inexpensive method for achieving high-dimensional multiplexing that can be implemented in manifold molecular diagnostic applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Fan2020,

title = {Epigenomic Reprogramming toward Mesenchymal-Epithelial Transition in Ovarian-Cancer-Associated Mesenchymal Stem Cells Drives Metastasis},

author = {H. Fan and H.I. Atiya and Y. Wang and T.R. Pisanic and T.H. Wang and I.M. Shih and K.K. Foy and L. Frisbie and R.J. Buckanovich and A.A. Chomiak and R.L. Tiedemann and S.B. Rothbart and C. Chandler and H. Shen and L.G. Coffman},

url = {https://www.sciencedirect.com/science/article/pii/S2211124720314625},

doi = {https://doi.org/10.1016/j.celrep.2020.108473},

year  = {2020},

date = {2020-11-08},

journal = {Cell Reports},

volume = {33},

number = {10},

pages = {108473},

abstract = {A role for cancer cell epithelial-to-mesenchymal transition (EMT) in cancer is well established. Here, we show that, in addition to cancer cell EMT, ovarian cancer cell metastasis relies on an epigenomic mesenchymal-to-epithelial transition (MET) in host mesenchymal stem cells (MSCs). These reprogrammed MSCs, termed carcinoma-associated MSCs (CA-MSCs), acquire pro-tumorigenic functions and directly bind cancer cells to serve as a metastatic driver/chaperone. Cancer cells induce this epigenomic MET characterized by enhancer-enriched DNA hypermethylation, altered chromatin accessibility, and differential histone modifications. This phenomenon appears clinically relevant, as CA-MSC MET is highly correlated with patient survival. Mechanistically, mirroring MET observed in development, MET in CA-MSCs is mediated by WT1 and EZH2. Importantly, EZH2 inhibitors, which are clinically available, significantly inhibited CA-MSC-mediated metastasis in mouse models of ovarian cancer.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Miller2020,

title = {Leveraging locus-specific epigenetic heterogeneity to improve the performance of blood-based DNA methylation biomarkers},

author = {B.F Miller and T.R. Pisanic II and G. Margolin and H.M. Petrykowska and P. Athamanolap and A. Goncearenco and A. Osei-Tutu and C.M. Annunziata and T.H. Wang and L. Elnitski},

url = {https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-020-00939-w},

doi = {https://doi.org/10.1101/579839.},

year  = {2020},

date = {2020-10-21},

journal = {Clinical Epigenetics},

volume = {12},

number = {1},

pages = {1-19},

abstract = {Background

Variation in intercellular methylation patterns can complicate the use of methylation biomarkers for clinical diagnostic applications such as blood-based cancer testing. Here, we describe development and validation of a methylation density binary classification method called EpiClass (available for download at https://github.com/Elnitskilab/EpiClass) that can be used to predict and optimize the performance of methylation biomarkers, particularly in challenging, heterogeneous samples such as liquid biopsies. This approach is based upon leveraging statistical differences in single-molecule sample methylation density distributions to identify ideal thresholds for sample classification.



Results

We developed and tested the classifier using reduced representation bisulfite sequencing (RRBS) data derived from ovarian carcinoma tissue DNA and controls. We used these data to perform in silico simulations using methylation density profiles from individual epiallelic copies of ZNF154, a genomic locus known to be recurrently methylated in numerous cancer types. From these profiles, we predicted the performance of the classifier in liquid biopsies for the detection of epithelial ovarian carcinomas (EOC). In silico analysis indicated that EpiClass could be leveraged to better identify cancer-positive liquid biopsy samples by implementing precise thresholds with respect to methylation density profiles derived from circulating cell-free DNA (cfDNA) analysis. These predictions were confirmed experimentally using DREAMing to perform digital methylation density analysis on a cohort of low volume (1-ml) plasma samples obtained from 26 EOC-positive and 41 cancer-free women. EpiClass performance was then validated in an independent cohort of 24 plasma specimens, derived from a longitudinal study of 8 EOC-positive women, and 12 plasma specimens derived from 12 healthy women, respectively, attaining a sensitivity/specificity of 91.7%/100.0%. Direct comparison of CA-125 measurements with EpiClass demonstrated that EpiClass was able to better identify EOC-positive women than standard CA-125 assessment. Finally, we used independent whole genome bisulfite sequencing (WGBS) datasets to demonstrate that EpiClass can also identify other cancer types as well or better than alternative methylation-based classifiers.



Conclusions

Our results indicate that assessment of intramolecular methylation density distributions calculated from cfDNA facilitates the use of methylation biomarkers for diagnostic applications. Furthermore, we demonstrated that EpiClass analysis of ZNF154 methylation was able to outperform CA-125 in the detection of etiologically diverse ovarian carcinomas, indicating broad utility of ZNF154 for use as a biomarker of ovarian cancer.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hashemi2020,

title = {A Novel Platform Using RNA Signatures To Accelerate Antimicrobial Susceptibility Testing in Neisseria gonorrhoeae},

author = {M.M. Hashemi and N. Ram-Mohan and X. Yang and N. Andini and N. R. Gessner and K.C. Carroll and T.H. Wang and S. Yang

},

url = {https://jcm.asm.org/content/58/12/e01152-20},

doi = {DOI: 10.1128/JCM.01152-20},

year  = {2020},

date = {2020-09-23},

journal = {J Clin Microbiol.},

volume = {58},

number = {12},

pages = {e01152-20},

abstract = {The rise of antimicrobial-resistant pathogens can be attributed to the lack of a rapid pathogen identification (ID) or antimicrobial susceptibility testing (AST), resulting in delayed therapeutic decisions at the point of care. Gonorrhea is usually empirically treated, with no AST results available before treatment, thus contributing to the rapid rise in drug resistance. Here, we present a rapid AST platform using RNA signatures for Neisseria gonorrhoeae. Transcriptome sequencing (RNA-seq) followed by bioinformatic tools was applied to explore potential markers in the transcriptome profile of N. gonorrhoeae upon minutes of azithromycin exposure. Validation of candidate markers using quantitative real-time PCR (qRT-PCR) showed that two markers (arsR [NGO1562] and rpsO) can deliver accurate AST results across 14 tested isolates. Further validation of our susceptibility threshold in comparison to MIC across 64 more isolates confirmed the reliability of our platform. Our RNA markers combined with emerging molecular point-of-care systems has the potential to greatly accelerate both ID and AST to inform treatment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Yang2020b,

title = {RNA markers for ultra-rapid molecular antimicrobial susceptibility testing in fluoroquinolone-treated Klebsiella pneumoniae},

author = {X. Yang and M.M. Hashemi and N. Andini and M.M. Li and S. Kuang and K.C. Carroll and T.H. Wang and S. Yan},

url = {https://academic.oup.com/jac/article/75/7/1747/5810132},

year  = {2020},

date = {2020-09-03},

journal = {Journal of Antimicrobial Chemotherapy},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Li2020,

title = {Electrode-free Concentration and Recovery of DNA at Physiologically Relevant Ionic Concentrations},

author = {S. Li and A. Li and K. Hsieh and S.M. Friedrich and T.H. Wang},

url = {https://pubs.acs.org/doi/abs/10.1021/acs.analchem.0c00831},

year  = {2020},

date = {2020-09-03},

journal = {Analytical Chemistry},

volume = {92},

number = {8},

pages = {6150–6157},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Bell2020,

title = {Single-cell lineage tracing of developing retinal systems},

author = {C. Bell and W. Fang and C. Berlinicke and A. Kaushik and P. Zhang and T.H. Wang and R. Kalhor and H. Ji and D.J. Zack},

url = {https://iovs.arvojournals.org/article.aspx?articleid=2768344},

year  = {2020},

date = {2020-09-03},

journal = {Investigative Ophthalmology & Visual Science},

volume = {61},

number = {7},

pages = {4014},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhang2020,

title = {ddRFC: A scalable multiplexed droplet digital nucleic acid amplification test platform},

author = {Y. Zhang and P. Zhang and L. Chen and A. Kaushik and K. Hu and T.H. Wang},

url = {https://www.sciencedirect.com/science/article/pii/S0956566320304917},

year  = {2020},

date = {2020-09-03},

journal = {Biosensors and Bioelectronics},

volume = {167},

number = {112499},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Beh2020,

title = {Microfluidic-prepared, monodisperse, X-ray-visible, embolic microspheres for non-oncological embolization applications},

author = {C.W. Beh and Y.i Fu and C.R. Weiss and C. Hu and A. Arepally and H.Q. Mao and T.H. Wang and D.L. Kraitchman},

url = {https://pubs.rsc.org/en/content/articlelanding/2020/lc/d0lc00098a#!divAbstract},

doi = {https://doi.org/10.1039/D0LC00098A},

year  = {2020},

date = {2020-09-01},

journal = {Lab on a Chip},

volume = {20},

pages = {3591-3600},

abstract = {Embolotherapy using particle embolics is normally performed with exogenous contrast to assist in visualization. However, the exact location of the embolics cannot be identified after contrast washout. We developed a novel, pseudo-check valve-integrated microfluidic device, that partitions barium- impregnated alginate from crosslinking solution, thereby preventing nozzle failure. This enables rapid and continuous generation of inherently X-ray-visible embolic microspheres (XEMs) with uniform size. The XEMs are visible under clinical X-ray and cone beam CT both in vitro and in vivo. In particular, we demonstrated the embolization properties of these XEMs in large animals, performing direct intra- and post-procedural assessment of embolic delivery. The persistent radiopacity of these XEMs enables real-time evaluation of embolization precision and offers great promise for non-invasive follow-up examination without exogenous contrast. We also demonstrated that bariatric arterial embolization with XEMs significantly suppresses weight gain in swine, as an example of a non-oncological application of embolotherapy.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gaddes2020,

title = {Facile Coupling of Droplet Magnetofluidic-Enabled Automated Sample Preparation to Digital Nucleic Acid Amplification Testing and Analysis},

author = {D.E. Gaddes and P,W. Lee and A.Y. Trick and P. Athamanolap and C.M. O’Keefe and C. Puleo and K. Hsieh and T.H. Wang},

doi = {https://doi.org/10.1021/acs.analchem.0c02454},

year  = {2020},

date = {2020-09-01},

journal = {Analytical Chemistry},

volume = {92},

number = {19},

pages = {13254-13261},

abstract = {Digital nucleic acid amplification testing (dNAAT) and analysis techniques, such as digital polymerase chain reaction (PCR), have become useful clinical diagnostic tools. However, nucleic acid (NA) sample preparation preceding dNAAT is generally laborious and performed manually, thus creating the need for a simple sample preparation technique and a facile coupling strategy for dNAAT. Therefore, we demonstrate a simple workflow which automates magnetic bead-based extraction of NAs with a one-step transfer to dNAAT. Specifically, we leverage droplet magnetofluidics (DM) to automate the movement of magnetic beads between small volumes of reagents commonly employed for NA extraction and purification. Importantly, the buffer typically used to elute the NAs off the magnetic beads is replaced by a carefully selected PCR solution, enabling direct transfer from sample preparation to dNAAT. Moreover, we demonstrate the potential for multiplexing using a digital high-resolution melt (dHRM) after the digital PCR (dPCR). The utility of this workflow is demonstrated with duplexed detection of bacteria in a sample imitating a coinfection. We first purify the bacterial DNA into a PCR solution using our DM-based sample preparation. We then transfer the purified bacterial DNA to our microfluidic nanoarray to amplify 16S rRNA using dPCR and then perform dHRM to identify the two bacterial species.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Liu2020,

title = {Detection of Promoter DNA Methylation in Urine and Plasma Aids the Detection of Non–Small Cell Lung Cancer},

author = {B. Liu and J. Ricarte-Filho and A. Mallisetty and C. Villani and A.E. Kottorou and K.P. Rodgers and C. Chen and T. Ito and K. Holmes and N. Gastala and K. Valyi-Nagy and O. David and R.C. Gaba and C. Ascoli and M. Pasquinelli and L.E. Feldman and M.G. Massad and T.H. Wang and I. Jusue-Torres and E. Benedetti and R.A. Winn and M.V. Brock and J.G. Herman and A. Hulbert},

url = {https://clincancerres.aacrjournals.org/content/26/16/4339.full},

doi = {DOI: 10.1158/1078-0432.CCR-19-2896},

year  = {2020},

date = {2020-08-12},

journal = {Clinical Cancer Research},

volume = {26},

number = {16},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Pisanic2020,

title = {The methylomic landscape of fallopian tube lesions associated with ovarian high-grade serous carcinoma},

author = {T R Pisanic and Y Wang and H Sun and M Considine and L Li and T H Wang and T L Wang and Ie M Shih},

doi = {DOI: 10.1158/1078-0432.CCR-20-0270},

year  = {2020},

date = {2020-08-12},

journal = {Clinical Cancer Research},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hsieh2020,

title = {Applying Biosensor Development Concepts to Improve Preamplification-Free CRISPR/Cas12a-Dx},

author = {K. Hsieh and G. Zhao and T.H. Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2020/an/d0an00664e#!divAbstract},

year  = {2020},

date = {2020-05-27},

journal = {Analyst},

volume = {145},

pages = {4880-4888},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Yang2020,

title = {Investigating cone photoreceptor development using patient-derived NRL null retinal organoids},

author = {A. Kallman and E.E. Capowski and J. Wang and A.M. Kaushik and A.D. Jansen and K.L. Edwards and L. Chen and C.A. Berlinicke and M.J. Phillips and E.A. Pierce and J. Qian and T.H. Wang and D.M. Gamm and D.J. Zack},

url = {https://www.nature.com/articles/s42003-020-0808-5},

year  = {2020},

date = {2020-02-21},

journal = {Communications Biology},

volume = {3},

number = {1},

pages = {1-13},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2019,

title = {Prognostic Value of Survival of MicroRNAs Signatures in Non-small Cell Lung Cancer},

author = {B Chen and T Gao and W Yuan and W Zhao and T H Wang and J Wu},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6843868/},

year  = {2019},

date = {2019-10-06},

journal = {Journal of Cancer},

volume = {10},

number = {23},

pages = {5793-5804},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{B2019,

title = {Rab8 GTPase regulates Klotho-mediated inhibition of cell growth and progression by directly modulating its surface expression in human non-small cell lung cancer},

author = {B Chen and S Huang and T R Pisanic and A Stark and Y Tao and B Cheng and Y Li and Y Wei and W Zhao and T H Wang and J Wu},

url = {https://www.sciencedirect.com/science/article/pii/S2352396419307108},

year  = {2019},

date = {2019-09-17},

journal = {EBioMedicine},

volume = {49},

pages = {118-132},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Athamanolap2019,

title = {Nanoarray Digital Polymerase Chain Reaction with High-Resolution Melt for Enabling Broad Bacteria Identification and Pheno–Molecular Antimicrobial Susceptibility Test},

author = {P. Athamanolap and K. Hsieh and C.M. O’Keefe and Y. Zhang and S. Yang and T.H. Wang},

url = {https://pubs.acs.org/doi/10.1021/acs.analchem.9b02344},

year  = {2019},

date = {2019-09-17},

journal = {Anal Chem},

volume = {91},

number = {20},

pages = {12784-12792},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Loo2019,

title = {Technological Advances in Multiscale Analysis of Single Cells in Biomedicine},

author = {J F C Loo and H P Ho and S K Kong and T H Wang and Y P Ho},

url = {https://onlinelibrary.wiley.com/doi/full/10.1002/adbi.201900138},

year  = {2019},

date = {2019-09-11},

journal = {Advanced Biosystems},

volume = {3},

number = {11},

pages = {1900138},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{O’Keefe2019,

title = {Highly Efficient Real-Time Droplet Analysis Platform for High-Throughput Interrogation of DNA Sequences by Melt},

author = {C.M. O’Keefe and A.M. Kaushik and and T.H. Wang},

url = {https://pubs.acs.org/doi/full/10.1021/acs.analchem.9b02346},

year  = {2019},

date = {2019-07-29},

journal = {Anal Chem},

volume = {91},

number = {17},

pages = {11275-11282},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhang2019,

title = {Rapid generation of chemical combinations on a magnetic digital microfluidic array},

author = {Y Zhang and T H Wang

},

url = {https://pubs.rsc.org/en/content/articlelanding/2019/ra/c9ra03469b#!divAbstract},

year  = {2019},

date = {2019-07-12},

journal = {RSC Advances},

volume = {9},

number = {38},

pages = {21741-21747},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6737,

title = {Customizing droplet contents and dynamic ranges via integrated programmable picodroplet assembler},

author = {P Zhang and A Kaushik and K Hsieh and T H Wang},

url = {https://doi.org/10.1038/s41378-019-0062-5},

doi = {10.1038/s41378-019-0062-5},

issn = {2055-7434},

year  = {2019},

date = {2019-07-01},

journal = {Microsystems & Nanoengineering},

volume = {5},

number = {1},

pages = {22},

abstract = {Droplet microfluidic technology is becoming increasingly useful for high-throughput and high-sensitivity detection of biological and biochemical reactions. Most current droplet devices function by passively discretizing a single sample subject to a homogeneous or random reagent/reaction condition into tens of thousands of picoliter-volume droplets for analysis. Despite their apparent advantages in speed and throughput, these droplet devices inherently lack the capability to customize the contents of droplets in order to test a single sample against multiple reagent conditions or multiple samples against multiple reagents. In order to incorporate such combinatorial capability into droplet platforms, we have developed the fully Integrated Programmable Picodroplet Assembler. Our platform is capable of generating customized picoliter-volume droplet groups from nanoliter-volume plugs which are assembled in situ on demand. By employing a combination of microvalves and flow-focusing-based discretization, our platform can be used to precisely control the content and volume of generated nanoliter-volume plugs, and thereafter the content and the effective dynamic range of picoliter-volume droplets. Furthermore, we can use a single integrated device for continuously generating, incubating, and detecting multiple distinct droplet groups. The device successfully marries the precise control and on-demand capability of microvalve-based platforms with the sensitivity and throughput of picoliter droplet platforms in a fully automated monolithic device. The device ultimately will find important applications in single-cell and single-molecule analyses.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6709,

title = {Widespread gene transfer to malignant gliomas with In vitro-to-In vivo correlation},

author = { K. Negron and N. Khalasawi and B. Lu and C. Y. Ho and J. Lee and S. Shenoy and H. Q. Mao and T. H. Wang and J. Hanes and J. S. Suk},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30978431},

doi = {10.1016/j.jconrel.2019.04.010},

issn = {1873-4995 (Electronic) 

0168-3659 (Linking)},

year  = {2019},

date = {2019-04-09},

journal = {J Control Release},

abstract = {Gene therapy of malignant gliomas has shown a lack of clinical success to date due in part to inability of conventional gene vectors to achieve widespread gene transfer throughout highly disseminated tumor areas within the brain. Here, we demonstrate that newly engineered polymer-based DNA-loaded nanoparticles (DNA-NP) possessing small particle diameters (~50nm) and non-adhesive surface polyethylene glycol (PEG) coatings efficiently penetrate brain tumor tissue as well as healthy brain parenchyma. Specifically, this brain-penetrating nanoparticle (BPN), following intracranial administration via convection enhanced delivery (CED), provides widespread transgene expression in heathy rodent striatum and an aggressive brain tumor tissue established orthotopically in rats. The ability of BPN to efficiently traverse both tissues is of great importance as the highly invasive glioma cells infiltrated into normal brain tissue are responsible for tumor recurrence. Of note, the transgene expression within the orthotopic tumor tissue occurred preferentially in glioma cells over microglial cells. We also show that three-dimensional (3D) multicellular spheroids established with malignant glioma cells, unlike conventional two-dimensional (2D) cell cultures, serve as an excellent in vitro model reliably predicting gene vector behaviors in vivo. Briefly, DNA-NP possessing greater surface PEG coverage exhibited more uniform and higher-level transgene expression both in the 3D model and in vivo, whereas the trend was opposite in 2D culture. The finding here alerts that gene transfer studies based primarily on 2D cultures should be interpreted with caution and underscores the relevance of 3D models for screening newly engineered gene vectors prior to their in vivo evaluation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6710,

title = {Promoter methylation of ADAMTS1 and BNC1 as potential biomarkers for early detection of pancreatic cancer in blood},

author = { M. A. L. Eissa and L. Lerner and E. Abdelfatah and N. Shankar and J. K. Canner and N. M. Hasan and V. Yaghoobi and B. Huang and Z. Kerner and F. Takaesu and C. Wolfgang and R. Kwak and M. Ruiz and M. Tam and T. R. 2nd Pisanic and C. A. Iacobuzio-Donahue and R. H. Hruban and J. He and T. H. Wang and L. D. Wood and A. Sharma and N. Ahuja},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30953539},

doi = {10.1186/s13148-019-0650-0},

issn = {1868-7083 (Electronic) 

1868-7075 (Linking)},

year  = {2019},

date = {2019-04-05},

journal = {Clin Epigenetics},

volume = {11},

number = {1},

pages = {59},

abstract = {BACKGROUND: Despite improvements in cancer management, most pancreatic cancers are still diagnosed at an advanced stage. We have recently identified promoter DNA methylation of the genes ADAMTS1 and BNC1 as potential blood biomarkers of pancreas cancer. In this study, we validate this biomarker panel in peripheral cell-free tumor DNA of patients with pancreatic cancer. RESULTS: Sensitivity and specificity for each gene are as follows: ADAMTS1 87.2% and 95.8% (AUC = 0.91; 95% CI 0.71-0.86) and BNC1 64.1% and 93.7% (AUC = 0.79; 95% CI 0.63-0.78). When using methylation of either gene as a combination panel, sensitivity increases to 97.3% and specificity to 91.6% (AUC = 0.95; 95% CI 0.77-0.90). Adding pre-operative CA 19-9 values to the combined two-gene methylation panel did not improve sensitivity. Methylation of ADAMTS1 was found to be positive in 87.5% (7/8) of stage I, 77.8% (7/9) of stage IIA, and 90% (18/20) of stage IIB disease. Similarly, BNC1 was positive in 62.5% (5/8) of stage I patients, 55.6% (5/9) of stage IIA, and 65% (13/20) of patients with stage IIB disease. The two-gene panel (ADAMTS1 and/or BNC1) was positive in 100% (8/8) of stage I, 88.9% (8/9) of stage IIA, and 100% (20/20) of stage IIB disease. The sensitivity and specificity of the two-gene panel for localized pancreatic cancer (stages I and II), where the cancer is eligible for surgical resection with curative potential, was 94.8% and 91.6% respectively. Additionally, the two-gene panel exhibited an AUC of 0.95 (95% CI 0.90-0.98) compared to 57.1% for CA 19-9 alone. CONCLUSION: The methylation status of ADAMTS1 and BNC1 in cfDNA shows promise for detecting pancreatic cancer during the early stages when curative resection of the tumor is still possible. This minimally invasive blood-based biomarker panel could be used as a promising tool for diagnosis and screening in a select subset of high-risk populations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6707,

title = {Emerging Analytical Techniques for Rapid Pathogen Identification and Susceptibility Testing},

author = { D. J. Shin and N. Andini and K. Hsieh and S. Yang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30939033},

doi = {10.1146/annurev-anchem-061318-115529},

issn = {1936-1335 (Electronic) 

1936-1327 (Linking)},

year  = {2019},

date = {2019-04-02},

journal = {Annu Rev Anal Chem (Palo Alto Calif)},

abstract = {In the face of looming threats from multi-drug resistant microorganisms, there is a growing need for technologies that will enable rapid identification and drug susceptibility profiling of these pathogens in health care settings. In particular, recent progress in microfluidics and nucleic acid amplification is pushing the boundaries of timescale for diagnosing bacterial infections. With a diverse range of techniques and parallel developments in the field of analytical chemistry, an integrative perspective is needed to understand the significance of these developments. This review examines the scope of new developments in assay technologies grouped by key enabling domains of research. First, we examine recent development in nucleic acid amplification assays for rapid identification and drug susceptibility testing in bacterial infections. Next, we examine advances in microfluidics that facilitate acceleration of diagnostic assays via integration and scale. Lastly, recent developments in biosensor technologies are reviewed. We conclude this review with perspectives on the use of emerging concepts to develop paradigm-changing assays. Expected final online publication date for the Annual Review of Analytical Chemistry Volume 12 is June 12, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6711,

title = {Methylation Biomarker Panel Performance in EsophaCap Cytology Samples for Diagnosing Barrett's Esophagus: A Prospective Validation Study},

author = { Z. Wang and S. Kambhampati and Y. Cheng and K. Ma and C. Simsek and A. H. Tieu and J. M. Abraham and X. Liu and V. Prasath and M. Duncan and A. Stark and A. Trick and H. L. Tsai and H. Wang and Y. He and M. A. Khashab and S. Ngamruengphong and E. J. Shin and T. H. Wang and S. J. Meltzer},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30670490},

doi = {10.1158/1078-0432.CCR-18-3696},

issn = {1078-0432 (Print) 

1078-0432 (Linking)},

year  = {2019},

date = {2019-04-01},

journal = {Clin Cancer Res},

volume = {25},

number = {7},

pages = {2127-2135},

abstract = {PURPOSE: Barrett's esophagus is the only known precursor of esophageal adenocarcinoma (EAC). Although endoscopy and biopsy are standard methods for Barrett's esophagus diagnosis, their high cost and risk limit their use as a screening modality. Here, we sought to develop a Barrett's esophagus detection method based on methylation status in cytology samples captured by EsophaCap using a streamlined sensitive technique, methylation on beads (MOB). EXPERIMENTAL DESIGN: We conducted a prospective cohort study on 80 patients (52 in the training set; 28 in the test set). We used MOB to extract and bisulfite-convert DNA, followed by quantitative methylation-specific PCR to assess methylation levels of 8 previously selected candidate markers. Lasso regression was applied to establish a prediction model in the training set, which was then tested on the independent test set. RESULTS: In the training set, five of eight candidate methylation biomarkers (p16, HPP1, NELL1, TAC1, and AKAP12) were significantly higher in Barrett's esophagus patients than in controls. We built a four-biomarker-plus-age lasso regression model for Barrett's esophagus diagnosis. The AUC was 0.894, with sensitivity 94.4% [95% confidence interval (CI), 71%-99%] and specificity 62.2% (95% CI, 44.6%-77.3%) in the training set. This model also performed with high accuracy for Barrett's esophagus diagnosis in an independent test set: AUC = 0.929 (P < 0.001; 95% CI, 0.810%-1%), with sensitivity=78.6% (95% CI, 48.8%-94.3%) and specificity = 92.8% (95% CI, 64.1%-99.6%). CONCLUSIONS: EsophaCap, in combination with an epigenetic biomarker panel and the MOB method, is a promising, well-tolerated, low-cost esophageal sampling strategy for Barrett's esophagus diagnosis. This approach merits further prospective studies in larger populations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6712,

title = {Long Interspersed Nuclear Element 1 Retrotransposons Become Deregulated during the Development of Ovarian Cancer Precursor Lesions},

author = {T.R. Pisanic and Y. Wang and H. Sun and M. Considine and L. Li, T.H. Wang and T.L. Wang and I.M. Shih},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30553834},

doi = {10.1016/j.ajpath.2018.11.005},

issn = {1525-2191 (Electronic) 0002-9440 (Linking)},

year  = {2019},

date = {2019-03-01},

journal = {Am J Pathol},

volume = {189},

number = {3},

pages = {513-520},

abstract = {There is growing evidence that most high-grade serous ovarian carcinomas likely arise from local dissemination of precursor lesions of the fallopian tube. Evolution of these lesions from early p53 signatures to latter-stage, serous tubal intraepithelial carcinomas (STICs) is characterized by cytologic atypia, accumulation of somatic mutations, and genomic instability, the etiologies of which remain unclear. Long interspersed element 1 (LINE-1) retrotransposon is expressed in many carcinomas, including high-grade serous ovarian carcinoma, where it contributes to genomic instability; however, the timing of LINE-1 activation during this evolution has yet to be elucidated. In this study, we assessed LINE-1 open reading frame 1 protein expression in 12 p53 signature lesions, 32 STICs, and 112 various types of ovarian cancers via immunohistochemical staining and examined LINE-1 promoter methylation in representative cases. We found that 78% and 57% of STICs, with and without concurrent ovarian carcinomas, respectively, exhibited intense LINE-1 immunoreactivity compared with adjacent, normal-appearing fallopian tube epithelium. Hypomethylation of the LINE-1 promoter was found in all STICs exhibiting overexpression. None of the 12 p53 signatures demonstrated significant LINE-1 expression. In ovarian cancer, 84 (75%) of 112 ovarian carcinomas overexpressed LINE-1. Our results indicate that LINE-1 retrotransposons often become deregulated during progression of ovarian cancer precursor lesions from the p53 signature to STIC stages and remain highly expressed in carcinoma.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6708,

title = {Nanotube assisted microwave electroporation for single cell pathogen identification and antimicrobial susceptibility testing},

author = { J. Gao and H. Li and P. Torab and K. E. Mach and D. W. Craft and N. J. Thomas and C. M. Puleo and J. C. Liao and T. H. Wang and P. K. Wong},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30794964},

doi = {10.1016/j.nano.2019.01.015},

issn = {1549-9642 (Electronic) 

1549-9634 (Linking)},

year  = {2019},

date = {2019-02-20},

journal = {Nanomedicine},

volume = {17},

pages = {246-253},

abstract = {A nanotube assisted microwave electroporation (NAME) technique is demonstrated for delivering molecular biosensors into viable bacteria for multiplex single cell pathogen identification to advance rapid diagnostics in clinical microbiology. Due to the small volume of a bacterial cell (~femtoliter), the intracellular concentration of the target molecule is high, which results in a strong signal for single cell detection without amplification. The NAME procedure can be completed in as little as 30 minutes and can achieve over 90% transformation efficiency. We demonstrate the feasibility of NAME for identifying clinical isolates of bloodborne and uropathogenic pathogens and detecting bacterial pathogens directly from patient's samples. In conjunction with a microfluidic single cell trapping technique, NAME allows single cell pathogen identification and antimicrobial susceptibility testing concurrently. Using this approach, the time for microbiological analysis reduces from days to hours, which will have a significant impact on the clinical management of bacterial infections.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6704,

title = {Versatile Analysis of DNA-Biomolecule Interactions in Solution by Hydrodynamic Separation and Single Molecule Detection},

author = { S. M. Friedrich and R. Bang and A. Li and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30668901},

doi = {10.1021/acs.analchem.8b04733},

issn = {1520-6882 (Electronic) 

0003-2700 (Linking)},

year  = {2019},

date = {2019-02-19},

journal = {Anal Chem},

volume = {91},

number = {4},

pages = {2822-2830},

abstract = {DNA can interact with a wide array of molecules with a range of binding affinities, stoichiometry, and size-scales. We present a sensitive, quantitative, and versatile platform for sensing and evaluating these diverse DNA-biomolecule interactions and DNA conformational changes in free solution. Single molecule free solution hydrodynamic separation utilizes differences in hydrodynamic mobility to separate bound DNA-biomolecule complexes from unbound DNA and determine the associated size change that results from binding. Single molecule detection enables highly quantitative analysis of the fraction of DNA in the bound and unbound state to characterize binding behavior including affinity, stoichiometry, and cooperativity. A stacked injection scheme increases throughput to enable practical analysis of DNA-biomolecule interactions using only picoliters of sample per measurement. To demonstrate analysis of DNA-protein interactions on a local scale, we investigate binding of the E. coli single stranded binding protein to two DNA oligos both individually and in direct competition. We show that stoichiometry and cooperativity is a function of DNA length and verify these differences in binding characteristics through direct competition. To demonstrate analysis of DNA-small molecule interactions and global conformational changes, we also assess DNA condensation with the polyamine spermidine. We use hydrodynamic mobility to evaluate the size of spermidine-condensed DNA and single molecule burst analysis to evaluate DNA packing within the condensed globules relative to free-coiled DNA. This platform thus presents a versatile tool capable of quantitative and sensitive evaluation of diverse biomolecular interactions, complex properties, and binding characteristics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6703,

title = {Multilayer microfluidic array for highly efficient sample loading and digital melt analysis of DNA methylation},

author = { C. M. O'Keefe and D. Giammanco and S. Li and T. R. Pisanic and T. J. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30623957},

doi = {10.1039/c8lc01189c},

issn = {1473-0189 (Electronic) 

1473-0189 (Linking)},

year  = {2019},

date = {2019-01-29},

journal = {Lab Chip},

volume = {19},

number = {3},

pages = {444-451},

abstract = {Liquid biopsies contain a treasure of genetic and epigenetic biomarkers that contain information for the detection and monitoring of human disease. DNA methylation is an epigenetic modification that is critical to determining cellular phenotype and often becomes altered in many disease states. In cancer, aberrant DNA methylation contributes to carcinogenesis and can profoundly affect tumor evolution, metastatic potential, and resistance to therapeutic intervention. However, current technologies are not well-suited for quantitative assessment of DNA methylation heterogeneity, especially in challenging samples such as liquid biopsies with low DNA input and high background. We present a multilayer microfluidic device for quantitative analysis of DNA methylation by digital PCR and high resolution melt (HRM). The multilayer design facilitates high-density array digitization aimed at maximizing sample loading efficiency. The platform achieves highly parallelized digital PCR-HRM-based discrimination of rare heterogeneous DNA methylation as low as 0.0001% methylated/unmethylated molecules of a classic tumor suppressor gene, CDKN2A (p14ARF).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6705,

title = {Optimizing peptide nucleic acid probes for hybridization-based detection and identification of bacterial pathogens},

author = { K. E. Mach and A. M. Kaushik and K. Hsieh and P. K. Wong and T. H. Wang and J. C. Liao},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30656297},

doi = {10.1039/c8an02194e},

issn = {1364-5528 (Electronic) 

0003-2654 (Linking)},

year  = {2019},

date = {2019-01-18},

journal = {Analyst},

abstract = {Point-of-care (POC) diagnostics for infectious diseases have the potential to improve patient care and antibiotic stewardship. Nucleic acid hybridization is at the core of many amplification-free molecular diagnostics and detection probe configuration is key to diagnostic performance. Modified nucleic acids such as peptide nucleic acid (PNA) offer advantages compared to conventional DNA probes allowing for faster hybridization, better stability and minimal sample preparation for direct detection of pathogens. Probes with tethered fluorophore and quencher allow for solution-based assays and eliminate the need for washing steps thereby facilitating integration into microfluidic devices. Here, we compared the sensitivity and specificity of double stranded PNA probes (dsPNA) and PNA molecular beacons targeting E. coli and P. aeruginosa for direct detection of bacterial pathogens. In bulk fluid assays, the dsPNAs had an overall higher fluorescent signal and better sensitivity and specificity than the PNA beacons for pathogen detection. We further designed and tested an expanded panel of dsPNA probes for detection of a wide variety of pathogenic bacteria including probes for universal detection of eubacteria, Enterobacteriaceae family, and P. mirablis. To confirm that the advantage translated to other assay types we compared the PNA beacon and dsPNA in a prototype droplet microfluidic device. Beyond the bulk fluid assay and droplet devices, use of dsPNA probes may be advantageous in a wide variety of assays that employ homogenous nucleic acid hybridization.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6646,

title = {Methylomic Analysis of Ovarian Cancers Identifies Tumor-Specific Alterations Readily Detectable in Early Precursor Lesions},

author = { T. R. 2nd Pisanic and L. M. Cope and S. F. Lin and T. T. Yen and P. Athamanolap and R. Asaka and K. Nakayama and A. N. Fader and T. H. Wang and I. M. Shih and T. L. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30108103},

doi = {10.1158/1078-0432.CCR-18-1199},

issn = {1078-0432 (Print) 

1078-0432 (Linking)},

year  = {2018},

date = {2018-12-15},

journal = {Clin Cancer Res},

volume = {24},

number = {24},

pages = {6536-6547},

abstract = {PURPOSE: High-grade serous ovarian carcinoma (HGSOC) typically remains undiagnosed until advanced stages when peritoneal dissemination has already occurred. Here, we sought to identify HGSOC-specific alterations in DNA methylation and assess their potential to provide sensitive and specific detection of HGSOC at its earliest stages. EXPERIMENTAL DESIGN: MethylationEPIC genome-wide methylation analysis was performed on a discovery cohort comprising 23 HGSOC, 37 non-HGSOC malignant, and 36 histologically unremarkable gynecologic tissue samples. The resulting data were processed using selective bioinformatic criteria to identify regions of high-confidence HGSOC-specific differential methylation. Quantitative methylation-specific real-time PCR (qMSP) assays were then developed for 8 of the top-performing regions and analytically validated in a cohort of 90 tissue samples. Lastly, qMSP assays were used to assess and compare methylation in 30 laser-capture microdissected (LCM) fallopian tube epithelia samples obtained from cancer-free and serous tubal intraepithelial carcinoma (STIC) positive women. RESULTS: Bioinformatic selection identified 91 regions of robust, HGSOC-specific hypermethylation, 23 of which exhibited an area under the receiver-operator curve (AUC) value >/= 0.9 in the discovery cohort. Seven of 8 top-performing regions demonstrated AUC values between 0.838 and 0.968 when analytically validated by qMSP in a 90-patient cohort. A panel of the 3 top-performing genes (c17orf64, IRX2, and TUBB6) was able to perfectly discriminate HGSOC (AUC 1.0). Hypermethylation within these loci was found exclusively in LCM fallopian tube epithelia from women with STIC lesions, but not in cancer-free fallopian tubes. CONCLUSIONS: A panel of methylation biomarkers can be used to accurately identify HGSOC, even at precursor stages of the disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6627,

title = {Fluorescence spectroscopic detection and measurement of single telomere molecules},

author = { C. W. Beh and Y. Zhang and Y. L. Zheng and B. Sun and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30010842},

doi = {10.1093/nar/gky627},

issn = {1362-4962 (Electronic) 

0305-1048 (Linking)},

year  = {2018},

date = {2018-11-02},

journal = {Nucleic Acids Res},

volume = {46},

number = {19},

pages = {e117},

abstract = {Telomeres are the end-caps of chromosomes that serve to protect the integrity of the genome. Below certain critical lengths, the telomeres can no longer fulfill their protective function, and chromosomal instability ensues. Telomeres shorten during normal cell division due to the end replication problem and are implicated in the development of various aging-associated diseases, including cancer. Telomere length has the potential to serve as a useful biomarker in the field of aging and cancer. However, existing methods of telomere measurement are either too laborious, unable to provide absolute measurement of individual telomere lengths, or limited to certain chromosomes or cell types. Here, we describe an easy single-molecule, fluorescence spectroscopic method for measuring the length of telomeres that permits the profiling of absolute telomere lengths in any DNA sample. We have demonstrated the accurate detection of telomeres as short as 100 bp using cloned telomere standards, and have profiled telomere lengths in human cancer cell lines and primary cells. Since this method allows direct comparison between samples, it could greatly improve the clinical utility of telomere biomarkers.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6502,

title = {Droplet microfluidics for high-sensitivity and high-throughput detection and screening of disease biomarkers},

author = { A. M. Kaushik and K. Hsieh and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/29797414},

doi = {10.1002/wnan.1522},

issn = {1939-0041 (Electronic) 

1939-0041 (Linking)},

year  = {2018},

date = {2018-11-01},

journal = {Wiley Interdiscip Rev Nanomed Nanobiotechnol},

volume = {10},

number = {6},

pages = {e1522},

abstract = {Biomarkers are nucleic acids, proteins, single cells, or small molecules in human tissues or biological fluids whose reliable detection can be used to confirm or predict disease and disease states. Sensitive detection of biomarkers is therefore critical in a variety of applications including disease diagnostics, therapeutics, and drug screening. Unfortunately for many diseases, low abundance of biomarkers in human samples and low sample volumes render standard benchtop platforms like 96-well plates ineffective for reliable detection and screening. Discretization of bulk samples into a large number of small volumes (fL-nL) via droplet microfluidic technology offers a promising solution for high-sensitivity and high-throughput detection and screening of biomarkers. Several microfluidic strategies exist for high-throughput biomarker digitization into droplets, and these strategies have been utilized by numerous droplet platforms for nucleic acid, protein, and single-cell detection and screening. While the potential of droplet-based platforms has led to burgeoning interest in droplets, seamless integration of sample preparation technologies and automation of platforms from biological sample to answer remain critical components that can render these platforms useful in the clinical setting in the near future. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6736,

title = {Ratiometric Fluorescence Coding for Multiplex Nucleic Acid Amplification Testing},

author = { Y. Zhang and L. Chen and K. Hsieh and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30251853},

doi = {10.1021/acs.analchem.8b03266},

issn = {1520-6882 (Electronic) 

0003-2700 (Linking)},

year  = {2018},

date = {2018-10-16},

journal = {Anal Chem},

volume = {90},

number = {20},

pages = {12180-12186},

abstract = {Although nucleic acid amplification testing (NAAT) has become the cornerstone for molecular diagnosis of diseases, expanding the multiplexed detection capacity of NAAT remains an important objective. To this end, encoding each nucleic acid target with a specific fluorescently labeled probe has been the most mature approach for multiplexed detection. Unfortunately, the number of targets that can be differentiated via this one-target-one-fluorophore multiplexed detection approach is restricted by spectral overlaps between fluorophores. In response, we present herein a new multiplexed detection approach termed ratiometric fluorescence coding, in which we encode each nucleic acid target with a specific ratio between two standard fluorophores. In ratiometric fluorescence coding, we employ the padlock probe chemistry to encode each nucleic acid target with a specific number of binding sites for two probes labeled with different fluorophores. Coupling the padlock probes with either rolling circle amplification (RCA) or hyperbranched rolling circle amplification (HRCA), we transform each nucleic acid target into a specific template that allows hybridization with the fluorescently labeled probes at predesigned ratios, thereby achieving multiplexed detection. For demonstration, we detected DNA targets from six infectious diseases and demonstrated the potential for further expanding the multiplexing capability of our approach. With further development, ratiometric fluorescence coding has the potential to enable highly multiplexed detection of nucleic acid targets and facilitate molecular diagnosis of diseases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6607,

title = {A "Culture" Shift: Broad Bacterial Detection, Identification, and Antimicrobial Susceptibility Testing Directly from Whole Blood},

author = { N. Andini and A. Hu and L. Zhou and S. Cogill and T. H. Wang and C. T. Wittwer and S. Yang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30087140},

doi = {10.1373/clinchem.2018.290189},

issn = {1530-8561 (Electronic) 

0009-9147 (Linking)},

year  = {2018},

date = {2018-10-01},

journal = {Clin Chem},

volume = {64},

number = {10},

pages = {1453-1462},

abstract = {BACKGROUND: The time required for bloodstream pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) does not satisfy the acute needs of disease management. Conventional methods take up to 3 days for ID and AST. Molecular diagnostics have reduced times for ID, but their promise to supplant culture is unmet because AST times remain slow. We developed a combined quantitative PCR (qPCR)-based ID+AST assay with sequential detection, ID, and AST of leading nosocomial bacterial pathogens. METHODS: ID+AST was performed on whole blood samples by (a) removing blood cells, (b) brief bacterial enrichment, (c) bacterial detection and ID, and (d) species-specific antimicrobial treatment. Broad-spectrum qPCR of the internal transcribed spacer between the 16S and 23S was amplified for detection. High-resolution melting identified the species with a curve classifier. AST was enabled by Ct differences between treated and untreated samples. RESULTS: A detection limit of 1 CFU/mL was achieved for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. All species were accurately identified by unique melting curves. Antimicrobial minimum inhibitory concentrations were identified with Ct differences of >/=1 cycle. Using an RNA target allowed reduction of AST incubation time from 60 min to 5 min. Rapid-cycle amplification reduced qPCR times by 83% to 30 min. CONCLUSIONS: Combined, sequential ID+AST protocols allow rapid and reliable detection, ID, and AST for the diagnosis of bloodstream infections, enabling conversion of empiric to targeted therapy by the second dose of antimicrobials.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6641,

title = {Direct-qPCR Assay for Coupled Identification and Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae},

author = { L. Chen and D. J. Shin and S. Zheng and J. H. Melendez and C. A. Gaydos and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/29999304},

doi = {10.1021/acsinfecdis.8b00104},

issn = {2373-8227 (Electronic) 

2373-8227 (Linking)},

year  = {2018},

date = {2018-09-14},

journal = {ACS Infect Dis},

volume = {4},

number = {9},

pages = {1377-1384},

abstract = {Multidrug-resistant gonorrhea has become an urgent issue for global public health. As the causative agent of gonorrhea, Neisseria gonorrhoeae, has been progressively developing resistance to nearly all prescribed antimicrobial drugs, monitoring its antimicrobial resistance on a broader scale has become a crucial agenda for effective antibiotic stewardship. Unfortunately, gold standard antimicrobial susceptibility testing (AST) relies on time and labor-intensive phenotypic assays, which lag behind the current diagnostic workflow for N. gonorrhoeae identification based on nucleic acid amplification tests (NAAT). Newer assay technologies based on NAAT can rapidly identify N. gonorrhoeae from clinical specimen but fundamentally lack the capacity to provide phenotypic AST information. Herein, we propose a direct-quantitative PCR (direct-qPCR) assay that enables pathogen-specific identification and phenotypic AST via quantitative measurement of N. gonorrhoeae growth directly from a liquid medium without any sample preprocessing. The assay has an analytical sensitivity of 10(2) CFU/mL and is highly specific to N. gonorrhoeae in the presence of urogenital flora and clinical swab eluent. We tested seven N. gonorrhoeae strains against three antibiotic agents, penicillin, tetracycline, and ciprofloxacin, and achieved 95.2% category agreement and 85.7% essential agreement with the FDA-approved E-test. The assay presented in this work has the unique ability to identify N. gonorrhoeae and provide phenotypic AST directly from the liquid medium with cell densities as low as 10(2) CFU/mL, demonstrating an accelerated, sensitive, and scalable workflow for performing both identification and AST of N. gonorrhoeae.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6702,

title = {Facile profiling of molecular heterogeneity by microfluidic digital melt},

author = { C. M. O'Keefe and T. R. 2nd Pisanic and H. Zec and M. J. Overman and J. G. Herman and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30263958},

doi = {10.1126/sciadv.aat6459},

issn = {2375-2548 (Electronic) 

2375-2548 (Linking)},

year  = {2018},

date = {2018-09-01},

journal = {Sci Adv},

volume = {4},

number = {9},

pages = {eaat6459},

abstract = {This work presents a digital microfluidic platform called HYPER-Melt (high-density profiling and enumeration by melt) for highly parallelized copy-by-copy DNA molecular profiling. HYPER-Melt provides a facile means of detecting and assessing sequence variations of thousands of individual DNA molecules through digitization in a nanowell microchip array, allowing amplification and interrogation of individual template molecules by detecting HRM fluorescence changes due to sequence-dependent denaturation. As a model application, HYPER-Melt is used here for the detection and assessment of intermolecular heterogeneity of DNA methylation within the promoters of classical tumor suppressor genes. The capabilities of this platform are validated through serial dilutions of mixed epialleles, with demonstrated detection limits as low as 1 methylated variant in 2 million unmethylated templates (0.00005%) of a classic tumor suppressor gene, CDKN2A (p14(ARF)). The clinical potential of the platform is demonstrated using a digital assay for NDRG4, a tumor suppressor gene that is commonly methylated in colorectal cancer, in liquid biopsies of healthy and colorectal cancer patients. Overall, the platform provides the depth of information, simplicity of use, and single-molecule sensitivity necessary for rapid assessment of intermolecular variation contributing to genetic and epigenetic heterogeneity for challenging applications in embryogenesis, carcinogenesis, and rare biomarker detection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6643,

title = {Simple and Precise Counting of Viable Bacteria by Resazurin-Amplified Picoarray Detection},

author = { K. Hsieh and H. C. Zec and L. Chen and A. M. Kaushik and K. E. Mach and J. C. Liao and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/29969556},

doi = {10.1021/acs.analchem.8b02096},

issn = {1520-6882 (Electronic) 

0003-2700 (Linking)},

year  = {2018},

date = {2018-08-07},

journal = {Anal Chem},

volume = {90},

number = {15},

pages = {9449-9456},

abstract = {Simple, fast, and precise counting of viable bacteria is fundamental to a variety of microbiological applications such as food quality monitoring and clinical diagnosis. To this end, agar plating, microscopy, and emerging microfluidic devices for single bacteria detection have provided useful means for counting viable bacteria, but they also have their limitations ranging from complexity, time, and inaccuracy. We present herein our new method RAPiD (Resazurin-Amplified Picoarray Detection) for addressing this important problem. In RAPiD, we employ vacuum-assisted sample loading and oil-driven sample digitization to stochastically confine single bacteria in Picoarray, a microfluidic device with picoliter-sized isolation chambers (picochambers), in <30 s with only a few minutes of hands-on time. We add AlamarBlue, a resazurin-based fluorescent dye for bacterial growth, in our assay to accelerate the detection of "microcolonies" proliferated from single bacteria within picochambers. Detecting fluorescence in picochambers as an amplified surrogate for bacterial cells allows us to count hundreds of microcolonies with a single image taken via wide-field fluorescence microscopy. We have also expanded our method to practically test multiple titrations from a single bacterial sample in parallel. Using this expanded "multi-RAPiD" strategy, we can quantify viable cells in E. coli and S. aureus samples with precision in approximately 3 h, illustrating RAPiD as a promising new method for counting viable bacteria for microbiological applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6700,

title = {Rapid Microbiology Screening in Pharmaceutical Workflows},

author = { C. Surrette and B. Scherer and A. Corwin and G. Grossmann and A. M. Kaushik and K. Hsieh and P. Zhang and J. C. Liao and P. K. Wong and T. H. Wang and C. M. Puleo},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30027813},

doi = {10.1177/2472630318779758},

issn = {2472-6311 (Electronic) 

2472-6303 (Linking)},

year  = {2018},

date = {2018-08-01},

journal = {SLAS Technol},

volume = {23},

number = {4},

pages = {387-394},

abstract = {Recently advances in miniaturization and automation have been utilized to rapidly decrease the time to result for microbiology testing in the clinic. These advances have been made due to the limitations of conventional culture-based microbiology methods, including agar plate and microbroth dilution, which have long turnaround times and require physicians to treat patients empirically with antibiotics before test results are available. Currently, there exist similar limitations in pharmaceutical sterility and bioburden testing, where the long turnaround times associated with standard microbiology testing drive costly inefficiencies in workflows. These include the time lag associated with sterility screening within drug production lines and the warehousing cost and time delays within supply chains during product testing. Herein, we demonstrate a proof-of-concept combination of a rapid microfluidic assay and an efficient cell filtration process that enables a path toward integrating rapid tests directly into pharmaceutical microbiological screening workflows. We demonstrate separation and detection of Escherichia coli directly captured and analyzed from a mammalian (i.e., CHO) cell culture with a 3.0 h incubation. The demonstration is performed using a membrane filtration module that is compatible with sampling from bioreactors, enabling in-line sampling and process monitoring.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}