Publications by authors named "Wenying Pan"

19 Publications

  • Page 1 of 1

A comprehensive characterization of the cell-free transcriptome reveals tissue- and subtype-specific biomarkers for cancer detection.

Nat Commun 2021 04 21;12(1):2357. Epub 2021 Apr 21.

GRAIL, Inc., Menlo Park, CA, USA.

Cell-free RNA (cfRNA) is a promising analyte for cancer detection. However, a comprehensive assessment of cfRNA in individuals with and without cancer has not been conducted. We perform the first transcriptome-wide characterization of cfRNA in cancer (stage III breast [n = 46], lung [n = 30]) and non-cancer (n = 89) participants from the Circulating Cell-free Genome Atlas (NCT02889978). Of 57,820 annotated genes, 39,564 (68%) are not detected in cfRNA from non-cancer individuals. Within these low-noise regions, we identify tissue- and cancer-specific genes, defined as "dark channel biomarker" (DCB) genes, that are recurrently detected in individuals with cancer. DCB levels in plasma correlate with tumor shedding rate and RNA expression in matched tissue, suggesting that DCBs with high expression in tumor tissue could enhance cancer detection in patients with low levels of circulating tumor DNA. Overall, cfRNA provides a unique opportunity to detect cancer, predict the tumor tissue of origin, and determine the cancer subtype.
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http://dx.doi.org/10.1038/s41467-021-22444-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060291PMC
April 2021

WWOX promotes apoptosis and inhibits autophagy in paclitaxel‑treated ovarian carcinoma cells.

Mol Med Rep 2021 02 10;23(2). Epub 2020 Dec 10.

Department of Gynecology and Obstetrics, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China.

Although paclitaxel (PTX) is a first‑line chemotherapeutic agent for the treatment of epithelial ovarian cancer (EOC), its clinical use is restricted by chemoresistance. Autophagy is believed to promote drug resistance, and WW domain‑containing oxidoreductase (WWOX) has been predicted to serve an essential role in apoptosis induction and to suppress autophagy in various tumor cell types. In the present study, the role of WWOX was demonstrated using PTX‑treated EOC cells. Cell viability and apoptosis were detected using Cell Counting Kit‑8, morphological and flow cytometric analyses. WWOX and phosphorylated (p)‑WWOX were highly expressed in PTX‑treated sensitive EOC cells (A2780), which was accompanied by activation of the apoptosis‑related proteins caspase‑3 and poly (ADP‑ribose) polymerase (PARP). Conversely, PTX‑resistant EOC cells (A2780/T) were characterized by reduced WWOX expression and constant phosphorylation levels, as well as undetectable levels of activated caspase‑3 and PARP when cells were treated with PTX. The altered expression of WWOX between the two cell types was further validated by reverse transcription‑-quantitative PCR. The apoptosis‑inducing role of WWOX was also confirmed by flow cytometry after WWOX overexpression was induced in PTX‑treated A2780 cells. These findings indicated that WWOX activation may inhibit chemoresistance and trigger cancer cell death. The upregulated expression levels of the autophagy‑related protein 12‑5 complex, Beclin‑1 and LC3, as well as the downregulation of P62, were also detected following PTX treatment, suggesting that PTX induced autophagic flux in both types of EOC cells. This conclusion was further supported by visualizing the accumulation of autophagosome and autolysosome vesicles, using confocal microscopy and transmission electron microscopy. PTX was also shown to inhibit mTOR signaling, indicated by a decreased level of p‑mTOR and increased expression of eukaryotic translation initiation factor 4E‑binding protein 1. Finally, the interaction between WWOX, mTOR and autophagy was investigated via WWOX transfection experimentation, and indicated that WWOX activated mTOR whilst inhibiting autophagy. These data indicated that WWOX may serve a critical role in PTX‑induced apoptosis and could suppress autophagy by downregulating essential autophagic effectors in EOC cells via mTOR signaling.
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http://dx.doi.org/10.3892/mmr.2020.11754DOI Listing
February 2021

Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle.

Nat Med 2020 10 14;26(10):1644-1653. Epub 2020 Sep 14.

Department of Bioengineering, Stanford University, Stanford, CA, USA.

In a human menstrual cycle the endometrium undergoes remodeling, shedding and regeneration, all of which are driven by substantial gene expression changes in the underlying cellular hierarchy. Despite its importance in human fertility and regenerative biology, our understanding of this unique type of tissue homeostasis remains rudimentary. We characterized the transcriptomic transformation of human endometrium at single-cell resolution across the menstrual cycle, resolving cellular heterogeneity in multiple dimensions. We profiled the behavior of seven endometrial cell types, including a previously uncharacterized ciliated cell type, during four major phases of endometrial transformation, and found characteristic signatures for each cell type and phase. We discovered that the human window of implantation opens with an abrupt and discontinuous transcriptomic activation in the epithelia, accompanied with a widespread decidualization feature in the stromal fibroblasts. Our study provides a high-resolution molecular and cellular characterization of human endometrial transformation across the menstrual cycle, providing insights into this essential physiological process.
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http://dx.doi.org/10.1038/s41591-020-1040-zDOI Listing
October 2020

Diagnostic value of miR-186-5p for carotid artery stenosis and its predictive significance for future cerebral ischemic event.

Diagn Pathol 2020 Jul 30;15(1):101. Epub 2020 Jul 30.

Department of Obstetrics & Gynecology, Binzhou Medical University Hospital, No. 661, Huanghe 2nd Road, Binzhou, 256603, Shandong, China.

Background: MicroRNAs (miRNAs) serve as novel promising biomarkers for the diagnosis and prognosis of many human diseases. This study investigated the diagnostic value of miR-186-5p for asymptomatic carotid artery stenosis (CAS), and its predictive value for future cerebral ischemic events (CIEs).

Methods: Sixty-seven cases with asymptomatic CAS and 60 healthy individuals were recruited. Serum levels of miR-186-5p were tested by using qRT-PCR. Receiving-operator characteristic (ROC) curve was drawn based on sensitivity and specificity analyses. All asymptomatic CAS cases were followed up for 5 years. Kaplan-Meier method was applied for the evaluation of the predictive value of miR-186-5p for the occurrence of CIE.

Results: The serum level of miR-186-5p was increased significantly in asymptomatic CAS patients. MiR-186-5p was the most significant factor associated with the high degree of carotid stenosis in asymptomatic CAS patients. In the ROC curve analysis, the AUC was 0.919, with the sensitivity of 89.6% and specificity of 81.7% at the cutoff value of 1.221. Kaplan-Meier method results revealed that high miR-186-5p level was associated with the occurrence of CIEs. High miR-186-5p level and high degree of carotid stenosis were independent factors for the occurrence of CIEs.

Conclusion: MiR-186-5p serves as a potential diagnostic biomarker for patients with asymptomatic CAS, and predicts the occurrence of future CIEs.
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http://dx.doi.org/10.1186/s13000-020-01007-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392647PMC
July 2020

Alignment-free filtering for cfNA fusion fragments.

Bioinformatics 2019 07;35(14):i225-i232

Grail, Inc, Menlo Park, CA, USA.

Motivation: Cell-free nucleic acid (cfNA) sequencing data require improvements to existing fusion detection methods along multiple axes: high depth of sequencing, low allele fractions, short fragment lengths and specialized barcodes, such as unique molecular identifiers.

Results: AF4 was developed to address these challenges. It uses a novel alignment-free kmer-based method to detect candidate fusion fragments with high sensitivity and orders of magnitude faster than existing tools. Candidate fragments are then filtered using a max-cover criterion that significantly reduces spurious matches while retaining authentic fusion fragments. This efficient first stage reduces the data sufficiently that commonly used criteria can process the remaining information, or sophisticated filtering policies that may not scale to the raw reads can be used. AF4 provides both targeted and de novo fusion detection modes. We demonstrate both modes in benchmark simulated and real RNA-seq data as well as clinical and cell-line cfNA data.

Availability And Implementation: AF4 is open sourced, licensed under Apache License 2.0, and is available at: https://github.com/grailbio/bio/tree/master/fusion.
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http://dx.doi.org/10.1093/bioinformatics/btz346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612805PMC
July 2019

Noninvasive blood tests for fetal development predict gestational age and preterm delivery.

Science 2018 06;360(6393):1133-1136

Departments of Bioengineering and Applied Physics, Stanford University and Chan Zuckerberg Biohub, Stanford, CA 94305, USA.

Noninvasive blood tests that provide information about fetal development and gestational age could potentially improve prenatal care. Ultrasound, the current gold standard, is not always affordable in low-resource settings and does not predict spontaneous preterm birth, a leading cause of infant death. In a pilot study of 31 healthy pregnant women, we found that measurement of nine cell-free RNA (cfRNA) transcripts in maternal blood predicted gestational age with comparable accuracy to ultrasound but at substantially lower cost. In a related study of 38 women (23 full-term and 15 preterm deliveries), all at elevated risk of delivering preterm, we identified seven cfRNA transcripts that accurately classified women who delivered preterm up to 2 months in advance of labor. These tests hold promise for prenatal care in both the developed and developing worlds, although they require validation in larger, blinded clinical trials.
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http://dx.doi.org/10.1126/science.aar3819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734383PMC
June 2018

Recurrently Mutated Genes Differ between Leptomeningeal and Solid Lung Cancer Brain Metastases.

J Thorac Oncol 2018 07 29;13(7):1022-1027. Epub 2018 Mar 29.

Department of Neurosurgery, Stanford University School of Medicine, Stanford, California. Electronic address:

When compared with solid brain metastases from NSCLC, leptomeningeal disease (LMD) has unique growth patterns and is rapidly fatal. Patients with LMD do not undergo surgical resection, limiting the tissue available for scientific research. In this study we performed whole exome sequencing on eight samples of LMD to identify somatic mutations and compared the results with those for 26 solid brain metastases. We found that taste 2 receptor member 31 gene (TAS2R31) and phosphodiesterase 4D interacting protein gene (PDE4DIP) were recurrently mutated among LMD samples, suggesting involvement in LMD progression. Together with a retrospective review of the charts of an additional 44 patients with NSCLC LMD, we discovered a surprisingly low number of KRAS mutations (n = 4 [7.7%]) but a high number of EGFR mutations (n = 33 [63.5%]). The median interval for development of LMD from NSCLC was shorter in patients with mutant EGFR (16.3 months) than in patients with wild-type EGFR (23.9 months) (p = 0.017). Targeted analysis of recurrent mutations thus presents a useful complement to the existing diagnostic tool kit, and correlations of EGFR in LMD and KRAS in solid metastases suggest that molecular distinctions or systemic treatment pressure underpin the differences in growth patterns within the brain.
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http://dx.doi.org/10.1016/j.jtho.2018.03.018DOI Listing
July 2018

Nigericin Inhibits Epithelial Ovarian Cancer Metastasis by Suppressing the Cell Cycle and Epithelial-Mesenchymal Transition.

Biochemistry (Mosc) 2017 Aug;82(8):933-941

Qilu Hospital, Shandong University, Department of Obstetrics and Gynecology, Jinan, Shandong, 250012, P.R. China.

Epithelial ovarian cancer (EOC) has the highest mortality among various types of gynecological malignancies. Most patients die of metastasis and recurrence due to cisplatin resistance. Thus, it is urgent to develop novel therapies to cure this disease. CCK-8 assay showed that nigericin exhibited strong cytotoxicity on A2780 and SKOV3 cell lines. Flow cytometry indicated that nigericin could induce cell cycle arrest at G0/G1 phase and promote cell apoptosis. Boyden chamber assay revealed that nigericin could inhibit migration and invasion in a dose-dependent manner by suppressing epithelial-mesenchymal transition (EMT) in EOC cells. These effects were mediated, at least partly, by the Wnt/β-catenin signaling pathway. Our results demonstrated that nigericin could inhibit EMT during cell invasion and metastasis through the canonical Wnt/β-catenin signaling pathway. Nigericin may prove to be a novel therapeutic strategy that is effective in patients with metastatic EOC.
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http://dx.doi.org/10.1134/S0006297917080089DOI Listing
August 2017

Simultaneously Monitoring Immune Response and Microbial Infections during Pregnancy through Plasma cfRNA Sequencing.

Clin Chem 2017 Nov 13;63(11):1695-1704. Epub 2017 Sep 13.

Departments of Bioengineering and Applied Physics, Stanford University, and Chan Zuckerberg Biohub, Stanford, CA;

Background: Plasma cell-free RNA (cfRNA) encompasses a broad spectrum of RNA species that can be derived from both human cells and microbes. Because cfRNA is fragmented and of low concentration, it has been challenging to profile its transcriptome using standard RNA-seq methods.

Methods: We assessed several recently developed RNA-seq methods on cfRNA samples. We then analyzed the dynamic changes of both the human transcriptome and the microbiome of plasma during pregnancy from 60 women.

Results: cfRNA reflects a well-orchestrated immune modulation during pregnancy: an up-regulation of antiinflammatory genes and an increased abundance of antimicrobial genes. We observed that the plasma microbiome remained relatively stable during pregnancy. The bacteria shows an increased prevalence and increased abundance at postpartum, which is likely to be associated with postpartum infection. We demonstrated that cfRNA-seq can be used to monitor viral infections. We detected a number of human pathogens in our patients, including an undiagnosed patient with a high load of human parvovirus B19 virus (B19V), which is known to be a potential cause of complications in pregnancy.

Conclusions: Plasma cfRNA-seq demonstrates the potential to simultaneously monitor immune response and microbial infections during pregnancy.
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http://dx.doi.org/10.1373/clinchem.2017.273888DOI Listing
November 2017

A pilot study on the use of cerebrospinal fluid cell-free DNA in intramedullary spinal ependymoma.

J Neurooncol 2017 Oct 12;135(1):29-36. Epub 2017 Sep 12.

Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Drive MC 5327, Stanford, CA, 94305, USA.

Cerebrospinal fluid (CSF) represents a promising source of cell-free DNA (cfDNA) for tumors of the central nervous system. A CSF-based liquid biopsy may obviate the need for riskier tissue biopsies and serve as a means for monitoring tumor recurrence or response to therapy. Spinal ependymomas most commonly occur in adults, and aggressive resection must be delicately balanced with the risk of injury to adjacent normal tissue. In patients with subtotal resection, recurrence commonly occurs. A CSF-based liquid biopsy matched to the patient's spinal ependymoma mutation profile has potential to be more sensitive then surveillance MRI, but the utility has not been well characterized for tumors of the spinal cord. In this study, we collected matched blood, tumor, and CSF samples from three adult patients with WHO grade II intramedullary spinal ependymoma. We performed whole exome sequencing on matched tumor and normal DNA to design Droplet Digital™ PCR (ddPCR) probes for tumor and wild-type mutations. We then interrogated CSF samples for tumor-derived cfDNA by performing ddPCR on extracted cfDNA. Tumor cfDNA was not reliably detected in the CSF of our cohort. Anatomic sequestration and low grade of intramedullary spinal cord tumors likely limits the role of CSF liquid biopsy.
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http://dx.doi.org/10.1007/s11060-017-2557-yDOI Listing
October 2017

Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA.

Proc Natl Acad Sci U S A 2017 09 22;114(36):9623-9628. Epub 2017 Aug 22.

Department of Bioengineering, Stanford University, Stanford, CA 94305;

Blood circulates throughout the human body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analyzing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.
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http://dx.doi.org/10.1073/pnas.1707009114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594678PMC
September 2017

Neurocognitive impairment is correlated with oxidative stress in patients with moderate-to-severe obstructive sleep apnea hypopnea syndrome.

Respir Med 2016 11 9;120:25-30. Epub 2016 Sep 9.

Sleep Center, The Second Affiliated Hospital of Soochow University, Soochow University, Suzhou, China; Department of Neurology, The Second Affiliated Hospital of Soochow University, Soochow University, Suzhou, China; Laboratory of Aging and Nervous Diseases, Institute of Neuroscience, Soochow University, Suzhou, China.

Background And Objectives: Patients with obstructive sleep apnea hypopnea syndrome (OSAHS) are associated with increased risk of neurocognitive impairment, which are largely recognized as mild cognitive impairment (MCI), and oxidative stress is postulated as one of the underlying mechanisms. This study aimed to investigate the relationship between MCI and oxidative stress biomarkers in OSAHS.

Methods: A total of 119 middle-aged patients with moderate-to-severe OSAHS were included. Based on the baseline Montreal Cognitive Assessment (MoCA, validated Chinese version), 86 and 33 patients presented with normal cognitive function (NC, MoCA ≥26) and mild cognitive impairment (MCI, MoCA <26), respectively. Overnight PSG, MoCA and serum levels of ischemia-modified albumin (IMA), malondialdehyde (MDA) and advanced oxidation protein products (AOPP) were collected and analyzed.

Results: Compared to NC group, patients with MCI were characterized with significantly greater waist-to-height ratio, AHI, ODI and time ratio of SpO<90%, and lower average SpO and time ratio of rapid eye movement (REM). All three oxidative stress biomarkers were markedly elevated in MCI group. Binary logistic regression analysis demonstrated that MCI is significantly correlated with serum levels of IMA, REM ratio and the age of patients.

Conclusions: The neurocognitive impairment in moderate-to-severe OSAHS patients is associated with significantly elevated oxidative stress. IMA might be a new useful biomarker correlated with mild cognitive impairment of the patients.
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http://dx.doi.org/10.1016/j.rmed.2016.09.009DOI Listing
November 2016

Tumor DNA in cerebral spinal fluid reflects clinical course in a patient with melanoma leptomeningeal brain metastases.

J Neurooncol 2016 05 9;128(1):93-100. Epub 2016 Mar 9.

Department of Neurosurgery, Stanford University, MSLS, 1205 Welch Rd, R307, Stanford, CA, 94305-5487, USA.

Cerebral spinal fluid (CSF) from brain tumor patients contains tumor cellular and cell-free DNA (cfDNA), which provides a less-invasive and routinely accessible method to obtain tumor genomic information. In this report, we used droplet digital PCR to test mutant tumor DNA in CSF of a patient to monitor the treatment response of metastatic melanoma leptomeningeal disease (LMD). The primary melanoma was known to have a BRAF (V600E) mutation, and the patient was treated with whole brain radiotherapy and BRAF inhibitors. We collected 9 CSF samples over 6 months. The mutant cfDNA fraction gradually decreased from 53 % (time of diagnosis) to 0 (time of symptom alleviation) over the first 6 time points. Three months after clinical improvement, the patient returned with severe symptoms and the mutant cfDNA was again detected in CSF at high levels. The mutant DNA fraction corresponded well with the patient's clinical response. We used whole exome sequencing to examine the mutation profiles of the LMD tumor DNA in CSF before therapeutic response and after disease relapse, and discovered a canonical cancer mutation PTEN (R130*) at both time points. The cellular and cfDNA revealed similar mutation profiles, suggesting cfDNA is representative of LMD cells. This study demonstrates the potential of using cellular or cfDNA in CSF to monitor treatment response for LMD.
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http://dx.doi.org/10.1007/s11060-016-2081-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412509PMC
May 2016

ORMDL3 contributes to the risk of atherosclerosis in Chinese Han population and mediates oxidized low-density lipoprotein-induced autophagy in endothelial cells.

Sci Rep 2015 Nov 25;5:17194. Epub 2015 Nov 25.

The Key Laboratory for Experimental Teratology of the Ministry of Education, Shandong University School of Medicine, Jinan, Shandong 250012, P.R. China.

ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) is a universally confirmed susceptibility gene for asthma and has recently emerged as a crucial modulator in lipid metabolism, inflammation and endoplasmic reticulum (ER) stress-the mechanisms also closely involved in atherosclerosis (AS). Here we first presented the evidence of two single nucleotide polymorphisms regulating ORMDL3 expression (rs7216389 and rs9303277) significantly associated with AS risk and the evidence of increased ORMDL3 expression in AS cases compared to controls, in Chinese Han population. Following the detection of its statistical correlation with AS, we further explored the functional relevance of ORMDL3 and hypothesized a potential role mediating autophagy as autophagy is activated upon modified lipid, inflammation and ER stress. Our results demonstrated that in endothelial cells oxidized low-density lipoprotein (ox-LDL) up-regulated ORMDL3 expression and knockdown of ORMDL3 alleviated not only ox-LDL-induced but also basal autophagy. BECN1 is essential for autophagy initiation and silencing of ORMDL3 suppressed ox-LDL-induced as well as basal BECN1 expression. In addition, deletion of ORMDL3 resulted in greater sensitivity to ox-LDL-induced cell death. Taken together, ORMDL3 might represent a causal gene mediating autophagy in endothelial cells in the pathogenesis of AS.
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http://dx.doi.org/10.1038/srep17194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4658630PMC
November 2015

Brain tumor mutations detected in cerebral spinal fluid.

Clin Chem 2015 Mar 20;61(3):514-22. Epub 2015 Jan 20.

Department of Bioengineering,

Background: Detecting tumor-derived cell-free DNA (cfDNA) in the blood of brain tumor patients is challenging, presumably owing to the blood-brain barrier. Cerebral spinal fluid (CSF) may serve as an alternative "liquid biopsy" of brain tumors by enabling measurement of circulating DNA within CSF to characterize tumor-specific mutations. Many aspects about the characteristics and detectability of tumor mutations in CSF remain undetermined.

Methods: We used digital PCR and targeted amplicon sequencing to quantify tumor mutations in the cfDNA of CSF and plasma collected from 7 patients with solid brain tumors. Also, we applied cancer panel sequencing to globally characterize the somatic mutation profile from the CSF of 1 patient with suspected leptomeningeal disease.

Results: We detected tumor mutations in CSF samples from 6 of 7 patients with solid brain tumors. The concentration of the tumor mutant alleles varied widely between patients, from <5 to nearly 3000 copies/mL CSF. We identified 7 somatic mutations from the CSF of a patient with leptomeningeal disease by use of cancer panel sequencing, and the result was concordant with genetic testing on the primary tumor biopsy.

Conclusions: Tumor mutations were detectable in cfDNA from the CSF of patients with different primary and metastatic brain tumors. We designed 2 strategies to characterize tumor mutations in CSF for potential clinical diagnosis: the targeted detection of known driver mutations to monitor brain metastasis and the global characterization of genomic aberrations to direct personalized cancer care.
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http://dx.doi.org/10.1373/clinchem.2014.235457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412506PMC
March 2015

Multiplexed microfluidic blotting of proteins and nucleic acids by parallel, serpentine microchannels.

Lab Chip 2015 Jan;15(1):105-12

Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China.

This work develops a high-throughput, high-efficiency and straightforward microfluidic blotting method for analyzing proteins and nucleic acids. Sample solutions containing antibodies (for protein detection) or hybridization probes (for nucleic acid detection) are introduced into the parallel, serpentine microchannels to specifically recognize the immobilized targets on the substrate, achieving the identification of multiple targets in multiple samples simultaneously. The loading control, molecular weight markers, and antigen/antibody titration are designed and integrated into the microfluidic chip, thus allowing for the quantification of proteins and nucleic acids. Importantly, we could easily distinguish the adjacent blotting bands inside parallel microchannels, which may be difficult to achieve in conventional blotting. The small dimensions of microfluidic channels also help to reduce the amount of probing molecules and to accelerate the biochemical reaction. Our microfluidic blotting could bypass the steps of blocking and washing, further reducing the operation time and complexity.
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http://dx.doi.org/10.1039/c4lc00901kDOI Listing
January 2015

Noninvasive in vivo monitoring of tissue-specific global gene expression in humans.

Proc Natl Acad Sci U S A 2014 May 5;111(20):7361-6. Epub 2014 May 5.

Departments of Bioengineering and

Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape circulating RNA in a cohort of human subjects. By focusing on genes whose expression is highly specific to certain tissues, we were able to identify the relative contributions of these tissues to circulating RNA and to monitor changes in tissue development and health. As one application of this approach, we performed a longitudinal study on pregnant women and analyzed their combined cell-free RNA transcriptomes across all three trimesters of pregnancy and after delivery. In addition to the analysis of mRNA, we observed and characterized noncoding species such as long noncoding RNA and circular RNA transcripts whose presence had not been previously observed in human plasma. We demonstrate that it is possible to track specific longitudinal phenotypic changes in both the mother and the fetus and that it is possible to directly measure transcripts from a variety of fetal tissues in the maternal blood sample. We also studied the role of neuron-specific transcripts in the blood of healthy adults and those suffering from the neurodegenerative disorder Alzheimer's disease and showed that disease specific neural transcripts are present at increased levels in the blood of affected individuals. Characterization of the cell-free transcriptome in its entirety may thus provide broad insights into human health and development without the need for invasive tissue sampling.
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http://dx.doi.org/10.1073/pnas.1405528111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034220PMC
May 2014

High-performance binary protein interaction screening in a microfluidic format.

Anal Chem 2012 Nov 23;84(21):9572-8. Epub 2012 Oct 23.

Department of Bioengineering, Stanford University, Stanford, California 94305, United States.

The standard procedure to increase microfluidic chip performance is to grow the number of parallel test systems on the chip. This process is accompanied by miniaturizing biochemical workflows and micromechanical elements, which is often a major challenge for both engineering fields. In this work, we show that it is possible to substantially increase the runtime performance of a microfluidic affinity assay for protein interactions by simultaneously engineering fluid logics and assay chemistry. For this, synergistic effects between the micro- and chemical architecture of the chip are exploited. The presented strategy of reducing the runtime rather than size and volume of the mechanical elements and biological reagent compartments will, in general, be of importance for future analytical test systems on microfluidic chips to overcome performance barriers.
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http://dx.doi.org/10.1021/ac302436yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3533494PMC
November 2012

Microfluidic Western blot.

Anal Chem 2010 May;82(10):3974-6

We develop a novel method for Western blot based on microfluidics, incorporating the internal molecular weight marker, loading control, and antibody titration in the same protocol. Compared with the conventional method which could detect only one protein, the microfluidic Western blot could analyze at least 10 proteins simultaneously from a single sample, and it requires only about 1% of the amount of antibody used in conventional Western blot.
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http://dx.doi.org/10.1021/ac1000493DOI Listing
May 2010