Zhiqi Tian

5-Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages

5-Hydroxymethylcytosine (5hmC) is an important mammalian DNA epigenetic modification that has been linked to gene regulation and cancer pathogenesis. Here we explored the diagnostic potential of 5hmC in circulating cell-free DNA (cfDNA) using a sensitive chemical labeling-based low-input shotgun sequencing approach. We sequenced cell-free 5hmC from 49 patients of seven different cancer types and found distinct features that could be used to predict cancer types and stages with high accuracy. Specifically, we discovered that lung cancer leads to a progressive global loss of 5hmC in cfDNA, whereas hepatocellular carcinoma and pancreatic cancer lead to disease-specific changes in the cell-free hydroxymethylome. Our proof-of-principle results suggest that cell-free 5hmC signatures may potentially be used not only to identify cancer types but also to track tumor stage in some cancers.

Versatile Structures of α-Synuclein

α-Synuclein (α-syn) is an intrinsically disordered protein abundantly distributed in presynaptic terminals. Aggregation of α-syn into Lewy bodies (LB) is a molecular hallmark of Parkinson’s disease (PD). α-Syn features an extreme conformational diversity, which adapts to different conditions and fulfills versatile functions. However, the molecular mechanism of α-syn transformation and the relation between different structural species and their functional and pathogenic roles in neuronal activities and PD remain unknown. In this mini-review, we summarize the recent discoveries of α-syn structures in the membrane-bound state, in cytosol, and in the amyloid state under physiological and pathological conditions. From the current knowledge on different structural species of α-syn, we intend to find a clue about its function and toxicity in normal neurons and under disease conditions, which could shed light on the PD pathogenesis.

SNARE‐Reconstituted Liposomes as Controllable Zeptoliter Nanoreactors for Macromolecules

Liposomes are synthetic phospholipid vesicles containing an aqueous lumen confined by a lipid bilayer. These vesicles have been used as nanoreactors because they offer confined environments that can result in significant changes to chemistry. However, a major limitation of using liposomes as nanocontainers is the impermeability of their lipid membranes. To overcome this, scientists have tested the use of porous liposomes generated by membrane phase changes or by pore formation proteins. In this study, the selective permeability of porous liposomes to molecules smaller than the threshold pore size is demonstrated for triggering internal reactions. Furthermore, in order to deliver macromolecules larger than the threshold pore size into lipid‐based nanoreactors, fusogenic soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins were employed to mediate membrane fusion between two liposomes to initiate reactions on demand without losing previously encapsulated macromolecules. This novel approach circumvents limitations associated with inserting molecules larger than the threshold pore size of porous lipid membranes.

Quantitation and mapping of the epigenetic marker 5-hydroxymethylcytosine

We here review primary methods used in quantifying and mapping 5-hydroxymethylcytosine (5hmC), including global quantification, restriction enzyme-based detection, and methods involving DNA-enrichment strategies and the genome-wide sequencing of 5hmC. As discovered in the mammalian genome in 2009, 5hmC, oxidized from 5-methylcytosine (5mC) by ten-eleven translocation (TET) dioxygenases, is increasingly being recognized as a biomarker in biological processes from development to pathogenesis, as its various detection methods have shown. We focus in particular on an ultrasensitive single-molecule imaging technique that can detect and quantify 5hmC from trace samples and thus offer information regarding the distance-based relationship between 5hmC and 5mC when used in combination with fluorescence resonance energy transfer.

Single-Molecule Fluorescence Measurement of SNARE-Mediated Vesicle Fusion

This chapter expounds the single vesicle-vesicle fluorescence resonance energy transfer (FRET) measurement to study the membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. The formation of a four-α-helix bundle of SNARE proteins can drive two membranes to a close proximity for fusion. Through single-molecule FRET-based microscopy, the lipid-mixing process at the single-vesicle level can be tracked in real time. This reconstitution system is applicable to study the molecular mechanism of SNAREs during different membrane fusion stages, such as docking, hemifusion, and full fusion. Four main parts are described in this chapter, including SNARE reconstitution, imaging preparation, data collection, and analysis.

Quantitative Analysis of Interactive Behavior of Mitochondria and Lysosomes Using Structured Illumination Microscopy

Super-resolution optical microscopy has extended the spatial resolution of cell biology from the cellular level to the nanoscale, enabling the observation of the interactive behavior of single mitochondria and lysosomes. Quantitative parametrization of interaction between mitochondria and lysosomes under super-resolution optical microscopy, however, is currently unavailable, which has severely limited our understanding of the molecular machinery underlying mitochondrial functionality. Here, we introduce an M-value to quantitatively investigate mitochondria and lysosome contact (MLC) and mitophagy under structured illumination microscopy. We found that the M-value for an MLC is typically less than 0.4, whereas in mitophagy it ranges from 0.5 to 1.0. This system permits further investigation of the detailed molecular mechanism governing the interactive behavior of mitochondria and lysosomes.

Super-Resolution Tracking of Mitochondrial Dynamics with An Iridium(III) Luminophore

Combining luminescent transition metal complex with super-resolution microscopy is an excellent strategy for the long-term visualization of the dynamics of subcellular structures in living cells. However, it remains unclear whether iridium(III) complexes are applicable for a particular type of super-resolution technique, structured illumination microscopy (SIM), to image subcellular structures. Herein, an iridium(III) dye, to track mitochondrial dynamics in living cells under SIM is described. The dye demonstrates excellent specificity and photostability and satisfactory cell permeability. While using SIM to image mitochondria, an ≈80 nm resolution is achieved that allows the clear observation of the structure of mitochondrial cristae. The dye is used to monitor and quantify mitochondrial dynamics relative to lysosomes, including fusion involved in mitophagy, and newly discovered mitochondria-lysosome contact (MLC) under different conditions. The MLC remains intact and fusion vanishes when five receptors, p62, NDP52, OPTN, NBR1, and TAX1BP1, are knocked out, suggesting that these two processes are independent.