Biology subjects

Dong, M.

Publications and source records attributed to Dong, M..

8 recordsLinked to original sources

bioRxiv2018

Flexibly-oriented double Cdc45-MCM-GINS intermediates during eukaryotic replicative helicase maturation

The core of the eukaryotic helicase MCM is loaded as an inactive double hexamer (DH). How it is assembled into two active Cdc45-MCM-GINS (CMG) helicases remains elusive. Here, we report that at the onset of S phase, both Cdc45 and GINS are loaded as dimers onto MCM DH, resulting in formation of double CMG (d-CMG). As S phase proceeds, d-CMGs gradually mature into two single CMG-centered replisome progression complexes (RPCs). Mass spectra reveal that RPA and DNA Pol /primase co-purify exclusively with RPCs, but not with d-CMGs. Consistently, d-CMGs are not able to catalyze either the unwinding or de novo DNA synthesis, while RPCs can do both. Using single-particle electron microscopy, we have obtained 2D class averages of d-CMGs. Compared to MCM DHs, they display heterogeneous, flexibly orientated and partially loosened conformations with changed interfaces. The dumbbell-shaped d-CMGs are mediated by Ctf4, while other types of d-CMGs are independent of Ctf4. These data suggest CMG dimers as bona fide intermediates during MCM maturation, providing an additional quality control for symmetric origin activation and bidirectional replication.

molecular biology↗

bioRxiv2018

Apigenin relaxes rat intrarenal arteries: involvement of Cl- channels and K+ channels

The vasodilator effect of apigenin (API) was demonstrated in a number of vascular beds. We aimed to characterize the vasospasmolytic and electrophysiological effects of apigenin (API) in intrarenal arteries (IRAs). The vascular tone of male rat isolated IRAs was recorded with a myograph. Transmembrane Cl- currents through Ca2+-activated Cl- channels (CaCCs), K+ currents through voltage-gated K+ (Kv) channels and inwardly rectifier K+ (Kir) channels were recorded with patch clamp in the freshly isolated arterial smooth muscle cells (ASMCs). Preincubation with API (10-100 M) concentration-dependently depressed the contractions induced by KCl, 9,11-dideoxy-9,11-methanoepoxy prostaglandin F2 (U46619), phenylephrine and vasopressin without significant preference and the IC50 values were 13.27-26.26 M. Acute application of API elicited instant relaxations in the IRAs precontracted with these vasoconstrictors and the RC50 values were 5.80-24.33 M. API relaxation was attenuated by chloride deprivation, CaCC blockers, Kv blocker and nitric oxide synthase inhibitor, but not by Kir blocker and cyclooxygenase inhibitor. At 10-100 M, API depressed CaCC currents and Kir currents while enhanced Kv currents of IRA ASMCs. The present results demonstrate that API counteracts various vasoconstrictors noncompetitively and nonspecifically and suggest that modulation of CaCCs, Kv and Kir channels of IRA ASMCs is involved in its vasospasmolytic effects.

pharmacology and toxicology↗

bioRxiv2018

Reactive Initiation Training is more effective than Shuttle Run to improve the on-court agility of novice badminton players

PurposeDespite its well-known importance in sports, agility is ambiguously defined and lack of research. Shuttle Run (SR) is commonly used to improve the on-court agility of badminton players. Reactive Initiation Training (RIT) contrasts SR in that it only demands rapid generation of initiation step toward the direction of shuttlecock. The current study compared SR with RIT to determine which one is more effective for improving on-court agility of novice badminton players.\n\nMethod20 novice badminton players were split in half to receive either RIT or SR on court for five days. Before and after training, participants were assessed on their ability to intercept the shuttlecocks randomly thrown by a coach to six corners of the court with and without visual occlusion of the coach. All trials of interception were recorded for video analysis of initiation time, running time and total time.\n\nResultsThe mean total times were greater with visual occlusion and varied systematically with the position of interception. Both training methods shortened the mean running time, however, only RIT additionally reduced the initiation time and its proportion on those time-consuming positions in the occluded condition.\n\nConclusionRIT is more effective than SR to improve the on-court agility of novice badminton players.

physiology↗

bioRxiv2018

Genetically encoded ratiometric indicators for potassium ion

Potassium ion (K+) homeostasis and dynamics play critical roles in regulating various biological activities, and the ability to monitor K+ spatial-temporal dynamics is critical to understanding these biological functions. Here we report the design and characterization of a Forster resonance energy transfer (FRET)-based genetically encoded K+ indicator, KIRIN1, constructed by inserting a bacterial cytosolic K+ binding protein (Kbp) between a fluorescent protein (FP) FRET pair, mCerulean3 and cp173Venus. Binding of K+ induces a conformational change in Kbp, resulting in an increase in FRET efficiency. KIRIN1 was able to detect K+ at physiologically relevant concentrations in vitro and is highly selective toward K+ over Na+. We further demonstrated that KIRIN1 allowed real-time imaging of pharmacologically induced depletion of cytosolic K+ in live cells, and KIRIN1 also enabled optical tracing of K+ efflux and reuptake in neurons upon glutamate stimulation in cultured primary neurons. These results demonstrate that KIRIN1 is a valuable tool to detect K+ in vitro and in live cells.

bioengineering↗

bioRxiv2017

Newly identified relatives of botulinum neurotoxins shed light on their molecular evolution

The evolution of bacterial toxins is a central question to understanding the origins of human pathogens and infectious disease. Through genomic data mining, we traced the evolution of the deadliest known toxin family, clostridial neurotoxins, comprised of tetanus and botulinum neurotoxins (BoNT). We identified numerous uncharacterized lineages of BoNT-related genes in environmental species outside of Clostridium, revealing insights into their molecular ancestry. Phylogenetic analysis pinpointed a sister lineage of BoNT-like toxins in the gram-negative organism, Chryseobacterium piperi, that exhibit distant homology at the sequence level but preserve overall domain architecture. Resequencing and assembly of the C. piperi genome confirmed the presence of BoNT-like proteins encoded within two toxin-rich gene clusters. A C. piperi BoNT-like protein was validated as a novel toxin that induced necrotic cell death in human kidney cells. Mutagenesis of the putative active site abolished toxicity and indicated a zinc metalloprotease-dependent mechanism. The C. piperi toxin did not cleave common SNARE substrates of BoNTs, indicating that BoNTs have diverged from related families in substrate specificity. The new lineages of BoNT-like toxins identified by computational methods represent evolutionary missing links, and suggest an origin of clostridial neurotoxins from ancestral toxins present in environmental bacteria.\n\nSignificance statementThe origins of bacterial toxins that cause human disease is a key question in our understanding of pathogen evolution. To explore this question, we searched genomes for evolutionary relatives of the deadliest biological toxins known to science, botulinum neurotoxins. Genomic and phylogenetic analysis revealed a group of toxins in the Chryseobacterium piperi genome that are a sister lineage to botulinum toxins. Genome sequencing of this organism confirmed the presence of toxin-rich gene clusters, and a predicted C. piperi toxin was shown to induce necrotic cell death in human cells. These newly predicted toxins are missing links in our understanding of botulinum neurotoxin evolution, revealing its origins from an ancestral family of toxins that may be widespread in the environment.

bioinformatics↗

bioRxiv2017

Single Molecule Sequencing of Cell-free DNA from Maternal Plasma for Noninvasive Trisomy Detection

The demand of non-invasive prenatal testing for autosomal aneuploidy using cell-free fetal DNA (cffDNA) in maternal plasma is a highly sought-after diagnostic, with a rapidly growing market. Current approaches developed by next generation sequencing (NGS) need PCR amplifcation during sample preparation, which results in amplification bias in GC-rich areas of the human genome. With these approaches, the minimum fetal fraction in maternal plasma is 4% for the small differences in circulating cfDNA between trisomic and disomic pregnancies to be detectable. In this paper, we performed single molecule sequencing of cell-free DNA from maternal plasma for noninvasive trisomy 13, 18 and 21 detections using the GenoCare platform. We found that single molecule sequencing is sensitive enough to detect these chromosome abnormalities when the fetal DNA fraction is as low as 2%. Compared to the Hiseq2500 platform, no significant GC bias was observed. The improved sensitivity and unbiased GC readout make GenoCare a promising platform for autosomal aneuploidy detections, even in the very early stage of pregnancy.

genomics↗

bioRxiv2017

Direct in vivo mapping of functional suppressors in glioblastoma genome

Glioblastoma (GBM) is one of the deadliest cancers, with limited effective treatments and single-digit five-year survival 1-7. A causative understanding of genetic factors that regulate GBM formation is of central importance 8-19. However, a global, quantitative and functional understanding of gliomagenesis in the native brain environment has been lacking due to multiple challenges. Here, we developed an adeno-associated virus (AAV) mediated autochthonous CRISPR screen and directly mapped functional suppressors in the GBM genome. Stereotaxic delivery of an AAV library targeting significantly mutated genes into fully immunocompetent conditional Cas9 mice robustly led to gliomagenesis, resulting in tumors that recapitulate features of human GBM. Targeted capture sequencing revealed deep mutational profiles with diverse patterns across mice, uncovering in vivo roles of previously uncharacterized factors in GBM such as immune regulator B2m, zinc finger protein Zc3h13, transcription repressor Cic, epigenetic regulators Mll2/3 and Arid1b, alongside canonical tumor suppressors Nf1 and Pten. Comparative cancer genomics showed that the mutation frequencies across all genes tested in mice significantly correlate with those in human from two independent patient cohorts. Co-mutation analysis identified frequently co-occurring driver combinations, which were validated using AAV minipools, such as Mll2, B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1. Distinct from Nf1-oncotype tumors, Rb1-oncotype tumors exhibit undifferentiated histopathology phenotype and aberrant activation of developmental reprogramming signatures such as Homeobox gene clusters. The secondary addition of Zc3h13 or Pten mutations drastically altered the gene expression profiles of Rb1 mutants and rendered them more resistant to the GBM chemotherapeutic temozolomide. Our study provides a systematic functional landscape of GBM suppressors directly in vivo, opening new paths for high-throughput molecular mapping and cancer phenotyping.

systems biology↗

bioRxiv2017

Pooled AAV-CRISPR Screen with Targeted Amplicon Sequencing

High-resolution, high-throughput direct in vivo screening of functional genetic factors in native tissues has long been challenging. Adeno-associated viruses (AAV) are powerful carriers of transgenes and have been shown to mediate efficient genome editing in various organs in mice. Here, we developed a new technological approach, Pooled AAV-CRISPR Screen with Targeted Amplicon Sequencing (PASTAS), and demonstrated its application for directly mapping functional cancer driver variants in the mouse liver in an autochthonous manner. Intravenous delivery of an AAV-CRISPR library targeting a set of the most frequently mutated tumor suppressor genes into fully immunocompetent conditional Cas9 knock-in mice consistently generated highly complex autochthonous liver tumors. The molecular landscapes of these genetically diverse tumors were mapped out by deep direct readout of Cas9-generated variants at predicted sgRNA cut sites using molecular inversion probe sequencing. Co-occurrence and correlation analyses as well as validation with lower complexity minipools further confirmed the potency of various co-mutated drivers. The PASTAS method can be applied to virtually any gene sets, any cancer types, or any type of in vivo genetic studies other than cancer.

systems biology↗