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Liu, H.

Publications and source records attributed to Liu, H..

At least 883 records · Page 49Linked to original sources
bioRxiv2020

Layer-specific Loss and Compensation of Parvocellular Response in Subcortical Pathways of Adult Human Amblyopia

Abnormal visual experience in critical period causes amblyopia or lazy eye, reducing visual abilities even with corrected optics. A long-standing question is where in the human visual system does the amblyopic deficit arise. In particular, whether amblyopia induces selective deficits of the magnocellular (M) or the parvocellular (P) geniculostriate pathways, and whether the more ancient retinotectal pathway is also affected. Technical limitations to non-invasively measure layer-specific activity in human lateral geniculate nucleus (LGN) and superior colliculus (SC) hampered efforts in addressing these questions. In the current study, using lamina-resolved 3T and 7T fMRI and visual stimuli selectively activating the M and P pathways, we investigated layer-specific response properties of the LGN and the SC of amblyopia patients and normal controls. With stimuli presented to the amblyopic eye, there was a stronger response loss in the P layers than in the M layers of the LGN. Compared to normal controls, amblyopic eyes response to the P stimulus was selectively reduced in the superficial SC, while the fellow eyes response was robustly increased in the deep SC. Selective P response deficits of amblyopia were also observed in the visual pulvinar, early visual cortex, and ventral but not dorsal visual streams. These results provide strong in vivo evidence in adult amblyopic patients for selective deficits of parvocellular functions in the visual thalamus, and additionally reveal response deficits to the amblyopic eye and neural compensation to the fellow eye in the retinotectal pathway. HighlightsO_LIParvocellular response loss in the LGN P layers, visual pulvinar and ventral visual stream C_LIO_LISelective amblyopic deficits of the parvocellular pathway C_LIO_LIAmblyopic eyes response decreased in the superficial SC C_LIO_LIFellow eyes response increased in the deep SC C_LIO_LIAmblyopic deficits and neural compensation in the retinotectal pathway C_LI

neuroscience↗
bioRxiv2020

Clinical Characteristics and Risk of Second Primary Lung Cancer After Cervix Cancer: A Population-Based Study

BackgroundLung cancer as a second primary malignancy is increasingly common, but the clinical characteristics of second primary non-small cell lung cancer after cervix cancer (CC-NSCLC) in comparison with first primary non-small cell lung cancer (NSCLC1) is unknown. MethodsThe Surveillance, Epidemiology and EndResults (SEER) cancer registry between 1998 and 2010 was used to conduct a large population-based cohort analysis. Demographic and clinical characteristics as well as prognostic data were systematically analyzed. We further compared overall survival (OS) in the two cohorts. Risk factors of secondary primary lung cancer in cervical cancer patients were also analyzed. Results557 (3.52%) had developed second primary lung cancer after cervix cancer and 451 were eligible for inclusion in the final analyses. In comparison to NSCLC1, patients with CC-NSCLC had a higher rate of squamous cell carcinoma (SCC) (36.59% vs. 19.07%, p<0.01). Median OS was longer for CC-NSCLC than for NSCLC1 before propensity score matching (PSM) (16 vs. 13months) but there was no significant difference after PSM. High-risk factors in cervical cancer to developing CC-NSCLC include: 50-79years old, black race (OR 1.417; 95%CI 1.095-1.834; p<0.05)and history of radiotherapy (OR 1.392; 95%CI 1.053-1.841; p<0.05). Conclusion50-79years old, black race and history of radiotherapy were independent risk factors of second primary lung cancer in cervical cancer patient. CC-NSCLC patients had distinctive clinical characteristics and a better prognosis compared with NSCLC1 patients.

cancer biology↗
bioRxiv2020

Evolution of cellular diversity in primary motor cortex of human, marmoset monkey, and mouse

The primary motor cortex (M1) is essential for voluntary fine motor control and is functionally conserved across mammals. Using high-throughput transcriptomic and epigenomic profiling of over 450,000 single nuclei in human, marmoset monkey, and mouse, we demonstrate a broadly conserved cellular makeup of this region, whose similarity mirrors evolutionary distance and is consistent between the transcriptome and epigenome. The core conserved molecular identity of neuronal and non-neuronal types allowed the generation of a cross-species consensus cell type classification and inference of conserved cell type properties across species. Despite overall conservation, many species specializations were apparent, including differences in cell type proportions, gene expression, DNA methylation, and chromatin state. Few cell type marker genes were conserved across species, providing a short list of candidate genes and regulatory mechanisms responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allowed the Patch-seq identification of layer 5 (L5) corticospinal Betz cells in non-human primate and human and characterization of their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell type diversity in M1 across mammals and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

neuroscience↗
bioRxiv2020

Structure of the ForT/PRPP complex uncovers the mechanism of C-C bond formation in C-nucleotide antibiotic biosynthesis

C-C bond formation is at the heart of anabolism and organic chemistry, but relatively few enzymatic strategies for catalyzing this reaction are known. The enzyme ForT catalyzes C-C bond formation between 5-phosphoribosyl-1-pyrophosphate (PRPP) and 4-amino-1H-pyrazole-3,5-dicarboxylate to make a key intermediate in the biosynthesis of the C-nucleotide formycin A 5-phosphate; we now report the 2.5 [A] resolution structure of the ForT/PRPP complex and thus locate the active site. Site-directed mutagenesis has identified those residues critical for PRPP recognition and catalysis. Structural conservation with GHMP kinases suggests that stabilization of the negatively charged pyrophosphate leaving group is crucial for catalysis in ForT. A mechanism for this new class of C-C bond forming enzymes is proposed. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/009662v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1817d54org.highwire.dtl.DTLVardef@4b3558org.highwire.dtl.DTLVardef@19b248eorg.highwire.dtl.DTLVardef@7d0c62_HPS_FORMAT_FIGEXP M_FIG C_FIG A new class of enzymes catalyse C-C bond formation by irreversible CO2 and pyrophosphate production.

biochemistry↗
bioRxiv2020

The Forensic Landscape and the Population Genetic Analyses of Hainan Li Based on Massively Parallel Sequencing DNA Profiling

Due to the formation of the Qiongzhou Strait by climate change and marine transition, Hainan island isolated from the mainland southern China during the Last Glacial Maximum. Hainan island, located at the southernmost part of China and separated from the Leizhou Peninsula by the Qiongzhou Strait, laid on one of the modern human northward migration routes from Southeast Asia to East Asia. The Hlai-language speaking Li minority, the second largest population after Han Chinese in Hainan island, is the direct descendants of the initial migrants in Hainan island and has unique ethnic properties and derived characteristics, however, the forensic associated studies on Hainan Li population are still insufficient. Hence, 136 Hainan Li individuals were genotyped in this study using the MPS-based ForenSeq DNA Signature Prep Kit (DNA Primer Set A) to characterize the forensic genetic polymorphism landscape, and DNA profiles were obtained from 152 different molecular genetic markers (27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 iiSNPs). A total of 419 distinct length variants and 586 repeat sequence sub-variants, with 31 novel alleles (at 17 loci), were identified across the 58 STR loci from the DNA profiles of Hainan Li population. We evaluated the forensic characteristics and efficiencies of DAPA, it demonstrated that the STRs and iiSNPs in DAPA were highly polymorphic in Hainan Li population and could be employed in forensic applications. In addition, we set up three Datasets, which included the genetic data of (I). iiSNPs (27 populations, 2640 individuals), (II). Y-STRs (42 populations, 8281 individuals), and (III). Y-haplogroups (123 populations, 4837 individuals) along with the population ancestries and language families, to perform population genetic analyses separately from different perspectives. In conclusion, the phylogenetic analyses indicated that Hainan Li, with a southern East Asia origin and Tai-Kadai language-speaking language, is an isolated population relatively. But the genetic pool of Hainan Li influenced by the limited gene flows from other Tai-Kadai populations and Hainan populations. Furthermore, the establishment of isolated population models will be beneficial to clarify the exquisite population structures and develop specific genetic markers for subpopulations in forensic genetic fields.

genetics↗
bioRxiv2020

Structure-Based Design, Synthesis and Biological Evaluation of Peptidomimetic Aldehydes as a Novel Series of Antiviral Drug Candidates Targeting the SARS-CoV-2 Main Protease

SARS-CoV-2 is the etiological agent responsible for the COVID-19 outbreak in Wuhan. Specific antiviral drug are urgently needed to treat COVID-19 infections. The main protease (Mpro) of SARS-CoV-2 is a key CoV enzyme that plays a pivotal role in mediating viral replication and transcription, which makes it an attractive drug target. In an effort to rapidly discover lead compounds targeting Mpro, two compounds (11a and 11b) were designed and synthesized, both of which exhibited excellent inhibitory activity with an IC50 value of 0.05 M and 0.04 M respectively. Significantly, both compounds exhibited potent anti-SARS-CoV-2 infection activity in a cell-based assay with an EC50 value of 0.42 M and 0.33 M, respectively. The X-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a and 11b were determined at 1.5 [A] resolution, respectively. The crystal structures showed that 11a and 11b are covalent inhibitors, the aldehyde groups of which are bound covalently to Cys145 of Mpro. Both compounds showed good PK properties in vivo, and 11a also exhibited low toxicity which is promising drug leads with clinical potential that merits further studies.

biochemistry↗
bioRxiv2020

Computational simulations reveal the binding dynamics between human ACE2 and the receptor binding domain of SARS-CoV-2 spike protein

A novel coronavirus (the SARS-CoV-2) has been identified in January 2020 as the causal pathogen for COVID-19 pneumonia, an outbreak started near the end of 2019 in Wuhan, China. The SARS-CoV-2 was found to be closely related to the SARS-CoV, based on the genomic analysis. The Angiotensin converting enzyme 2 protein (ACE2) utilized by the SARS-CoV as a receptor was found to facilitate the infection of SARS-CoV-2 as well, initiated by the binding of the spike protein to the human ACE2. Using homology modeling and molecular dynamics (MD) simulation methods, we report here the detailed structure of the ACE2 in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The predicted model is highly consistent with the experimentally determined complex structures. Plausible binding modes between human ACE2 and the RBD were revealed from all-atom MD simulations. The simulation data further revealed critical residues at the complex interface and provided more details about the interactions between the SARS-CoV-2 RBD and human ACE2. Two mutants mimicking rat ACE2 were modeled to study the mutation effects on RBD binding to ACE2. The simulations showed that the N-terminal helix and the K353 of the human ACE2 alter the binding modes of the CoV2-RBD to the ACE2.

biophysics↗
bioRxiv2020

Glycyrrhizic acid improves cognitive levels of aging mice by regulating T/B cell proliferation

Dear Editor,Licorice is the root of Glycyrrhiza glabra L. (Leguminosae), which grows in various warm climates such as the Middle East, Asia, and Southern Europe. It is one of the oldest known medicinal herbs and is referred to as "the father of herbal medicine." Glycyrrhizic acid (GA) (Fig. 1A), a triterpenoid saponin, is a major component of licorice. It has a variety of pharmacological activities such as anti-inflammatory, antioxidant, anticancer, neuroprotective, and immune effects, among others.[1]. Previous studies indicated that GA produces robust neuroprotection via the modulation of anti-apoptotic and pro-apoptotic factors, primarily through the ERK signaling pathway and its anti-inflammatory properties against high-mobility group box 1 phosphorylation and the suppression of inflammatory cytokine induction [2-4]. These results were based on pathological models. Although numerous pathways have been implicated in the neuroprotective effects of GA, the molecular mechanisms are not yet completely understood. In this study, we aimed to investigate the effects of GA in preventing age-related immune involution and cognitive disorders, the relationship between immune involution and cognition, and the underlying molecular mechanisms. O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/008821v1_fig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@5a123dorg.highwire.dtl.DTLVardef@163a085org.highwire.dtl.DTLVardef@1bfadb6org.highwire.dtl.DTLVardef@125b2ae_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Glycyrrhizic acid ameliorated cognitive declines in aged mice, improved CBF and increased T and B cell numbers. A. The chemical structure of glycyrrhizic acid (GA). B. Escape latencies from six consecutive daily tests. C. The first platform-finding latencies in the probe trial. D. The time spent in the target quadrant in the probe trial. E. The target platform crossing times in the probe trial. F. Images of CBF in C57BL/6 mice. G. Quantification of blood perfusion in the ipsilateral hemisphere. H. Bubble diagram showing GA treatment influence on hematopoietic cell lineage. I. Heatmap showing actively expressed genes related to T cells in the hematopoietic cell lineage of control and GA-treated C57BL/6 mice. J. Representative FACS plots showing CD3e+ T and CD45R/B220+ B cells in the spleens of C57BL mice; bar graphs showing the statistical analysis of T and B cells in the spleens of C57BL mice. K. Representative FACS plots showing CD3e+ T and CD45R/B220+ B cells in the blood of C57BL mice; Bar graphs showing the statistical results of T and B cell analyses in the blood of C57BL mice. GA (5mg/kg, once every other day); Data represent mean {+/-} SEM (n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctrl. C_FIG

animal behavior and cognition↗
bioRxiv2020

Metformin improves cognition of aged mice by promoting cerebral angiogenesis and neurogenesis

The cerebral microvasculature is essential for preservation of normal cerebral function. Age-related decreases of neurogenesis and cognitive function are accompanied by reduced blood flow and a decline in neural stem cell (NSC) number. Here, we report that metformin administered by tail vein injection enhanced cognition in aged but not young mice in a dose-dependent manner. Further, metformin restored cerebral blood flow and brain vascular density and promoted neurogenic potential of the subependymal zone/subventricular zone both in vivo and in vitro. RNA-Seq result indicated that metformin could enhance glycolysis in blood, with an increase in relative mRNA expression of the enzyme in the glycolysis pathway from hippocampal tissue of metformin-treated mice. Mechanistically, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the glycolysis pathway, may contribute to angiogenic and neurogenic potentials of NSCs. Interestingly, examination of peripheral blood mononuclear cells from people of various ages showed that mRNA expression of GAPDH gradually decreased with age, while its expression level positively correlated with cognitive levels. Our results indicate that metformin represents a candidate pharmacological approach for recruitment of NSCs in aged mouse brain by enhancing glycolysis and promoting neurovascular generation, a strategy that might be of therapeutic value for anti-aging in humans. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/006767v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1f8fc8aorg.highwire.dtl.DTLVardef@18be6c8org.highwire.dtl.DTLVardef@1dc815forg.highwire.dtl.DTLVardef@1b19557_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗
bioRxiv2020

FEP-based screening prompts drug repositioning against COVID-19

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global crisis. There is no therapeutic treatment specific for COVID-19. It is highly desirable to identify potential antiviral agents against SARS-CoV-2 from existing drugs available for other diseases and, thus, repurpose them for treatment of COVID-19. In general, a drug repurposing effort for treatment of a new disease, such as COVID-19, usually starts from a virtual screening of existing drugs, followed by experimental validation, but the actual hit rate is generally rather low with traditional computational methods. Here we report a new virtual screening approach with accelerated free energy perturbation-based absolute binding free energy (FEP-ABFE) predictions and its use in identifying drugs targeting SARS-CoV-2 main protease (Mpro). The accurate FEP-ABFE predictions were based on the use of a new restraint energy distribution (RED) function designed to accelerate the FEP-ABFE calculations and make the practical FEP-ABFE-based virtual screening of the existing drug library possible for the first time. As a result, out of twenty-five drugs predicted, fifteen were confirmed as potent inhibitors of SARS-CoV-2 Mpro. The most potent one is dipyridamole (Ki=0.04 M) which has showed promising therapeutic effects in subsequently conducted clinical studies for treatment of patients with COVID-19. Additionally, hydroxychloroquine (Ki=0.36 M) and chloroquine (Ki=0.56 M) were also found to potently inhibit SARS-CoV-2 Mpro for the first time. We anticipate that the FEP-ABFE prediction-based virtual screening approach will be useful in many other drug repurposing or discovery efforts. Significance StatementDrug repurposing effort for treatment of a new disease, such as COVID-19, usually starts from a virtual screening of existing drugs, followed by experimental validation, but the actual hit rate is generally rather low with traditional computational methods. It has been demonstrated that a new virtual screening approach with accelerated free energy perturbation-based absolute binding free energy (FEP-ABFE) predictions can reach an unprecedently high hit rate, leading to successful identification of 16 potent inhibitors of SARS-CoV-2 main protease (Mpro) from computationally selected 25 drugs under a threshold of Ki = 4 M. The outcomes of this study are valuable for not only drug repurposing to treat COVID-19, but also demonstrating the promising potential of the FEP-ABFE prediction-based virtual screening approach.

biophysics↗
bioRxiv2020

Siderophores provoke extracellular superoxide production by carbon-starving Arthrobacter strains when carbon sources recover

Superoxide and other reactive oxygen species (ROS) in the environment shape microbial communities1 and drive transformation of metals2,3 and inorganic/organic matter4,5. Taxonomically diverse bacteria and phytoplankton can produce extracellular superoxide during laboratory cultivation6-11. Understanding the physiological reasons for extracellular superoxide production by aerobes in the environment is a crucial question yet not fully solved. Here, we showed that iron-starving Arthrobacter sp. QXT-31 (referred to as A. QXT-31 hereafter) secreted a type of siderophore (deferoxamine, DFO), which provoked extracellular superoxide production by carbon-starving A. QXT-31 when carbon sources were recovered. Several other siderophores also demonstrated similar effects. RNA-Seq data hinted that DFO stripped iron from iron-bearing proteins in the electron transfer chain (ETC) of metabolically active A. QXT-31, resulting in electron leakage from the electron-rich (resulting from carbons source metabolism) ETC and superoxide production. Considering that most aerobes secrete siderophore(s)12 and often suffer from carbon starvation in the environment, certain aerobes are expected to produce extracellular superoxide when carbon source(s) recover/fluctuate, thus influencing the microbial community and cycling of many elements. In addition, an artificial iron-chelator (diethylenetriamine pentaacetic acid, DTPA) was widely used in microbial superoxide quantification. Our results showed that DTPA provoked superoxide production by A. QXT-31 and highlighted its potential interference in microbial superoxide quantification.

microbiology↗
bioRxiv2020

ZmCLA4 regulates leaf angle through multiple plant hormone-mediated signal pathways in maize

Leaf angle in cereals is an important agronomic trait contributing to plant architecture and grain yield by determining the plant compactness. Although ZmCLA4 was identified to shape plant architecture by affecting leaf angle, the detailed regulatory mechanism of ZmCLA4 in maize remains unclear. ZmCLA4 was identified as a transcriptional repressor using the Gal4-LexA/UAS system and transactivation analysis in yeast. The DNA affinity purification (DAP)-seq assay showed that ZmCLA4 not only acts as a repressor containing the EAR motif (CACCGGAC), but was also found to have two new motifs, CCGARGS and CDTCNTC. On analyzing the ZmCLA4-bound targeted genes, we found that ZmCLA4, as a cross node of multiple plant hormone-mediated pathways, directly bound to ARF22 and IAA26 to regulate auxin transport and mediated brassinosteroid signaling by directly binding to BZR3 and 14-3-3. ZmCLA4 bound two WRKY genes involved with abscisic acid, two genes (CYP75B1, CYP93D1) involved with jasmonic acid, B3 involved in the response to ethylene, and thereby negatively regulated leaf angle formation. We built a new regulatory network for the ZmCLA4 gene controlling leaf angle in maize, which contributed to the understanding of ZmCLA4s regulatory mechanism and will improve grain yields by facilitating optimization of plant architecture.

molecular biology↗
bioRxiv2020

Blunted Diurnal Firing in Lateral Habenula Projections to Dorsal Raphe Nucleus and Delayed Photoentrainment in Stress-Susceptible Mice.

Daily rhythms are disrupted in patients suffering from mood disorders. The lateral habenula (LHb) and dorsal raphe nucleus (DRN) contribute to circadian timekeeping and regulate mood. Thus, pathophysiology in these nuclei may be responsible for aberrations in daily rhythms during mood disorders. Using the chronic social defeat stress (CSDS) paradigm and in-vitro slice electrophysiology we measured the effects of stress on diurnal rhythms in firing of LHb cells projecting to the DRN (cellsLHb[->]DRN) and DRN cells alone. We also performed optogenetic experiments to investigate if increased firing in cellsLHb[->]DRN during exposure to subthreshold social defeat stress (SSDS), induces stress-susceptibility. Last we investigated whether exposure to CSDS affected the ability of mice to phototentrain to a new LD cycle. The cellsLHb[->]DRN and DRN cells alone of stress-susceptible mice express greater blunted diurnal firing compared to stress-naive (control) and stress-resilient mice. Day-time optogenetic activation of cellsLHb[->]DRN during SSDS induces stress-susceptibility which shows the direct correlation between increased activity in this circuit and putative mood disorders. Finally, we found that stress-susceptible mice are slower, while stress-resilient mice are faster, at photoentraining to a new LD cycle. Our findings suggest that CSDS induces blunted daily rhythms in firing in cellsLHb[->]DRN and slow rate of photoentrainment in susceptible-mice. In contrast, resilientmice may undergo homeostatic adaptations that maintain daily rhythms in firing in cellsLHb[->]DRN and also show rapid photoentrainment to a new LD-cycle.

neuroscience↗
bioRxiv2020

An Experimental Validation of the Computer Fluid Dynamics of Normal Human Nasal Airflow Using a Precise 3-D Printing Model

Computer Fluid Dynamics (CFD) is a popular method for studying airflow of nasal cavities. However, the data of CFD studies has rarely been validated through experiments. To test the accuracy of CFD computation, we studied the consistency of the air pressure of nasal cavities in the CFD and the experiment. A proportional resin model of a normal human subjects nasal cavities was created by a 3-d printer with a precision value of 0.1mm. The pressure of 63 check points in the nasal cavities in different breathing states was measured. The experimental data was compared with the data obtained by CFD simulation. At the flow rates of 180 ml s-1 and 560 ml s-1, the pressure in all check points remained highly consistent with the CFD data. At 1100 ml s-1 flow rate, there was a significant deviation in the posterior segment of the nasal cavity during exhalation. The method used in this study to measure the pressure in the nasal cavities can be used in experimental validation of CFD data. The computational methods and the boundary conditions used in this study resulted in a high agreement between the results of the CFD simulation and the experiment. Author SummaryIn the contemporary era, Computer fluid dynamics (CFD) is the mainstream method for studying air flow. Due to the complex anatomical structure of the nasal cavity, the CFD results of the nasal flow have rarely been experimentally verified. This study provides a method to verify the methods and results of nasal CFD. We printed an accurate model of a normal persons nasal cavity with a high-precision 3D printer. In this nasal cavity model, we set 63 small holes to detect the air pressure of the places we concerned. Three different nasal flow quantity are used to represent different breathing conditions: high (1100 ml s-1), medium (560 ml s-1), and low (180 ml s-1). In medium and low nasal flow quantities, our CFD results are in good agreement with the experimental pressure values. On this basis, we analyzed the characteristics of nasal airflow in normal people. The method used in this study to measure the pressure in the nasal cavities can be used in experimental measurements of the partial resistance of the nasal cavity. With proper modification, it can be applied to the clinical practice for nasal resistance, giving more help for the design of the operation plan.

biophysics↗
bioRxiv2020

Adaptive evolution of DNA methylation reshaped gene regulation in maize

DNA methylation is a ubiquitous chromatin feature -- in maize, more than 25% of cytosines in the genome are methylated. Recently, major progress has been made in describing the molecular mechanisms driving methylation, yet variation and evolution of the methylation landscape during maize domestication remain largely unknown. Here we leveraged whole-genome sequencing (WGS) and whole genome bisulfite sequencing (WGBS) on populations of modern maize, landrace, and teosinte (Zea mays ssp. parviglumis) to investigate the adaptive and phenotypic consequences of methylation variations in maize. By using a novel estimation approach, we inferred the methylome site frequency spectrum (mSFS) to estimate forward and backward epimutation rates and selection coefficients. We found weak evidence for direct selection on DNA methylation in any context, but thousands of differentially methylated regions (DMRs) were identified in population-wide that are correlated with recent selection. Further investigation revealed that DMRs are enriched in 5 untranslated regions, and that maize hypomethylated DMRs likely helped rewire distal gene regulation. For two trait-associated DMRs, vgt1-DMR and tb1-DMR, our HiChIP data indicated that the interactive loops between DMRs and respective downstream genes were present in B73, a modern maize line, but absent in teosinte. Functional analyses suggested that these DMRs likely served as cis-acting elements that modulated gene regulation after domestication. Our results enable a better understanding of the evolutionary forces acting on patterns of DNA methylation and suggest a role of methylation variation in adaptive evolution.

plant biology↗
bioRxiv2020

MGMT genomic rearrangements contribute to chemotherapy resistance in gliomas

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.

cancer biology↗
bioRxiv2020

Systematic genome-wide querying of coding and non-coding functional elements in E. coli using CRISPRi

Genome-wide repression screens using CRISPR interference (CRISPRi) have enabled the high-throughput identification of essential genes in bacteria. However, there is a lack of functional studies leveraging CRISPRi to systematically explore targeting of both the coding and non-coding genome in bacteria. Here we perform CRISPRi screens in Escherichia coli MG1655 K-12 targeting ~13,000 genomic features, including nearly all protein-coding genes, non-coding RNAs, promoters, and transcription factor binding sites (TFBSs) using a ~33,000-member sgRNA library, which represents the most compact and comprehensive genome-wide CRISPRi library in E. coli to date. Our data reveal insights into the conditional essentiality of the genome with key refinements to screen design and profiling. First, we demonstrate that strong fitness defects associated with essential cellular processes can be resolved using inducible time-series measurements. We show that knockdowns of different classes of genes exhibit distinct, transient responses that are correlated to gene function with genes involved in translation exhibiting the strongest responses. We also query feature essentiality across several biochemical conditions and show that several genes, sRNAs, and operons exhibit conditional phenotypes not reported by previous high-throughput efforts. Second, we evaluate systematically targeting non-genic features (promoters and TFBSs) in the E. coli genome. We show that promoter-targeting guides can be used to add phenotypic confidence to promoter annotations and verify computationally predicted promoters. In contrast to prior studies, we find that promoter knockdowns exhibit a strong targeting orientation dependency where targeting the non-template strand of the promoter closest to the target gene is more effective in knocking down gene expression than other promoter targeting orientations. Unlike eukaryotic genomes, we note that interpreting the effects of TFBS targeting is particularly challenging due to the small size of such features and their proximity to and overlap with other genomic features. Together, this work reveals novel conditionally essential gene phenotypes, provides a characterized set of sgRNAs for future E. coli CRISPRi screens, and highlights considerations for CRISPRi library design and screening for microbial genome characterization.

microbiology↗