Most low GC Gram-positive bacteria possess an essential walKR two-component system (TCS) for signal transduction involved in regulating cell wall homoeostasis. Despite the well-established ...intracellular regulatory mechanism, the role of this TCS in extracellular signal recognition and factors that modulate the activity of this TCS remain largely unknown. Here we identify the extracellular receptor of the kinase 'WalK' (erWalK) as a key hub for bridging extracellular signal input and intracellular kinase activity modulation in Staphylococcus aureus. Characterization of the crystal structure of erWalK revealed a canonical Per-Arnt-Sim (PAS) domain for signal sensing. Single amino-acid mutation of potential signal-transduction residues resulted in severely impaired function of WalKR. A small molecule derived from structure-based virtual screening against erWalK is capable of selectively activating the walKR TCS. The molecular level characterization of erWalK will not only facilitate exploration of natural signal(s) but also provide a template for rational design of erWalK inhibitors.
Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, here, we ...introduce m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. We mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. We observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.m6A-SAC-seq uses chemical labeling to quantify m6A at single-base resolution in the mammalian transcriptome.
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications ...are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa.
Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9-3.7 ppm) 6mA modifications above background.
Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon–carbon bond formation reactions between organic compounds. ...However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein–small molecule conjugation. The identified simple compound–Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Members of the mammalian AlkB family are known to mediate nucleic acid demethylation
. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism
, but its function and ...mechanism of action are unknown. Here we report an approach to site-specifically detect N
-methyladenosine (m
A), N
-methylcytidine (m
C), N
-methylguanosine (m
G) and N
,N
-dimethylguanosine (m
G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m
G and m
A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA
. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.
Full text
Available for:
GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
N6-Methyladenine (m
dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m
dA events are still lacking. Single-molecule ...real-time (SMRT) sequencing has enabled the detection of m
dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m
dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m
dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m
dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m
dA at single-nucleotide and single-molecule resolution. For human lymphoblastoid cells (hLCLs), it was necessary to integrate SMRT sequencing data with independent sequencing data. The joint analyses suggest putative m
dA events are enriched in the promoters of young full-length LINE-1 elements (L1s), but call for validation by additional methods. These analyses demonstrate a general method for rigorous mapping and characterization of m
dA events in eukaryotic genomes.
Abstract
Six kinds of dithiocarbamates (DTCs) were synthesized from three linear amines with different amino numbers, two polyether amines with different molecular weights, and one branched amine ...with benzene rings, respectively. The conditions affecting oil removal rate and floc rising time of DTC were studied using simulated oily wastewater. Furthermore, the effects of the molecular structure of DTC on oil removal efficiency, floc morphology, floc rising time, and floc adhesion were investigated. When the conditions were optimal, the oil removal efficiency of DTC synthesized from polyethylene polyamine was 95.14%, which was higher than other DTCs. Meanwhile, the ferrous ion was the most suitable chelating metal ion for DTC than other transition metal ions. The increase of amino groups in the initiators improves the oil removal efficiency of DTC, while the linear structural DTC exhibits a low oil removal efficiency due to a lack of network structural flocs. The introduction of polyether structure helps reduce the volume of the flocs and make them compact, but it also increases the adhesion of the floc on the metal surface. The introduction of bisphenol A phenol amino resin structure induces the generation of the flocs in oil wastewater and improves the oil removal efficiency.
RNA-based therapeutics have emerged as a promising approach for the treatment of various diseases, including cancer, genetic disorders, and infectious diseases. However, the delivery of RNA molecules ...into target cells has been a major challenge due to their susceptibility to degradation and inefficient cellular uptake. To overcome these hurdles, DNA-based nano technology offers an unprecedented opportunity as a potential delivery platform for RNA therapeutics. Due to its excellent characteristics such as programmability and biocompatibility, these DNA-based nanostructures, composed of DNA molecules assembled into precise and programmable structures, have garnered significant attention as ideal building materials for protecting and delivering RNA payloads to the desired cellular destinations. In this review, we highlight the current progress in the design and application of three DNA-based nanostructures: DNA origami, lipid-nanoparticle (LNP) technology related to frame guided assembly (FGA), and DNA hydrogel for the delivery of RNA molecules. Their biomedical applications are briefly discussed and the challenges and future perspectives in this field are also highlighted.
This study provides a reference for estimating the building envelope thermal performance at the initial stage of design for nearly zero-energy buildings in different climate zones. A simplified model ...of heat load prediction, which combines the quasi-steady-state thermal balance calculation procedure in ISO 52016 and the variable-base degree-days method, was proposed. Therefore, a building energy performance evaluation tool BPT V1.0 was developed. Subsequently, the simplified model was validated through comparative analysis with the Building Energy Simulation Test (BESTEST) standard procedure. To conduct a feasibility analysis of the development tool, case studies were performed on the performance evaluation of building envelopes of residential and office buildings in different climate zones in China. Compared to the simulation results from EnergyPlus, the deviation of heat load calculated by BPT V1.0 was within 10%, which further verifies the applicability of the tool under different climatic conditions. Annual heat load under different thermal performance building envelopes was calculated through BPT V1.0. The building energy efficiency improvement rates were found to range from 30 to 60% in nearly zero-energy buildings in different climate zones in China. The study results can provide a reference for energy managers and a basis for estimating the building energy efficiency performance with different envelope thermal properties in the region.
DNA methylation plays a pivotal role in various biological processes and is highly related to multiple diseases. The exact functions of DNA methylation are still puzzling due to its uneven ...distribution, dynamic conversion, and complex interactions with other substances. Current methods such as chemical- and enzyme-based sequencing techniques have enabled us to pinpoint DNA methylation at single-base resolution, which necessitated the manipulation of DNA methylation at comparable resolution to precisely illustrate the correlations and causal relationships between the functions of DNA methylation and its spatiotemporal patterns. Here a perspective on the past, recent process, and future of precise DNA methylation tools is provided. Specifically, genome-wide and site-specific manipulation of DNA methylation methods is discussed, with an emphasis on their principles, limitations, applications, and future developmental directions.
PDF
