Direct identification of pathogens via microbial cellular DNA in whole blood by MeltArray.

Rapid identification of pathogens is critical for early and appropriate treatment of bloodstream infections. The various culture-independent assays that have been developed often have long turnaround times, low sensitivity and narrow pathogen coverage. Here, we propose a new multiplex PCR assay, MeltArray, which uses intact microbial cells as the source of genomic DNA (gDNA). The successive steps of the MeltArray assay, including selective lysis of human cells, microbial cell sedimentation, microbial cellular DNA extraction, target-specific pre-amplification and multiplex PCR detection, allowed the detection of 35 major bloodstream infectious pathogens in whole blood within 5.5 h. The limits of detection varied depending on the pathogen and ranged from 1 to 5 CFU/mL. Of 443 blood culture samples, including 373 positive blood culture samples and 70 negative blood culture samples, the MeltArray assay showed a sensitivity of 93.8% (350/373, 95% confidence interval [CI] = 90.7%-96.0%), specificity of 98.6% (69/70, 95% CI = 91.2%-99.9%), positive predictive value of 99.7% (95% CI = 98.1%-99.9%), and negative predictive value of 75.0% (95% CI = 64.7%-83.2%). The MeltArray detection results of 16 samples differed from MALDI-TOF and were confirmed by Sanger sequencing. Further testing of 110 whole blood samples from patients with suspected bloodstream infections using blood culture results revealed that the MeltArray assay had a clinical sensitivity of 100% (9/9, 95% CI = 62.8%-100.0%), clinical specificity of 74.5% (70/94, 95% CI = 64.2%-82.7%), positive predictive value of 27.3% (95% CI = 13.9%-45.8%), and negative predictive value of 100.0% (95% CI = 93.5%-100.0%). Compared with metagenomic next-generation sequencing, the MeltArray assay displayed a positive agreement of 85.7% (6/7, 95% CI = 42.0%-99.2%) and negative agreement of 100.0% (4/4, 95% CI = 39.6%-100.0%). We conclude that the MeltArray assay can be used as a rapid and reliable tool for direct identification of pathogens in bloodstream infections.

Study on persulfate activated by Ce-modified tea waste biochar to degrade tetracycline.

In this study, the Ce-modified tea residue biochar (Ce-TBC) was successfully generated and applied to the biochar/persulfate system (Ce-TBC/PDS), the mechanism of the removal of tetracycline (TC) using Ce-TBC/PDS was elaborated. Under the optimal experimental conditions (Ce-TBC = 0.8 g L-1, PDS = 4 mM, TC = 10 mg L-1), the removal efficiency of TC was 91.28%, and after 5 cycles, the elimination rate of Ce-TBC/PDS still reached up to 80%. The mechanism of TC removal by Ce-TBC/PDS was analyzed by scanning electron microscopy (SEM), X-ray diffractometer (XRD), Fourier infrared transform spectrometer (FT-IR), and X-ray photoelectron spectrometer (XPS) characterization, and influence factor experiments. The results showed that the introduction of CeOx increased the oxygen vacancies on the TBC surface and promoted the interconversion between Ce3+ and Ce4+ for better activation of PDS and generation of active species. Free radical quenching experiments and paramagnetic resonance spectrometry (EPR) analysis showed that the non-radical pathway 1O2 played a dominant role in the Ce-TBC/PDS system. The present work provided an efficient means of PDS activator and recycling of tea waste.

Insight into atrazine removal by fallen leaf biochar prepared at different pyrolysis temperatures: Batch experiments, column adsorption and DFT calculations.

The environmental pollution caused by atrazine in the agricultural production cannot be ignored. In this study, the fallen leaf biochar (LBC) was prepared at three different temperatures (500 °C, 600 °C, and 700 °C) using a simple pyrolysis method (500 LBC, 600 LBC, and 700 LBC) for atrazine adsorption. Batch experiments showed that the performance of LBC in atrazine adsorption improved with rising pyrolysis temperature, and the highest adsorption amount of 700 LBC reached 84.32 mg g-1. Kinetic and isotherm models showed that the adsorption behaviors were both monolayer and multilayer chemisorption. The findings of the characterizations (Elemental analysis, BET, XRD, Raman, FT-IR, and XPS) confirmed that the degree of aromatization determined the adsorption capacity of LBC to atrazine, and π-π electron donor-acceptor interaction was the main adsorption mechanism. Density functional theory (DFT) calculations showed that the highly aromatized biochar was more effective for atrazine adsorption, manifested as smaller molecular distances, higher adsorption energies, more stable complex structures, and stronger π-electron conjugation. In the column adsorption experiments, reducing the inlet flow rate or increasing the bed height extended the breakthrough time and exhaustion time of the breakthrough curves, and 700 LBC still showed good adsorption performance after five cycles. Overall, fallen leaf biochar as a reuse product of resource showed good potential for application in atrazine adsorption, which can be used for atrazine-contaminated water remediation.

Study on visible light photocatalytic performance of BiVO4 modified by graphene analogue boron nitride.

In this study, a variety of boron nitride (BN) modified BiVO4 (BN-BiVO4) composites with visible-light response were prepared and used to degrade tetracyclines (TCs), including tetracycline (TC) and oxytetracycline (OTC). When treating the TCs solution under visible light irradiation, 4BN-BiVO4 displayed high photocatalytic performance (87.1% for TC and 86.2% for OTC), which were 3.6 and 2.3 times than that of BiVO4, respectively. Photoluminescence spectroscopy (PL) and transient photocurrent proved that the combination of BN and BiVO4 effectively promotes the efficient separation of photogenerated electrons and holes in the material, resulting in enhanced photocatalytic activity. Further, radical trapping experiments in combination with electron spin resonance (ESR) revealed that ·OH radicals and holes were the predominant reactive species. Ultimately, the possible photocatalytic mechanism for TCs degradation was proposed on the basis of the experiments and characterization analysis. This study offers a new promising approach for the design of photocatalysts with visible-light response for efficient TCs elimination.

Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling.

Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl-CoA (Ac-CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry-based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac-CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl-CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub-acetylomes of many other HAT enzymes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling HAT protein substrates with azide/alkyne-biotin Alternate Protocol: Labeling protein substrates of HATs with azide/alkyne-TAMRA for in-gel visualization Support Protocol 1: Expression and purification of HAT mutants Support Protocol 2: Synthesis of Ac-CoA surrogates Basic Protocol 2: Streptavidin enrichment of biotinylated HAT substrates Basic Protocol 3: Chemoproteomic identification of HAT substrates Basic Protocol 4: Validation of specific HAT substrates with western blotting.

Chemoproteomic Profiling of Protein Substrates of a Major Lysine Acetyltransferase in the Native Cellular Context.

The family of lysine acetyltransferases (KATs) regulates epigenetics and signaling pathways in eukaryotic cells. So far, knowledge of different KAT members contributing to the cellular acetylome is limited, which limits our understanding of biological functions of KATs in physiology and disease. Here, we found that a clickable acyl-CoA reporter, 3-azidopropanoyl CoA (3AZ-CoA), presented remarkable cell permeability and effectively acylated proteins in cells. We rationally engineered the major KAT member, histone acetyltransferase 1 (HAT1), to generate its mutant forms that displayed excellent bio-orthogonal activity for 3AZ-CoA in substrate labeling. We were able to apply the bio-orthogonal enzyme-cofactor pair combined with SILAC proteomics to achieve HAT1 substrate targeting, enrichment, and proteomic profiling in living cells. A total of 123 protein substrates of HAT1 were disclosed, underlining the multifactorial functions of this important enzyme than hitherto known. This study demonstrates the first example of utilizing bio-orthogonal reporters as a chemoproteomic strategy for substrate mapping of individual KAT isoforms in the native biological contexts.

Identification of lysine isobutyrylation as a new histone modification mark.

Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.

Comparison of transcriptional change of B. melitensis M5-90 after macrophage infection highlights the role of ribosome gene L31 in virulence.

Brucella, a facultative intracellular bacterium, can survive and replicate in various cell types such as epithelial cell, fibroblasts and macrophage. Macrophage is the most important sites for the survival of Brucella in vivo. The mechanisms of pathogenesis are difficult to address, since the unknown virulence genes are still exist. RNA-seq is available to study transcriptional changes that occur during disease as a way to identify important virulence-related genes. Here we described and analyzed the transcriptional change of avirulent strain Brucella melitensis M5-90 (B. melitensis M5-90) during macrophage infection using RNA-seq technology. We detected 601 significant changed genes of which 428 were upregulated after infection. The upregulated gene L31 which involved in ribosome KEGG pathway was selected to illustrate its effect on virulence in a vaccine strain B. melitensis M5-90 and a virulent strain B. melitensis M28. Deletion of L31 significant attenuates the spleen colonization in model of M5-90 or M28 infection mouse at 7, 21 and 35 days post-infection (P < 0.05). We further examine the role of L31 in a macrophage cell infection model, and the result showed a significant reduction of intracellular M28ΔL31 cells at 48 h post-infection (P < 0.001). In total, our study provided a view of transcriptional landscape of B. melitensis M5-90 intracellular, and found L31 gene is required for the full virulence of B. melitensis.

Improving FnCas12a Genome Editing by Exonuclease Fusion.

Among current reported Cas12a orthologs, Francisella novicida Cas12a (FnCas12a) is less restricted by protospacer adjacent motif (PAM). However, the activity of FnCas12a nuclease is relatively low or undetectable in human cells, limiting its application as desirable genome engineering tools. Here, we describe TEXT (Tethering EXonuclease T5 with FnCas12a)-a fusion strategy that significantly increased the knockout efficiency of FnCas12a in human cells at multiple genomic loci in three different cell lines. TEXT results in higher insertion and deletion efficiency than FnCas12a under different spacer lengths from 18 nt to 23 nt. Deep sequencing shows that TEXT substantially increased the deletion frequency and deletion size at the targeted locus. Compared to other Cas12a orthologs, including AsCas12a and LbCas12a, TEXT achieves the highest on-targeting efficiency and shows minimal off-targeting effects at all tested sites. TEXT enhances the activity of FnCas12a nuclease and expands its targeting scope and efficiency in human cell genome engineering.

Bioorthogonal Reporters for Detecting and Profiling Protein Acetylation and Acylation.

Protein acylation, exemplified by lysine acetylation, is a type of indispensable and widespread protein posttranslational modification in eukaryotes. Functional annotation of various lysine acetyltransferases (KATs) is critical to understanding their regulatory roles in abundant biological processes. Traditional radiometric and immunosorbent assays have found broad use in KAT study but have intrinsic limitations. Designing acyl-coenzyme A (CoA) reporter molecules bearing chemoselective chemical warhead groups as surrogates of the native cofactor acetyl-CoA for bioorthogonal labeling of KAT substrates has come into a technical innovation in recent years. This chemical biology platform equips molecular biologists with empowering tools in acyltransferase activity detection and substrate profiling. In the bioorthogonal labeling, protein substrates are first enzymatically modified with a functionalized acyl group. Subsequently, the chemical warhead on the acyl chain conjugates with either an imaging chromophore or an affinity handle or any other appropriate probes through an orthogonal chemical ligation. This bioorganic strategy reformats the chemically inert acetylation and acylation marks into a chemically maneuverable functionality and generates measurable signals without recourse to radioisotopes or antibodies. It offers ample opportunities for facile sensitive detection of KAT activity with temporal and spatial resolutions as well as allows for chemoproteomic profiling of protein acetylation pertaining to specific KATs of interest on the global scale. We reviewed here the past and current advances in bioorthogonal protein acylations and highlighted their wide-spectrum applications. We also discussed the design of other related acyl-CoA and CoA-based chemical probes and their deployment in illuminating protein acetylation and acylation biology.

Improvement and application research of the SRM in alpine regions.

The simulation of snowmelt runoff in alpine mountainous areas is of great significance not only for the risk assessment of snowmelt flood in spring and summer, but also for the development and management of water resources in the basin. An improved snowmelt runoff model (SRM) is constructed based on the analysis of change characteristics of climate, runoff, and snow and ice cover in the middle and upper reaches of the Taxkorgan River in Xinjiang Province, China. Because of the large evaporation in the study basin, the evaporation loss is added to the model. The SRM and the improved SRM are calibrated and verified by using data such as temperature, precipitation, water vapor pressure, and snow-covered area (SCA) ratio in the study basin from 2002 to 2012. The results show that, compared with the SRM, the average Nash-Sutcliffe coefficient (NSE) of annual runoff simulation increases from 0.80 to 0.86 in the calibration and increases from 0.74 to 0.83 in the validation through the improved model, and the average runoff error reduces from - 12.8 to 1.32% in the calibration and reduces from - 20.0 to - 11.51% in the validation. After adding the measured flow rate for real-time correction, the average NSE of annual runoff simulation increases from 0.91 to 0.93 and the average annual runoff error reduces from - 7.76 to - 3.91% in the calibration. The average NSE increases from 0.85 to 0.89 and the average runoff error reduces from - 12.35 to - 2.76% in the validation. It indicates that the SRM structure with increased evaporation loss is more in line with the actual situation. The short-term simulation effect of the model is greatly improved by adding the measured flow rate for real-time correction. At the same time, the improved SRM and the hypothetical climate change scenario are used to analyze the impact analysis of the snowmelt runoff simulation in the partial wet year. The results show that in the case of rising temperature, the ice and snow ablation period is prolonged, and the annual runoff also changes significantly in time distribution. It is of guiding significance for the influence of climate change on the runoff of recharged rivers with ice-snow meltwater in the other alpine regions.

Transcription Factors AhR/ARNT Regulate the Expression of CYP6CY3 and CYP6CY4 Switch Conferring Nicotine Adaptation.

Nicotine is one of the most toxic secondary plant metabolites in nature and it is highly toxic to herbivorous insects. The overexpression of CYP6CY3 and its homologous isozyme CYP6CY4 in Myzus persicae nicotianae is correlated with nicotine tolerance. The expanded (AC)n repeat in promoter is the cis element for CYP6CY3 transcription. These repeat sequences are conserved in the CYP6CY3 gene from Aphis gossypii and the homologous P450 genes in Acyrthosiphon pisum. The potential transcriptional factors that may regulate CYP6CY3 were isolated by DNA pulldown and sequenced in order to investigate the underlying transcriptional regulation mechanism of CYP6CY3. These identified transcriptional factors, AhR and ARNT, whose abundance was highly correlated with an abundance of the CYP6CY3 gene, were validated. RNAi and co-transfection results further confirm that AhR and ARNT play a major role in the transcriptional regulation of the CYP6CY3 gene. When the CYP6CY3 transcript is destabilized by AhR/ARNT RNAi, the transcription of the CYP6CY4 is dramatically up-regulated, indicating a compensatory mechanism between the CYP6CY3 and CYP6CY4 genes. Our present study sheds light on the CYP6CY3 and CYP6CY4 mediated nicotine adaption of M. persicae nicotianae to tobacco. The current studies shed light on the molecular mechanisms that underlie the genotypic and phenotypic changes that are involved in insect host shifts and we conclude that AhR/ARNT regulate the expression of CYP6CY3 and CYP6CY4 cooperatively, conferring the nicotine adaption of M. persicae nicotianae to tobacco.

A novel small RNA Bmsr1 enhances virulence in Brucella melitensis M28.

Brucellosis, caused by Brucella spp., is one of the most serious zoonotic bacterial diseases. Small RNAs (sRNAs) are recognized as a key player in bacterial post-transcription regulation, since they participate in many biological processes with high efficiency and may govern the intracellular biochemistry and virulence of some pathogenic bacteria. Here, a novel small regulatory RNA, Bmsr1 (Brucella melitensis M28 small RNA 1), was identified in a virulent Brucella melitensis M28 strain based on bioinformatic analysis, reverse transcription PCR (RT-PCR), and Northern blot. The Bmsr1 expression level was highly induced after infection of macrophage cells RAW264.7 at 48 h, suggesting a role for Bmsr1 during in vitro infection. Indeed, bmsr1 deletion mutant of M28 attenuated its intracellular survival in RAW264.7 at 24 h and 48 h post-infection. In a mouse model of chronic infection, bmsr1 deletion strain displayed decreased colonization in the spleen while Bmsr1-overexpressed strain showed higher colonization levels than wild type pathogen. Isobaric tags for relative and absolute quantification (iTRAQ) revealed that 314 proteins were differentially expressed in M28Δbmsr1 compared with wild type. Functional annotation analysis demonstrated that most of those proteins are involved in biological processes and those proteins in the ribosome and nitrogen metabolism pathways were enriched. iTRAQ results combined with target prediction identified several potential target genes related to virulence, including virB2, virB9, virB10, virB11, and vjbR and many metabolism genes. Taken together, this study revealed the contribution of a novel sRNA Bmsr1 to virulence of B. melitensis M28, probably by influencing genes involved in T4SS, virulence regulator VjbR and other metabolism genes.