Assessment of chlorine and hydrogen peroxide on airborne bacteria: Disinfection efficiency and induction of antibiotic resistance.

Airborne pathogens severely threaten public health worldwide. Air disinfection is essential to ensure public health. However, excessive use of disinfectants may endanger environmental and ecological security due to the residual disinfectants and their by-products. This study systematically evaluated disinfection efficiency, induction of multidrug resistance, and the underlying mechanisms of disinfectants (NaClO and H2O2) on airborne bacteria. The results showed that airborne bacteria were effectively inactivated by atomized NaClO (>160 µg/L) and H2O2 (>320 µg/L) after 15 min. However, some bacteria still survived after disinfection by atomized NaClO (0-80 µg/L) and H2O2 (0-160 µg/L), and they exhibited significant increases in antibiotic resistance. The whole-genome sequencing of the resistant bacteria revealed distinct mutations that were responsible for both antibiotic resistance and virulence. This study also provided evidences and insights into possible mechanisms underlying the induction of antibiotic resistance by air disinfection, which involved intracellular reactive oxygen species formation, oxidative stress responses, alterations in bacterial membranes, activation of efflux pumps, and the thickening of biofilms. The present results also shed light on the role of air disinfection in inducing antibiotic resistance, which could be a crucial factor contributing to the global spread of antibiotic resistance through the air.

Structural and functional insights into the helicase protein E5 of Mpox virus.

Mpox virus (MPXV) can cause mpox in humans. Due to its quick and wide spread in the past two years, mpox has turned into a significant public health concern. Helicase E5 is a multi-domain protein; its primer synthesis and DNA unwinding activity are required for genome uncoating and DNA replication of MPXV. However, the in vitro DNA unwinding activity has never been demonstrated. Here, we report the structural and biochemical studies of MPXV E5, showing that the full-length protein adopts an auto-inhibited conformation. Truncation of the N-terminus can recover the in vitro unwinding activity of E5 towards the forked DNA. Further structural analysis reveals that MPXV E5 shares a conserved mechanism in DNA unwinding and primer synthesis with the homologous proteins. These findings not only advance our understanding on the function of MPXV E5, but also provide a solid basis for the development of anti-poxvirus drugs.

Abnormal levels of expression of microRNAs in peripheral blood of patients with traumatic brain injury are induced by microglial activation and correlated with severity of injury.

BACKGROUND: Microglia play a crucial role in regulating the progression of traumatic brain injury (TBI). In specific, microglia can self-activate and secrete various substances that exacerbate or alleviate the neuroimmune response to TBI. In addition, microRNAs (miRNAs) are involved in the functional regulation of microglia. However, molecular markers that reflect the dynamics of TBI have not yet been found in peripheral tissues. METHODS: Paired samples of peripheral blood were collected from patients with TBI before and after treatment. Next-generation sequencing and bioinformatics analysis were used to identify the main pathways and biological functions of TBI-related miRNAs in the samples. Moreover, lipopolysaccharide-treated human microglia were used to construct a cellular immune-activation model. This was combined with analysis of peripheral blood samples to screen for highly expressed miRNAs derived from activated microglia after TBI treatment. Quantitative reverse-transcriptase polymerase chain reaction was used to determine the expression levels of these miRNAs, allowing their relationship with the severity of TBI to be examined. Receiver operating characteristic (ROC) curves were constructed to analyse the clinical utility of these miRNAs for determining the extent of TBI. RESULTS: Sequencing results showed that 37 miRNAs were differentially expressed in peripheral blood samples from patients with TBI before and after treatment, with 17 miRNAs being upregulated and 20 miRNAs being downregulated after treatment. The expression profiles of these miRNAs were verified in microglial inflammation models and in the abovementioned peripheral blood samples. The results showed that hsa-miR-122-5p and hsa-miR-193b-3p were highly expressed in the peripheral blood of patients with TBI after treatment and that the expression levels of these miRNAs were correlated with the patients' scores on the Glasgow Coma Scale. ROC curve analysis revealed that abnormally high levels of expression of hsa-miR-122-5p and hsa-miR-193b-3p in peripheral blood have some clinical utility for distinguishing different extents of TBI and thus could serve as biomarkers of TBI. CONCLUSION: Abnormally high levels of expression of hsa-miR-122-5p and hsa-miR-193b-3p in the peripheral blood of patients with TBI were due to the activation of microglia and correlated with the severity of TBI. This discovery may help to increase understanding of the molecular pathology of TBI and guide the development of new strategies for TBI therapy based on microglial function.

Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity.

CRISPR-Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR-CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR-Cas12f1 working mechanism and expand the mini-CRISPR toolbox.

Research on fast marking method for indicator diagram of pumping well based on K-means clustering.

Indicator diagram is the key basis for fault diagnosis of pumping wells in oil exploitation. With the rapid development of machine learning, the fault diagnosis of indicator diagram based on deep learning has garnered increasing attention. This kind of methods train neural network models with marked samples, and then inputs images into the trained models and outputs their categories. At present, the preparation of indicator diagram sample set relies on experts' analysis of indicator diagram images one by one. However, it involves extensive manual work and manual marking is prone to errors, so the marked samples are often insufficient in quantity. In order to quickly mark a large number of indicator diagram samples, the oil well data was plotted into standardized indicator diagram, and then three feature extraction methods for indicator diagrams were proposed: feature extraction based on original vector, feature extraction based on three-dimensional pixel tensor, feature extraction based on convolutional neural network. These methods convert the indicator diagram into corresponding feature vectors, which are then clustered using the K-means clustering algorithm, enabling the corresponding indicator diagrams to be classified into different categories based on the clustering results. Using 20,000 randomly selected pieces of data from 100 pumping wells, this study clusters the sample set using the three proposed methods. The results indicated that the time consumption were 0.2, 8.3, and 0.7 h, with accuracy rates of 98%, 92%, and 95%, respectively. For indicator diagrams, the clustering method based on the original vector has outstanding performance in terms of efficiency and accuracy. This provides an automatic tool for the preparation of the pumping well fault diagnosis dataset, and its efficiency can be increased by tens of times compared with manual marking.

Structures and implications of the C962R protein of African swine fever virus.

African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. Although it has been extensively studied in the past, no vaccine or other useful treatment against ASFV is available. The genome of ASFV encodes more than 170 proteins, but the structures and functions for the majority of the proteins remain elusive, which hindered our understanding on the life cycle of ASFV and the development of ASFV-specific inhibitors. Here, we report the structural and biochemical studies of the highly conserved C962R protein of ASFV, showing that C962R is a multidomain protein. The N-terminal AEP domain is responsible for the DNA polymerization activity, whereas the DNA unwinding activity is catalyzed by the central SF3 helicase domain. The middle PriCT2 and D5_N domains and the C-terminal Tail domain all contribute to the DNA unwinding activity of C962R. C962R preferentially works on forked DNA, and likely functions in Base-excision repair (BER) or other repair pathway in ASFV. Although it is not essential for the replication of ASFV, C962R can serve as a model and provide mechanistic insight into the replicative primase proteins from many other species, such as nitratiruptor phage NrS-1, vaccinia virus (VACV) and other viruses.

Structural and functional studies of PCNA from African swine fever virus.

Proliferating cell nuclear antigen (PCNA) belongs to the DNA sliding clamp family. Via interacting with various partner proteins, PCNA plays critical roles in DNA replication, DNA repair, chromatin assembly, epigenetic inheritance, chromatin remodeling, and many other fundamental biological processes. Although PCNA and PCNA-interacting partner networks are conserved across species, PCNA of a given species is rarely functional in heterologous systems, emphasizing the importance of more representative PCNA studies. Here, we report two crystal structures of PCNA from African swine fever virus (ASFV), which is the only member of the Asfarviridae family. Compared to the eukaryotic and archaeal PCNAs and the sliding clamp structural homologs from other viruses, AsfvPCNA possesses unique sequences and/or conformations at several regions, such as the J-loop, interdomain-connecting loop (IDCL), P-loop, and C-tail, which are involved in partner recognition or modification of sliding clamps. In addition to double-stranded DNA binding, we also demonstrate that AsfvPCNA can modestly enhance the ligation activity of the AsfvLIG protein. The unique structural features of AsfvPCNA can serve as a potential target for the development of ASFV-specific inhibitors and help combat the deadly virus. IMPORTANCE Two high-resolution crystal structures of African swine fever virus proliferating cell nuclear antigen (AsfvPCNA) are presented here. Structural comparison revealed that AsfvPCNA is unique at several regions, such as the J-loop, the interdomain-connecting loop linker, and the P-loop, which may play important roles in ASFV-specific partner selection of AsfvPCNA. Unlike eukaryotic and archaeal PCNAs, AsfvPCNA possesses high double-stranded DNA-binding affinity. Besides DNA binding, AsfvPCNA can also modestly enhance the ligation activity of the AsfvLIG protein, which is essential for the replication and repair of ASFV genome. The unique structural features make AsfvPCNA a potential target for drug development, which will help combat the deadly virus.

Crystal structures and identification of novel Cd2+-specific DNA aptamer.

Cadmium (Cd) is one of the most toxic heavy metals. Exposure to Cd can impair the functions of the kidney, respiratory system, reproductive system and skeletal system. Cd2+-binding aptamers have been extensively utilized in the development of Cd2+-detecting devices; however, the underlying mechanisms remain elusive. This study reports four Cd2+-bound DNA aptamer structures, representing the only Cd2+-specific aptamer structures available to date. In all the structures, the Cd2+-binding loop (CBL-loop) adopts a compact, double-twisted conformation and the Cd2+ ion is mainly coordinated with the G9, C12 and G16 nucleotides. Moreover, T11 and A15 within the CBL-loop form one regular Watson-Crick pair and stabilize the conformation of G9. The conformation of G16 is stabilized by the G8-C18 pair of the stem. By folding and/or stabilizing the CBL-loop, the other four nucleotides of the CBL-loop also play important roles in Cd2+ binding. Similarly to the native sequence, crystal structures, circular dichroism spectrum and isothermal titration calorimetry analysis confirm that several variants of the aptamer can recognize Cd2+. This study not only reveals the underlying basis for the binding of Cd2+ ions with the aptamer, but also extends the sequence for the construction of novel metal-DNA complex.

Aggregation kinetics of polystyrene nanoplastics in gastric environments: Effects of plastic properties, solution conditions, and gastric constituents.

Nanoplastics are inevitably ingested into human gastric environment, wherein their aggregation kinetics and interactions with gastric constituents remain unclear. This study investigated the early-stage (20 min) and long-term (1-6 h) aggregation kinetics of four commonly-found polystyrene nanoplastics (PSNPs) including NP100 (100-nm), A-NP100 (100-nm, amino-modified), C-NP100 (100-nm, carboxyl-modified), and NP500 (500-nm) under gastric conditions. Five simulated human gastric fluids (SGFs) including SGF1-3 (0-3.2 g/L pepsin and 34.2 mM NaCl), SGF4 (400 mM glycine), and SGF5 (nine constituents), three pH (2, fasted state; 3.5, late-fed state; and 5, early-fed state), and 1-100 mg/L PSNPs were examined. Aggregation rates ranked NP100 > A-NP100 ≈ C-NP100 > NP500, SGF5 > SGF4 > SGF3 > SGF2 > SGF1, and pH 2 > 3.5 > 5. Increasing PSNP concentration enhanced aggregation rate up to 13.82 nm/s. Aggregation behavior generally followed the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Pepsin, glycine, and proteose-peptone strongly influenced PSNP stability via electrostatic interaction and steric hindrance imparted by protein corona. Freundlich isotherm suggested that PSNPs adsorbed organic constituents following lysozyme > porcine bile > proteose-peptone > pepsin > glycine > D-glucose, inducing changes in constituent structure and PSNP properties. These findings provide insights on the transport of nanoplastics in the gastric environments.

MiR-373/miR-520s-CD44 Axis Significantly Inhibits the Growth and Invasion of Human Glioblastoma Cells.

BACKGROUND: The expression and regulation of microRNAs (miRNAs) play an important role in glioblastoma (GBM) tumorigenesis, progression and prognosis. Little is known about the role of the miRNA regulatory network of GBM risk-related genes in GBM growth and invasiveness. METHODS: The UALCAN and Oncomine gene expression dataset were used to explore gene expression profiles in human GBM. The Kaplan-Meier method was performed to evaluate the prognostic values of the GBM-related genes. Multiple bioinformatics databases were analysed to predict the GBM-related genes targeted by miRNAs. A luciferase reporter assay and other molecular cell function experiments were conducted to reveal the mechanisms of interaction between the identified miRNAs and their targets. RESULTS: The CD44 expression is significantly higher in GBM tissues than that in normal tissues, and negatively correlated with survival duration in GBM patients. In normal physiological conditions, CD44 expression is lower in various parts of the central nervous system than in other organ systems. The mRNA encoding CD44 is a direct target of miR-373 and miR-520s, and this finding was verified by molecular biology experiments. We further found that miR-373 and miR-520s expression was negatively associated with CD44 expression in GBM specimens, and that the miR-373 or miR-520s-CD44 interaction network significantly affected the growth and invasiveness of GBM cells. CONCLUSION: The miR-373 and miR-520s exert their functions by suppressing CD44 expression in GBM cells, and their expression, together with that of CD44, could thus serve as a valuable biomarker of GBM prognosis.

Structures and implications of the nuclease domain of human parvovirus B19 NS1 protein.

Infection of human parvovirus B19 (B19V) can cause a variety of diseases, such as hydrops fetalis, erythema infectiosum in children and acute arthropathy in women. Although B19V infection mainly occurs during childhood, about 50 % of adults are still susceptible to B19V infection. As the major replication protein of B19V, deletion of NS1 completely abolishes the infectivity of the virus. The nuclease domain of NS1 (NS1_Nuc) is responsible for DNA Ori binding and nicking that is critical for B19V viral DNA replication. NS1 has various variants, the structure and function for the majority of the variants are poorly studied. Here, we report two high-resolution crystal structures of NS1_Nuc, revealed the detailed conformations of many key residues. Structural comparison indicates that these residues are important for ssDNA or dsDNA binding by NS1. NS1 belongs to the HUH-endonuclease superfamily and it shares conserved ssDNA cleavage mechanism with other HUH-endonuclease members. However, our structural analyses, mutagenesis and in vitro assay results all suggested that NS1_Nuc utilizes one unique model in ssDNA binding.

Crystal structures and insights into precursor tRNA 5'-end processing by prokaryotic minimal protein-only RNase P.

Besides the canonical RNA-based RNase P, pre-tRNA 5'-end processing can also be catalyzed by protein-only RNase P (PRORP). To date, various PRORPs have been discovered, but the basis underlying substrate binding and cleavage by HARPs (homolog of Aquifex RNase P) remains elusive. Here, we report structural and biochemical studies of HARPs. Comparison of the apo- and pre-tRNA-complexed structures showed that HARP is able to undergo large conformational changes that facilitate pre-tRNA binding and catalytic site formation. Planctomycetes bacterium HARP exists as dimer in vitro, but gel filtration and electron microscopy analysis confirmed that HARPs from Thermococcus celer, Thermocrinis minervae and Thermocrinis ruber can assemble into larger oligomers. Structural analysis, mutagenesis and in vitro biochemical studies all supported one cooperative pre-tRNA processing mode, in which one HARP dimer binds pre-tRNA at the elbow region whereas 5'-end removal is catalyzed by the partner dimer. Our studies significantly advance our understanding on pre-tRNA processing by PRORPs.

Structure-guided development of Pb2+-binding DNA aptamers.

Owing to its great threat to human health and environment, Pb2+ pollution has been recognized as a major public problem by the World Health Organization (WHO). Many DNA aptamers have been utilized in the development of Pb2+-detection sensors, but the underlying mechanisms remain elusive. Here, we report three Pb2+-complexed structures of the thrombin binding aptamer (TBA). These high-resolution crystal structures showed that TBA forms intramolecular G-quadruplex and Pb2+ is bound by the two G-tetrads in the center. Compared to K+-stabilized G-quadruplexes, the coordinating distance between Pb2+ and the G-tetrads are much shorter. The T3T4 and T12T13 linkers play important roles in dimerization and crystallization of TBA, but they are changeable for Pb2+-binding. In combination with mutagenesis and CD spectra, the G8C mutant structure unraveled that the T7G8T9 linker of TBA is also variable. In addition to expansion of the Pb2+-binding aptamer sequences, our study also set up one great example for quick and rational development of other aptamers with similar or optimized binding activity.

Colloidal stability of nanosized activated carbon in aquatic systems: Effects of pH, electrolytes, and macromolecules.

Nanosized activated carbon (NAC) is a novel adsorbent with great potential for water reclamation. However, its transport and reactivity in aqueous environments may be greatly affected by its stability against aggregation. This study investigated the colloidal stability of NAC in model aqueous systems with broad background solution chemistries including 7 electrolytes (NaCl, NaNO3, Na2SO4, KCl, CaCl2, MgCl2, and BaCl2), pH 4-9, and 6 macromolecules (humic acid (HA), fulvic acid (FA), cellulose (CEL), bovine serum albumin (BSA), alginate (ALG), and extracellular polymeric substance (EPS)), along with natural water samples collected from pristine to polluted rivers. The results showed that higher solution pH stabilized NAC by raising the critical coagulation concentration from 28 to 590 mM NaCl. Increased cation concentration destabilized NAC by charge screening, with the cationic influence following Ba2+ > Ca2+ > Mg2+ >> Na+ > K+. Its aggregation behavior could be predicted with the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory with a Hamaker constant (ACWC) of 4.3 × 10-20 J. The presence of macromolecules stabilized NAC in NaCl solution and most CaCl2 solution following EPS > BSA > CEL > HA > FA > ALG, due largely to enhanced electrical repulsion and steric hindrance originated from adsorbed macromolecules. However, ALG and HA strongly destabilized NAC via cation bridging at high Ca2+ concentrations. Approximately half of NAC particles remained stably suspended for ∼10 d in neutral freshwater samples. The results demonstrated the complex effects of water chemistry on fate and transport of NAC in aquatic environments.

Simultaneous PAHs degradation, odour mitigation and energy harvesting by sediment microbial fuel cell coupled with nitrate-induced biostimulation.

The study investigates a bioremediation process of polycyclic aromatic hydrocarbons (PAHs) removal and odour mitigation combined with energy harvesting. Sediment microbial fuel cells (SMFCs) were constructed with the addition of nitrate in the sediment to simultaneously remove acid-volatile sulphide (AVS) and PAHs. With the combined nitrate-SMFC treatment, over 90% of the AVS was removed from the sediment in 6 weeks of the SMFC operation and a maximum of 94% of AVS removal efficiency was reached at Week 10. The highest removal efficiencies of phenanthrene, pyrene, and benzo[a]pyrene was 93%, 80%, and 69%, respectively. The maximum voltage attained for the combined nitrate-SMFC treatment was 341 mV. Illumina HiSeq sequencing revealed that the autotrophic denitrifiers Thiobacillus are the dominant genus. In electricity generation, both sulphide-oxidation and PAH-oxidation are the possible pathways. Besides, the addition of nitrate stimulated the growth of Pseudomonas which is responsible for the electricity generation and direct biodegradation of the PAHs, indicating a synergistic effect. The developed bioremediation process demonstrated the potential in the in-situ bioremediation process utilizing SMFC combined with nitrate-induced bioremediation.

Structural basis for guide RNA trimming by RNase D ribonuclease in Trypanosoma brucei.

Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs.

Corrigendum: Molecular Mechanism and Approach in Progression of Meningioma.

[This corrects the article DOI: 10.3389/fonc.2020.538845.].

HGF/c-Met Axis: The Advanced Development in Digestive System Cancer.

Numerous studies have indicated that abnormal activation of the HGF/c-Met signaling pathway can lead to cell proliferation, invasiveness, and metastasis of cancers of the digestive system. Moreover, overexpression of c-Met has been implicated in poor prognosis of patients with these forms of cancer, suggesting the possibility for HGF/c-Met axis as a potential therapeutic target. Despite the large number of clinical and preclinical trials worldwide, no significant positive success in the use of anti-HGF/c-Met treatments on cancers of the digestive system has been achieved. In this review, we summarize advanced development of clinical research on HGF/c-Met antibody and small-molecule c-Met inhibitors of cancers of the digestive system and provide a possible direction for future research.

Molecular Mechanism and Approach in Progression of Meningioma.

Meningioma is the most common tumor of the central nervous system, most of which is benign. Even after complete resection, a high rate of recurrence of meningioma is observed. From in-depth study of its pathogenesis, it has been found that a number of chromosomal variations and abnormal molecular signals are closely related to the occurrence and development of malignancy in meningioma, which may provide the theoretical basis and potential direction for accurate and targeted treatment. We have reviewed advances in chromosomal variations and molecular mechanisms involved in the progression of meningioma, and have highlighted the association with malignant biological behavior including cell proliferation, angiogenesis, increased invasiveness, and inhibition of apoptosis. In addition, the chemotherapy of meningioma is summarized and discussed.

A unique DNA-binding mode of African swine fever virus AP endonuclease.

African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. ASFV is primarily replicated in the cytoplasm of pig macrophages, which is oxidative and caused constant damage to ASFV genome. ASFV AP endonuclease (AsfvAP) catalyzes DNA cleavage reaction at the abasic site and is a key enzyme of ASFV base excision repair (BER) system. Although it plays an essential role in ASFV survival in host cells, the basis underlying substrate binding and cleavage by AsfvAP remains unclear. Here, we reported the structural and functional studies of AsfvAP, showing that AsfvAP adopts a novel DNA-binding mode distinct from other APs. AsfvAP possesses many unique structural features, including one narrower nucleotide-binding pocket at the active site, the C16-C20 disulfide bond-containing region, and histidine-rich loop. As indicated by our mutagenesis, in vitro binding and cleavage assays, these features are important for AsfvAP to suit the acidic and oxidative environment. Owing to their functional importance, these unique features could serve as targets for designing small molecule inhibitors that could disrupt the repair process of ASFV genome and help fight against this deadly virus in the future.