An Enhanced Vacuum-Assisted Resin Transfer Molding Process and Its Pressure Effect on Resin Infusion Behavior and Composite Material Performance.

In this paper, an enhanced VARTM process is proposed and its pressure effect on resin infusion behavior and composite material performance is studied to reveal the control mechanism of the fiber volume fraction and void content. The molding is vacuumized during the resin injection stage while it is pressurized during the mold filling and curing stages via a VARTM pressure control system designed in this paper. Theoretical calculations and simulation methods are used to reveal the resin's in-plane, transverse, and three-dimensional flow patterns in multi-layer media. For typical thin-walled components, the infiltration behavior of resin in isotropic porous media is studied, elucidating the control mechanisms of fiber volume fraction and void content. The experiments demonstrate that the enhanced VARTM process significantly improves mold filling efficiency and composite's performance. Compared to the regular VARTM process, the panel thickness is reduced by 4% from 1.7 mm, the average tensile strength is increased by 7.3% to 760 MPa, the average flexural strength remains at approximately 720 MPa, porosity is decreased from 1.5% to below 1%, and the fiber volume fraction is increased from 55% to 62%.

High-resolution crystal structure of RNA kinase ArK1 from G. acetivorans.

U47 phosphorylation (Up47) is a novel tRNA modification discovered recently; it can confer thermal stability and nuclease resistance to tRNAs. U47 phosphorylation is catalyzed by Archaeal RNA kinase (Ark1) in an ATP-dependent manner. However, the structural basis for tRNA and/or ATP binding by Ark1 is unclear. Here, we report the expression, purification, and crystallization studies of Ark1 from G. acetivorans (GaArk1). In addition to the Apo-form structure, one GaArk1-ATP complex was also determined in atomic resolution and revealed the detailed basis for ATP binding by GaArk1. The GaArk1-ATP complex represents the only ATP-bound structure of the Ark1 protein. The majority of the ATP-binding residues are conserved, suggesting that GaArk1 and the homologous proteins share similar mechanism in ATP binding. Sequence and structural analysis further indicated that endogenous guanosine will only inhibit the activities of certain Ark1 proteins, such as Ark1 from T. kodakarensis.

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.

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.

Fluorescent Aptaswitch for Detection of Lead Ions.

Detection of metal ions has essential roles in biology, food industry, and environmental sciences. In this work, we developed a Pb2+ detection strategy based on a fluorophore-tagged Pb2+-binding aptamer. The DNA aptamer changes its conformation on binding Pb2+, switching from an "off" state (low fluorescence) to an "on" state (high fluorescence). This method provides a quantitative readout with a detection limit of 468 nM, is highly specific to Pb2+ when tested against other metal ions, and is functional in complex biofluids. Such metal sensing DNA aptamers could be coupled with other biomolecules for sense-and-actuate mechanisms in biomedical and environmental applications.

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.

Crystallization and Structural Determination of 8-17 DNAzyme.

DNAzymes are a group of DNA molecules that can catalyze various chemical reactions. Owing to their great application potentials, DNAzymes have received significant attention. However, due to their intrinsic difficulties in crystallization and structural determination, only very limited structural information of DNAzymes is available to date. Using co-crystallization with the African Swine Fever Virus Polymerase X (AsfvPolX) protein, we have recently solved a complex structure of the 8-17 DNAzyme, which represents the first structure of the catalytically active RNA-cleaving DNAzyme. In this chapter, we describe the detailed protocols including gene construction, AsfvPolX expression and purification, crystallization, structure determination, and in vitro cleavage assay. While the specific methods described herein were originally designed for the 8-17 DNAzyme, they can also be utilized to solve other DNAzyme structures.

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.

Improved support vector machine algorithm based on the influence of Gestational Diabetes Mellitus on the outcome of perinatal outcome by ultrasound imaging.

OBJECTIVES: In order to understand the incidence and epidemiological characteristics of gestational diabetes mellitus, the ultrasound imaging of support vector machine processing algorithm was used to clarify the outcome of maternal and neonatal gestational diabetes mellitus. METHODS: This study selected clinical data of 12,190 pregnant women who were hospitalized for delivery, and were divided into diabetic group (1268 cases) and control group (10922 cases) according to the diagnosis of gestational diabetes. The study was conducted from January 1, 2012 to December 31, 2019. Colour Doppler ultrasound was performed to record fatal umbilical artery and brain the middle arteries and uterine arteries which are effective indicators of measuring fatal intrauterine conditions. Chi-square test was used to compare the rates between groups, and multivariate logistic regression was used for labour outcomes. RESULTS: The incidence of diabetes during pregnancy is about 10.4% (1268/12190). Senior citizens and women suffering from obesity increase the risk of gestational diabetes, maternal hypertension disorders in pregnancy, premature rupture of membranes, oligohydramnios, fatal distress, multiple births, malpresentation risk increased significantly (P <0.05) than the control group. In gestational diabetes caesarean section rate was significantly higher (61.0% vs46.4%). Caesarean new born 5-minute Apgar score was significantly lower than the control group (P <0.05). CONCLUSION: In maternal gestational diabetes in high risk pregnancies, complications of pregnancy significantly increase the importance of enhancing weight management and blood glucose monitoring to reduce complications.

Learning Local-Global Multiple Correlation Filters for Robust Visual Tracking with Kalman Filter Redetection.

Visual object tracking is a significant technology for camera-based sensor networks applications. Multilayer convolutional features comprehensively used in correlation filter (CF)-based tracking algorithms have achieved excellent performance. However, there are tracking failures in some challenging situations because ordinary features are not able to well represent the object appearance variations and the correlation filters are updated irrationally. In this paper, we propose a local-global multiple correlation filters (LGCF) tracking algorithm for edge computing systems capturing moving targets, such as vehicles and pedestrians. First, we construct a global correlation filter model with deep convolutional features, and choose horizontal or vertical division according to the aspect ratio to build two local filters with hand-crafted features. Then, we propose a local-global collaborative strategy to exchange information between local and global correlation filters. This strategy can avoid the wrong learning of the object appearance model. Finally, we propose a time-space peak to sidelobe ratio (TSPSR) to evaluate the stability of the current CF. When the estimated results of the current CF are not reliable, the Kalman filter redetection (KFR) model would be enabled to recapture the object. The experimental results show that our presented algorithm achieves better performances on OTB-2013 and OTB-2015 compared with the other latest 12 tracking algorithms. Moreover, our algorithm handles various challenges in object tracking well.

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.

Short DNA Oligonucleotide as a Ag+ Binding Detector.

Ag+ has been known to mediate several natural metallo-base pairs. Based on the unique structural information of a short 8-mer DNA strand (5'-GCACGCGC-3') induced by Ag+, we constructed several fluorescent DNA beacons for the detection of Ag+ according to the increase in the fluorescence emission on Ag+ binding. This Ag+ detection assay is quick, sensitive, and easy to adapt and can function in a wide range of temperatures from 5 to 65 °C.

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.

Structural studies reveal a ring-shaped architecture of deep-sea vent phage NrS-1 polymerase.

NrS-1 is the first known phage that can infect Epsilonproteobacteria, one of the predominant primary producers in the deep-sea hydrothermal vent ecosystems. NrS-1 polymerase is a multidomain enzyme and is one key component of the phage replisome. The N-terminal Prim/Pol and HBD domains are responsible for DNA polymerization and de novo primer synthesis activities of NrS-1 polymerase. However, the structure and function of the C-terminus (CTR) of NrS-1 polymerase are poorly understood. Here, we report two crystal structures, showing that NrS-1 CTR adopts one unique hexameric ring-shaped conformation. Although the central helicase domain of NrS-1 CTR shares structural similarity with the superfamily III helicases, the folds of the Head and Tail domains are completely novel. Via mutagenesis and in vitro biochemical analysis, we identified many residues important for the helicase and polymerization activities of NrS-1 polymerase. In addition to NrS-1 polymerase, our study may also help us identify and understand the functions of multidomain polymerases expressed by many NrS-1 related phages.

Structure of the error-prone DNA ligase of African swine fever virus identifies critical active site residues.

African swine fever virus (ASFV) is contagious and can cause highly lethal disease in pigs. ASFV DNA ligase (AsfvLIG) is one of the most error-prone ligases identified to date; it catalyzes DNA joining reaction during DNA repair process of ASFV and plays important roles in mutagenesis of the viral genome. Here, we report four AsfvLIG:DNA complex structures and demonstrate that AsfvLIG has a unique N-terminal domain (NTD) that plays critical roles in substrate binding and catalytic complex assembly. In combination with mutagenesis, in vitro binding and catalytic assays, our study reveals that four unique active site residues (Asn153 and Leu211 of the AD domain; Leu402 and Gln403 of the OB domain) are crucial for the catalytic efficiency of AsfvLIG. These unique structural features can serve as potential targets for small molecule design, which could impair genome repair in ASFV and help combat this virus in the future.

Construction and structure studies of DNA-bipyridine complexes as versatile scaffolds for site-specific incorporation of metal ions into DNA.

The facile construction of metal-DNA complexes using 'Click' reactions is reported here. A series of 2'-propargyl-modified DNA oligonucleotides were initially synthesized as structure scaffolds and were then modified through 'Click' reaction to incorporate a bipyridine ligand equipped with an azido group. These metal chelating ligands can be placed in the DNA context in site-specific fashion to provide versatile templates for binding various metal ions, which are exchangeable using a simple EDTA washing-and-filtration step. The constructed metal-DNA complexes were found to be thermally stable. Their structures were explored by solving a crystal structure of a propargyl-modified DNA duplex and installing the bipyridine ligands by molecular modeling and simulation. These metal-DNA complexes could have wide applications as novel organometallic catalysts, artificial ribonucleases, and potential metal delivery systems.

High-resolution DNA quadruplex structure containing all the A-, G-, C-, T-tetrads.

DNA can form diverse structures, which predefine their physiological functions. Besides duplexes that carry the genetic information, quadruplexes are the most well-studied DNA structures. In addition to their important roles in recombination, replication, transcription and translation, DNA quadruplexes have also been applied as diagnostic aptamers and antidisease therapeutics. Herein we further expand the sequence and structure complexity of DNA quadruplex by presenting a high-resolution crystal structure of DNA1 (5'-AGAGAGATGGGTGCGTT-3'). This is the first quadruplex structure that contains all the internal A-, G-, C-, T-tetrads, A:T:A:T tetrads and bulged nucleotides in one single structure; as revealed by site-specific mutagenesis and biophysical studies, the central ATGGG motif plays important role in the quadruplex formation. Interestingly, our structure also provides great new insights into cation recognition, including the first-time reported Pb2+, by tetrad structures.

Structural basis for multiple gene regulation by human DUX4.

DUX4 plays critical role in the molecular pathogenesis of the neuromuscular disorder facioscapulohumeral muscular dystrophy and acute lymphoblastic leukemia in humans. As a master transcription regulator, DUX4 can also bind the promoters and activate the transcription of hundreds ZGA-associated genes. Here we report on the structural and biochemical studies of DUX4 double homeodomains (DUX4-DH), representing the only structures contain both homeodomain 1 (HD1) and homeodomain 2 (HD2). HD1 and HD2 adopt classical homeobox fold; via the helix inserted into the major groove and the N-terminal extended loop inserted into the minor groove, HD1 and HD2 recognize the box1 (5'-TAA-3') and box2 (5'-TGA-3') nucleotides of the consensus sequence, respectively. Among the box1 and box2 linking nucleotides (CCTAA), the two adenine residues are reported to be highly conserved; however, they are not directly recognized by DUX4-DH in the structures. Besides different nucleotides, our ITC analysis indicated that DUX4-DH can also tolerate various changes in the linker length. Our studies not only revealed the basis for target DNA recognition by DUX4, but also advanced our understanding on multiple gene activation by DUX4.