Nanobody in a Double "Y"-Shaped Assembly: A Promising Candidate for Lateral Flow Immunoassays.

Derived from camelid heavy-chain antibodies, nanobodies (Nbs) are the smallest natural antibodies and are an ideal tool in biological studies because of their simple structure, high yield, and low cost. Nbs possess significant potential for developing highly specific and user-friendly diagnostic assays. Despite offering considerable advantages in detection applications, knowledge is limited regarding the exclusive use of Nbs in lateral flow immunoassay (LFIA) detection. Herein, we present a novel double "Y" architecture, achieved by using the SpyTag/SpyCatcher and Im7/CL7 systems. The double "Y" assemblies exhibited a significantly higher affinity for their epitopes, as particularly evident in the reduced dissociation rate. An LFIA employing double "Y" assemblies was effectively used to detect the severe acute respiratory syndrome coronavirus-2 N protein, with a detection limit of at least 500 pg/mL. This study helps broaden the array of tools available for the development of Nb-based diagnostic techniques.

LncRNA-protein interaction prediction with reweighted feature selection.

LncRNA-protein interactions are ubiquitous in organisms and play a crucial role in a variety of biological processes and complex diseases. Many computational methods have been reported for lncRNA-protein interaction prediction. However, the experimental techniques to detect lncRNA-protein interactions are laborious and time-consuming. Therefore, to address this challenge, this paper proposes a reweighting boosting feature selection (RBFS) method model to select key features. Specially, a reweighted apporach can adjust the contribution of each observational samples to learning model fitting; let higher weights are given more influence samples than those with lower weights. Feature selection with boosting can efficiently rank to iterate over important features to obtain the optimal feature subset. Besides, in the experiments, the RBFS method is applied to the prediction of lncRNA-protein interactions. The experimental results demonstrate that our method achieves higher accuracy and less redundancy with fewer features.

Research on the Recognition and Tracking of Group-Housed Pigs' Posture Based on Edge Computing.

The existing algorithms for identifying and tracking pigs in barns generally have a large number of parameters, relatively complex networks and a high demand for computational resources, which are not suitable for deployment in embedded-edge nodes on farms. A lightweight multi-objective identification and tracking algorithm based on improved YOLOv5s and DeepSort was developed for group-housed pigs in this study. The identification algorithm was optimized by: (i) using a dilated convolution in the YOLOv5s backbone network to reduce the number of model parameters and computational power requirements; (ii) adding a coordinate attention mechanism to improve the model precision; and (iii) pruning the BN layers to reduce the computational requirements. The optimized identification model was combined with DeepSort to form the final Tracking by Detecting algorithm and ported to a Jetson AGX Xavier edge computing node. The algorithm reduced the model size by 65.3% compared to the original YOLOv5s. The algorithm achieved a recognition precision of 96.6%; a tracking time of 46 ms; and a tracking frame rate of 21.7 FPS, and the precision of the tracking statistics was greater than 90%. The model size and performance met the requirements for stable real-time operation in embedded-edge computing nodes for monitoring group-housed pigs.

A virus-target host proteins recognition method based on integrated complexes data and seed extension.

BACKGROUND: Target drugs play an important role in the clinical treatment of virus diseases. Virus-encoded proteins are widely used as targets for target drugs. However, they cannot cope with the drug resistance caused by a mutated virus and ignore the importance of host proteins for virus replication. Some methods use interactions between viruses and their host proteins to predict potential virus-target host proteins, which are less susceptible to mutated viruses. However, these methods only consider the network topology between the virus and the host proteins, ignoring the influences of protein complexes. Therefore, we introduce protein complexes that are less susceptible to drug resistance of mutated viruses, which helps recognize the unknown virus-target host proteins and reduce the cost of disease treatment. RESULTS: Since protein complexes contain virus-target host proteins, it is reasonable to predict virus-target human proteins from the perspective of the protein complexes. We propose a coverage clustering-core-subsidiary protein complex recognition method named CCA-SE that integrates the known virus-target host proteins, the human protein-protein interaction network, and the known human protein complexes. The proposed method aims to obtain the potential unknown virus-target human host proteins. We list part of the targets after proving our results effectively in enrichment experiments. CONCLUSIONS: Our proposed CCA-SE method consists of two parts: one is CCA, which is to recognize protein complexes, and the other is SE, which is to select seed nodes as the core of protein complexes by using seed expansion. The experimental results validate that CCA-SE achieves efficient recognition of the virus-target host proteins.

YdiV regulates Escherichia coli ferric uptake by manipulating the DNA-binding ability of Fur in a SlyD-dependent manner.

Iron is essential for all bacteria. In most bacteria, intracellular iron homeostasis is tightly regulated by the ferric uptake regulator Fur. However, how Fur activates the iron-uptake system during iron deficiency is not fully elucidated. In this study, we found that YdiV, the flagella gene inhibitor, is involved in iron homeostasis in Escherichia coli. Iron deficiency triggers overexpression of YdiV. High levels of YdiV then transforms Fur into a novel form which does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent manner. Thus, the cooperation of YdiV, SlyD and Fur activates the gene expression of iron-uptake systems under conditions of iron deficiency. Bacterial invasion assays also demonstrated that both ydiV and slyD are necessary for the survival and growth of uropathogenic E. coli in bladder epithelial cells. This reveals a mechanism where YdiV not only represses flagella expression to make E. coli invisible to the host immune system, but it also promotes iron acquisition to help E. coli overcome host nutritional immunity.

Protein functional module identification method combining topological features and gene expression data.

BACKGROUND: The study of protein complexes and protein functional modules has become an important method to further understand the mechanism and organization of life activities. The clustering algorithms used to analyze the information contained in protein-protein interaction network are effective ways to explore the characteristics of protein functional modules. RESULTS: This paper conducts an intensive study on the problems of low recognition efficiency and noise in the overlapping structure of protein functional modules, based on topological characteristics of PPI network. Developing a protein function module recognition method ECTG based on Topological Features and Gene expression data for Protein Complex Identification. CONCLUSIONS: The algorithm can effectively remove the noise data reflected by calculating the topological structure characteristic values in the PPI network through the similarity of gene expression patterns, and also properly use the information hidden in the gene expression data. The experimental results show that the ECTG algorithm can detect protein functional modules better.

T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis.

The assembly of DNA fragments with homologous arms is becoming popular in routine cloning. For an in vitro assembly reaction, a DNA polymerase is often used either alone for its 3'-5' exonuclease activity or together with a 5'-3' exonuclease for its DNA polymerase activity. Here, we present a 'T5 exonuclease DNA assembly' (TEDA) method that only uses a 5'-3' exonuclease. DNA fragments with short homologous ends were treated by T5 exonuclease and then transformed into Escherichia coli to produce clone colonies. The cloning efficiency was similar to that of the commercial In-Fusion method employing a proprietary DNA polymerase, but higher than that of the Gibson method utilizing T5 exonuclease, Phusion DNA polymerase, and DNA ligase. It also assembled multiple DNA fragments and did simultaneous site-directed mutagenesis at multiple sites. The reaction mixture was simple, and each reaction used 0.04 U of T5 exonuclease that cost 0.25 US cents. The simplicity, cost effectiveness, and cloning efficiency should promote its routine use, especially for labs with a budget constraint. TEDA may trigger further development of DNA assembly methods that employ single exonucleases.

Crystal structures of porcine STINGCBD-CDN complexes reveal the mechanism of ligand recognition and discrimination of STING proteins.

The cyclic dinucleotide (CDN)-stimulator of interferon genes (STING) pathway plays an important role in the detection of viral and bacterial pathogens in animals. Previous studies have shown that the metazoan second messenger cyclic [G(2',5')pA(3',5')p] (2',3'-cGAMP) generated by cyclic GMP-AMP synthase cGAS binds STING with high affinity compared with bacterial CDNs such as c-di-GMP, c-di-AMP, and 3',3'-cGAMP. Despite recent progress indicating that the CDN-binding domain (CBD) of dimeric STING binds asymmetric 2',3'-cGAMP preferentially over symmetric 3',3'-CDNs, it remains an open question whether STING molecules, such as human STING, adopt a symmetric dimeric conformation to efficiently engage its asymmetric ligand. Here, structural studies of the CBD from porcine STING (STINGCBD) in complex with CDNs at 1.76-2.6 Å resolution revealed that porcine STINGCBD, unlike its human and mouse counterparts, can adopt an asymmetric ligand-binding pocket to accommodate the CDNs. We observed that the extensive interactions and shape complementarity between asymmetric 2',3'-cGAMP and the ligand-binding pocket make it the most preferred ligand for porcine STING and that geometry constraints limit the binding between symmetric 3',3'-CDN and porcine STING. The ligand-discrimination mechanism of porcine STING observed here expands our understanding of how the CDN-STING pathway is activated and of its role in antiviral defense.

The archaeal ATPase PINA interacts with the helicase Hjm via its carboxyl terminal KH domain remodeling and processing replication fork and Holliday junction.

PINA is a novel ATPase and DNA helicase highly conserved in Archaea, the third domain of life. The PINA from Sulfolobus islandicus (SisPINA) forms a hexameric ring in crystal and solution. The protein is able to promote Holliday junction (HJ) migration and physically and functionally interacts with Hjc, the HJ specific endonuclease. Here, we show that SisPINA has direct physical interaction with Hjm (Hel308a), a helicase presumably targeting replication forks. In vitro biochemical analysis revealed that Hjm, Hjc, and SisPINA are able to coordinate HJ migration and cleavage in a concerted way. Deletion of the carboxyl 13 amino acid residues impaired the interaction between SisPINA and Hjm. Crystal structure analysis showed that the carboxyl 70 amino acid residues fold into a type II KH domain which, in other proteins, functions in binding RNA or ssDNA. The KH domain not only mediates the interactions of PINA with Hjm and Hjc but also regulates the hexameric assembly of PINA. Our results collectively suggest that SisPINA, Hjm and Hjc work together to function in replication fork regression, HJ formation and HJ cleavage.

Structural and biochemical characterization of the catalytic domains of GdpP reveals a unified hydrolysis mechanism for the DHH/DHHA1 phosphodiesterase.

The Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterases (PDEs) that catalyze degradation of cyclic di-adenosine monophosphate (c-di-AMP) could be subdivided into two subfamilies based on the final product [5'-phosphadenylyl-adenosine (5'-pApA) or AMP]. In a previous study, we revealed that Rv2837c, a stand-alone DHH/DHHA1 PDE, employs a 5'-pApA internal flipping mechanism to produce AMPs. However, why the membrane-bound DHH/DHHA1 PDE can only degrade c-di-AMP to 5'-pApA remains obscure. Here, we report the crystal structure of the DHH/DHHA1 domain of GdpP (GdpP-C), and structures in complex with c-di-AMP, cyclic di-guanosine monophosphate (c-di-GMP), and 5'-pApA. Structural analysis reveals that GdpP-C binds nucleotide substrates quite differently from how Rv2837c does in terms of substrate-binding position. Accordingly, the nucleotide-binding site of the DHH/DHHA1 PDEs is organized into three (C, G, and R) subsites. For GdpP-C, in the C and G sites c-di-AMP binds and degrades into 5'-pApA, and its G site determines nucleotide specificity. To further degrade into AMPs, 5'-pApA must slide into the C and R sites for flipping and hydrolysis as in Rv2837c. Subsequent mutagenesis and enzymatic studies of GdpP-C and Rv2837c uncover the complete flipping process and reveal a unified catalytic mechanism for members of both DHH/DHHA1 PDE subfamilies.

Salmonella STM1697 coordinates flagella biogenesis and virulence by restricting flagellar master protein FlhD4C2 from recruiting RNA polymerase.

Salmonella reduces flagella biogenesis to avoid detection within host cells by a largely unknown mechanism. We identified an EAL-like protein STM1697 as required and sufficient for this process. STM1697 surges to a high level after Salmonella enters host cells and restrains the expression of flagellar genes by regulating the function of flagellar switch protein FlhD4C2, the transcription activator of all other flagellar genes. Unlike other anti-FlhD4C2 factors, STM1697 does not prevent FlhD4C2 from binding to target DNA. A 2.0 Å resolution STM1697-FlhD structure reveals that STM1697 binds the same region of FlhD as STM1344, but with weaker affinity. Further experiments show that STM1697 regulates flagella biogenesis by restricting FlhD4C2 from recruiting RNA polymerase and the regulatory effect of STM1697 on flagellar biogenesis and virulence are all achieved by interaction with FlhD. Finally, we describe a novel mechanism mediated by STM1697 in which Salmonella can inhibit the production of flagella antigen and escape from the host immune system.

Effects of PslG on the Surface Movement of Pseudomonas aeruginosa.

PslG attracted a lot of attention recently due to its great potential abilities in inhibiting biofilms of Pseudomonas aeruginosa However, how PslG affects biofilm development still remains largely unexplored. Here, we focused on the surface motility of bacterial cells, which is critical for biofilm development. We studied the effects of PslG on bacterial surface movement in early biofilm development at a single-cell resolution by using a high-throughput bacterial tracking technique. The results showed that compared with no exogenous PslG addition, when PslG was added to the medium, bacterial surface movement was significantly (4 to 5 times) faster and proceeded in a more random way with no clear preferred direction. A further study revealed that the fraction of walking mode increased when PslG was added, which then resulted in an elevated average speed. The differences of motility due to PslG addition led to a clear distinction in patterns of bacterial surface movement and retarded microcolony formation greatly. Our results provide insight into developing new PslG-based biofilm control techniques.IMPORTANCE Biofilms of Pseudomonas aeruginosa are a major cause for hospital-acquired infections. They are notoriously difficult to eradicate and pose serious health hazards to human society. So, finding new ways to control biofilms is urgently needed. Recent work on PslG showed that PslG might be a good candidate for inhibiting/disassembling biofilms of Pseudomonas aeruginosa through Psl-based regulation. However, to fully explore PslG functions in biofilm control, a better understanding of PslG-Psl interactions is needed. Toward this end, we examined the effects of PslG on the surface movement of Pseudomonas aeruginosa in this work. The significance of our work is in greatly enhancing our understanding of the inhibiting mechanism of PslG on biofilms by providing a detailed picture of bacterial surface movement at a single-cell level, which will allow a full understanding of PslG abilities in biofilm control and thus present potential applications in biomedical fields.

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase.

The intracellular infections of Mycobacterium tuberculosis, which is the causative agent of tuberculosis, are regulated by many cyclic dinucleotide signaling. Rv2837c from M. tuberculosis is a soluble, stand-alone DHH-DHHA1 domain phosphodiesterase that down-regulates c-di-AMP through catalytic degradation and plays an important role in M. tuberculosis infections. Here, we report the crystal structure of Rv2837c (2.0 Å), and its complex with hydrolysis intermediate 5'-pApA (2.35 Å). Our structures indicate that both DHH and DHHA1 domains are essential for c-di-AMP degradation. Further structural analysis shows that Rv2837c does not distinguish adenine from guanine, which explains why Rv2837c hydrolyzes all linear dinucleotides with almost the same efficiency. We observed that Rv2837c degraded other c-di-NMPs at a lower rate than it did on c-di-AMP. Nevertheless, our data also showed that Rv2837c significantly decreases concentrations of both c-di-AMP and c-di-GMP in vivo. Our results suggest that beside its major role in c-di-AMP degradation Rv2837c could also regulate c-di-GMP signaling pathways in bacterial cell.

Inhibiting Firefly Bioluminescence by Chalcones.

Chalcone refers to an aromatic ketone and an enone that constitutes the central core for various important biological compounds in drug discovery. Moreover, the firefly luciferase (Fluc) as the bioluminescent reporter has been widely used in life science research and high-throughput screening (HTS). However, Fluc might suffer from direct inhibition by HTS compounds resulting in the occurrence of "false positives." In the current research, we discovered a series of chalcone compounds as Fluc inhibitors with favorable potency both in vitro and in vivo. Moreover, our compound 3i showed remarkable systemic inhibition in transgenic mice. Both enzymatic kinetics study and cocrystal structure demonstrated that compound 3i is competitive for substrate aminoluciferin, while noncompetitive for ATP. Besides, compound 3i exhibited excellent selectivity as a promising quenching agent in a simulated dual-luciferase reporter assay. We believed that our research would contribute to improving scientists' awareness of the Fluc inhibitors, pay attention to the bias results, and even expand the utilization of bioluminescence in life science research.

cybLuc: An Effective Aminoluciferin Derivative for Deep Bioluminescence Imaging.

To enhance the efficiency of firefly luciferase/luciferin bioluminescence imaging, a series of N-cycloalkylaminoluciferins (cyaLucs) were developed by introducing lipophilic N-cycloalkylated substitutions. The experimental results demonstrate that these cyaLucs are effective substrates for native firefly luciferase (Fluc) and can produce elevated bioluminescent signals in vitro, in cellulo, and in vivo. It should be noted that, in animal studies, N-cyclobutylaminoluciferin (cybLuc) at 10 µM (0.1 mL), which is 0.01% of the standard dose of d-luciferin (dLuc) used in mouse imaging, can radiate 20-fold more bioluminescent light than d-luciferin (dLuc) or aminoluciferin (aLuc) at the same concentration. Longer in vivo emission imaging using cybLuc suggests that it can be used for long-time observation. Regarding the mechanism of cybLuc, our cocrystal structure data from firefly luciferase with oxidized cybLuc suggested that oxidized cybLuc fits into the same pocket as oxyluciferin. Most interestingly, our results demonstrate that the sensitivity of cybLuc in brain tumor imaging contributes to its extended application in deep tissues.

Crystal structure of the crenarchaeal ExoIII AP endonuclease SisExoIII reveals a conserved disulfide bond endowing the protein with thermostability.

AP endonuclease recognizes and cleaves apurinic/apyrimidinic (AP) sites and plays a critical role in base excision repair. Many ExoIII and EndoIV family AP endonucleases have been characterized both biochemically and structurally in Eukaryote and Bacteria. However, relatively fewer have been studied in Euryarchaeota and there is no such report on an AP endonuclease from Crenarchaeota. Here we report, for the first time, the crystal structure of a crenarchaeal ExoIII AP endonuclease, SisExoIII, from Sulfolobus islandicus REY15A. SisExoIII comprises a two-layer core formed by 10 ß-sheets and a shell formed by 9 surrounding α-helices. A disulfide bond connecting ß8 and ß9 is formed by Cys142 and Cys215. This intra-molecular linkage is conserved among crenarchaeal ExoIII homologs and site-directed mutagenesis revealed that it endows the protein with thermostability, however, disruption of the disulfide bond only has a slight effect on the AP endonuclease activity. We also observed that several key residues within the catalytic center including conserved Glu35 and Asn9 show different conformation compared with known ExoIII proteins and form various intra-molecular salt bridges. The protein possesses three putative DNA binding loops with higher flexibility and hydrophobicity than those of ExoIIIs from other organisms. These features may result in low AP endonuclease activity and defect of exonuclease activity of SisExoIII. The study has deepened our understanding in the structural basis of crenarchaeal ExoIII catalysis and clarified a role of the disulfide bond in maintaining protein thermostability.

Crystal structure of PvdO from Pseudomonas aeruginosa.

Pyoverdine I (PVDI) is a water-soluble fluorescein siderophore with strong iron chelating ability from the gram-negative pathogen Pseudomonas aeruginosa PAO1. Compared to common siderophores, PVDI is a relatively large compound whose synthesis requires a group of enzymes with different catalytic activities. In addition to four nonribosomal peptide synthetases (NRPS) which are responsible for the production of the peptide backbone of PVDI, several additional enzymes are associated with the modification of the side chains. PvdO is one of these enzymes and participates in PVDI precursor maturation in the periplasm. We determined the crystal structure of PvdO at 1.24 Å resolution. The PvdO structure shares a common fold with some FGly-generating enzymes (FGE) and is stabilized by Ca2+. However, the catalytic residues in FGE are not observed in PvdO, indicating PvdO adopts a unique catalytic mechanism.

Crystal structure and biochemical features of dye-decolorizing peroxidase YfeX from Escherichia coli O157 Asp143 and Arg232 play divergent roles toward different substrates.

YfeX from Escherichia coli O157 is a bacterial dye-decolorizing peroxidase that represents both dye-decoloring activity and typical peroxidase activity. We reported the crystal structure of YfeX bound to heme at 2.09 Å resolution. The YfeX monomer resembles a ferredoxin-like fold and contains two domains. The three conserved residues surrounding the heme group are His215, Asp143 and Arg232. His215 functions as the proximal axial ligand of the heme iron atom. Biochemical data show that the catalytic significance of the conserved Asp143 and Arg232 depends on the substrate types and that YfeX may adopt various catalytic mechanisms toward divergent substrates. In addition, it is observed that an access tunnel spans from the protein molecular surface to the heme distal region, it serves as the passageway for the entrance and binding of the H2O2.

Crystal structure of PnpCD, a two-subunit hydroquinone 1,2-dioxygenase, reveals a novel structural class of Fe2+-dependent dioxygenases.

Aerobic microorganisms have evolved a variety of pathways to degrade aromatic and heterocyclic compounds. However, only several classes of oxygenolytic fission reaction have been identified for the critical ring cleavage dioxygenases. Among them, the most well studied dioxygenases proceed via catecholic intermediates, followed by noncatecholic hydroxy-substituted aromatic carboxylic acids. Therefore, the recently reported hydroquinone 1,2-dioxygenases add to the diversity of ring cleavage reactions. Two-subunit hydroquinone 1,2-dioxygenase PnpCD, the key enzyme in the hydroquinone pathway of para-nitrophenol degradation, catalyzes the ring cleavage of hydroquinone to γ-hydroxymuconic semialdehyde. Here, we report three PnpCD structures, named apo-PnpCD, PnpCD-Fe(3+), and PnpCD-Cd(2+)-HBN (substrate analog hydroxyenzonitrile), respectively. Structural analysis showed that both the PnpC and the C-terminal domains of PnpD comprise a conserved cupin fold, whereas PnpC cannot form a competent metal binding pocket as can PnpD cupin. Four residues of PnpD (His-256, Asn-258, Glu-262, and His-303) were observed to coordinate the iron ion. The Asn-258 coordination is particularly interesting because this coordinating residue has never been observed in the homologous cupin structures of PnpCD. Asn-258 is proposed to play a pivotal role in binding the iron prior to the enzymatic reaction, but it might lose coordination to the iron when the reaction begins. PnpD also consists of an intriguing N-terminal domain that might have functions other than nucleic acid binding in its structural homologs. In summary, PnpCD has no apparent evolutionary relationship with other iron-dependent dioxygenases and therefore defines a new structural class. The study of PnpCD might add to the understanding of the ring cleavage of dioxygenases.

Membrane association of SadC enhances its diguanylate cyclase activity to control exopolysaccharides synthesis and biofilm formation in Pseudomonas aeruginosa.

Cyclic diguanosine monophosphate (c-di-GMP) is one of the most important bacterial second messengers that controls many bacterial cellular functions including lifestyle switch between plankton and biofilm. Surface attachment defective (SadC) is a diguanylate cyclase (DGC) involved in the biosynthesis of c-di-GMP in Pseudomonas aeruginosa, an opportunistic pathogen that can cause diverse infections. Here we report the crystal structure of GGDEF domain from SadC and the critical role of the trans-membrane (TM) domain of SadC with regard to biofilm formation, exopolysaccharide production and motility. We showed that over-expression of SadC in P. aeruginosa PAO1 totally inhibited swimming motility and significantly enhanced the production of exopolysaccharide Psl. SadC lacking TM domains (SadC300-487 ) could not localize on cytoplasmic membrane and form cluster, lost the ability to inhibit the swimming and twitching motility, and showed the attenuated activity to promote Psl production despite that SadC300-487 was able to catalyze the synthesize of c-di-GMP in vitro and in vivo. The GGDEF domain of SadC has a typical GGDEF structure and the α-helix connected the TM domains with SadC GGDEF domain is essential for SadC to form DGC oligomers. Our data imply that membrane association of SadC promotes its DGC activity by affecting the formation of active DGC oligomers.