Manipulating Interactions between T4 Phage Long Tail Fibers and Escherichia coli Receptors.

Bacteriophages are the most abundant and diverse biological entities on Earth. Phages exhibit strict host specificity that is largely conferred by adsorption. However, the mechanism underlying this phage host specificity remains poorly understood. In this study, we examined the interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the long tail fibers of bacteriophage T4. T4 phage uses OmpC of the K-12 strain, but not of the O157 strain, for adsorption, even though OmpCs from the two E. coli strains share 94% homology. We identified amino acids P177 and F182 in loop 4 of the K-12 OmpC as essential for T4 phage adsorption in the copresence of loops 1 and 5. Analyses of phage mutants capable of adsorbing to OmpC mutants demonstrated that amino acids at positions 937 and 942 of the gp37 protein, which is present in the distal tip (DT) region of the T4 long tail fibers, play an important role in adsorption. Furthermore, we created a T4 phage mutant library with artificial modifications in the DT region and isolated and characterized multiple phage mutants capable of adsorbing to OmpC of the O157 strain or lipopolysaccharide of the K-12 strain. These results shed light on the mechanism underlying the phage host specificity mediated by gp37 and OmpC and may be useful in the development of phage therapy via artificial modifications of the DT region of T4 phage. IMPORTANCE Understanding the host specificity of phages will lead to the development of phage therapy. The interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the gp37 protein present in the distal tip (DT) region of the long tail fibers of T4 bacteriophages largely determines their host specificity. Here, we elucidated the amino acid residues important for the interaction between gp37 and OmpC. This result suggests that the shapes of both proteins at the binding interface play important roles in their interactions, which are likely mediated by multiple residues of both binding partners. Additionally, we successfully isolated multiple phage mutants capable of adsorbing to a variety of E. coli receptors using a mutant T4 phage library with artificial modifications in the DT region, providing a foundation for the alteration of the host specificity.

Effects of toothbrush abrasion on surface and antibacterial properties of hydroxyapatite-tryptophan complex with gray titania.

Photocatalysts have attracted attention in the medical field for their antibacterial effects. However, typical photocatalysts are activated by ultraviolet rays, which may have adverse effects. Therefore, we focused on a new photocatalyst that is activated by visible light, hydroxyapatite (HAp), and amino acid complex with gray titania, and evaluated its antibacterial effects against Porphyromonas gingivalis and effect by toothbrushing. The test sample was a titanium alloy substrate, and four surface treatments were applied: (1) substrate only, (2) substrate with HAp complex, (3) substrate with HAp complex with gray titania, and (4) HAp-tryptophan complex with gray titania (TR). These surface treatments were evaluated with or without toothbrushing (8 total groups). Surface roughness (Sa), fluorescent X-ray analysis (XRF), and scanning electron microscopy (SEM) were used to evaluate surface properties. To investigate antibacterial effects, each sample was seeded with P. gingivalis, irradiated with red light, and total viable bacterial count was determined. For Sa measurement, TR showed no significant difference after toothbrushing. However, in XRF and SEM observation, TR exhibited peeling of the applied coating after toothbrushing. In the antibacterial test, TR showed a decrease in P. gingivalis under no toothbrushing condition. Conversely, with toothbrushing, the TR coating appeared to peel. However, no significant difference in P. gingivalis count was observed among all groups. HAp-tryptophan complex with gray titania coating showed an antibacterial effect against P. gingivalis when irradiated with visible light. However, toothbrushing can result in coat peeling and consequently reduce the antibacterial effect.

An ADP-ribosyltransferase Alt of bacteriophage T4 negatively regulates the Escherichia coli†MazF toxin of a toxin-antitoxin module.

Prokaryotic toxin-antitoxin (TA) systems are linked to many roles in cell physiology, such as plasmid maintenance, stress response, persistence and protection from phage infection, and the activities of toxins are tightly regulated. Here, we describe a novel regulatory mechanism for a toxin of Escherichia coli TA systems. The MazF toxin of MazE-MazF, which is one of the best characterized type II TA systems, was modified immediately after infection with bacteriophage T4. Mass spectrometry demonstrated that the molecular weight of this modification was 542 Da, corresponding to a mono-ADP-ribosylation. This modification disappeared in cells infected with T4 phage lacking Alt, which is one of three ADP-ribosyltransferases encoded by T4 phage and is injected together with phage DNA upon infection. In vivo and in vitro analyses confirmed that T4 Alt ADP-ribosylated MazF at an arginine residue at position 4. Finally, the ADP-ribosylation of MazF by Alt resulted in the reduction of MazF RNA cleavage activity in vitro, suggesting that it may function to inactivate MazF during T4 infection. This is the first example of the chemical modification of an E. coli toxin in TA systems to regulate activity.

Structural insights into the inhibition mechanism of bacterial toxin LsoA by bacteriophage antitoxin Dmd.

Bacteria have obtained a variety of resistance mechanisms including toxin-antitoxin (TA) systems against bacteriophages (phages), whereas phages have also evolved to overcome bacterial anti-phage mechanisms. Dmd from T4 phage can suppress the toxicities of homologous toxins LsoA and RnlA from Escherichia coli, representing the first example of a phage antitoxin against multiple bacterial toxins in known TA systems. Here, the crystal structure of LsoA-Dmd complex showed Dmd is inserted into the deep groove between the N-terminal repeated domain (NRD) and the Dmd-binding domain (DBD) of LsoA. The NRD shifts significantly from a 'closed' to an 'open' conformation upon Dmd binding. Site-directed mutagenesis of Dmd revealed the conserved residues (W31 and N40) are necessary for LsoA binding and the toxicity suppression as determined by pull-down and cell toxicity assays. Further mutagenesis identified the conserved Dmd-binding residues (R243, E246 and R305) of LsoA are vital for its toxicity, and suggested Dmd and LsoB may possess different inhibitory mechanisms against LsoA toxicity. Our structure-function studies demonstrate Dmd can recognize LsoA and inhibit its toxicity by occupying the active site possibly via substrate mimicry. These findings have provided unique insights into the defense and counter-defense mechanisms between bacteria and phages in their co-evolution.

Prokaryotic toxin-antitoxin systems: novel regulations of the toxins.

Toxin-antitoxin (TA) systems are widely conserved in prokaryotic plasmids and chromosomes and are linked to many roles in cell physiology, including plasmid maintenance, stress response, persistence and protection from phage infection. A TA system is composed of a stable toxin and a labile antitoxin that inhibits a harmful effect of the cognate toxin. When gene expression from the TA loci is repressed under certain conditions such as nutrient starvation, the toxin is freed from the rapidly degrading antitoxin and obstructs an essential cellular process, such as DNA replication, translation and peptidoglycan synthesis, which subsequently causes growth arrest. TA systems are classified into five types according to the nature and the function of antitoxins, and the activity of toxins is tightly regulated in a variety of ways. This short-review highlights several novel regulatory mechanisms for Escherichia coli toxins that we recently discovered.

RNase HI stimulates the activity of RnlA toxin in Escherichia coli.

A type II toxin-antitoxin system in Escherichia coli, rnlA-rnlB, functions as an anti-phage mechanism. RnlA is a toxin with an endoribonuclease activity and the cognate RnlB inhibits RnlA toxicity in E. coli cells. After bacteriophage T4 infection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs and consequently no T4 propagation, when T4 dmd is defective: Dmd is an antitoxin against RnlA for promoting own propagation. Previous studies suggested that the activation of RnlA after T4 infection was regulated by multiple components. Here, we provide the evidence that RNase HI is an essential factor for activation of RnlA. The dmd mutant phage could grow on ΔrnhA (encoding RNase HI) cells, in which RnlA-mediated mRNA cleavage activity was defective. RNase HI bound to RnlA in vivo and enhanced the RNA cleavage activity of RnlA in vitro. In addition, ectopic expression of RnlA in ΔrnlAB ΔrnhA cells has less effect on cell toxicity and RnlA-mediated mRNA degradation than in ΔrnlAB cells. This is the first example of a direct factor for activation of a toxin.

Structure-function studies of Escherichia coli RnlA reveal a novel toxin structure involved in bacteriophage resistance.

Escherichia coli RnlA-RnlB is a newly identified toxin-antitoxin (TA) system that plays a role in bacteriophage resistance. RnlA functions as a toxin with mRNA endoribonuclease activity and the cognate antitoxin RnlB inhibits RnlA toxicity in E. coli cells. Interestingly, T4 phage encodes the antitoxin Dmd, which acts against RnlA to promote its own propagation, suggesting that RnlA-Dmd represents a novel TA system. Here, we have determined the crystal structure of RnlA refined to 2.10 (Dmd-binding domain), which is an organization not previously observed among known toxin structures. Small-angle X-ray scattering (SAXS) analysis revealed that RnlA forms a dimer in solution via interactions between the DBDs from both monomers. The in vitro and in vivo functional studies showed that among the three domains, only the DBD is responsible for recognition and inhibition by Dmd and subcellular location of RnlA. In particular, the helix located at the C-terminus of DBD plays a vital role in binding Dmd. Our comprehensive studies reveal the key region responsible for RnlA toxicity and provide novel insights into its structure-function relationship.

Identification of the human PMR1 mRNA endonuclease as an alternatively processed product of the gene for peroxidasin-like protein.

The PMR1 endonuclease was discovered in Xenopus liver and identified as a member of the large and diverse peroxidase gene family. The peroxidase genes arose from multiple duplication and rearrangement events, and their high degree of sequence similarity confounded attempts to identify human PMR1. The functioning of PMR1 in mRNA decay depends on the phosphorylation of a tyrosine in the C-terminal polysome targeting domain by c-Src. The sequences of regions that are required for c-Src binding and phosphorylation of Xenopus PMR1 were used to inform a bioinformatics search that identified two related genes as potential candidates for human PMR1: peroxidasin homolog (PXDN) and peroxidasin homolog-like (PXDNL) protein. Although each of these genes is predicted to encode a large, multidomain membrane-bound peroxidase, alternative splicing of PXDNL pre-mRNA yields a transcript whose predicted product is a 57-kDa protein with 42% sequence identity to Xenopus PMR1. Results presented here confirm the existence of the predicted 57-kDa protein, show this is the only form of PXDNL detected in any of the human cell lines examined, and confirm its identity as human PMR1. Like the Xenopus protein, human PMR1 binds to c-Src, is tyrosine phosphorylated, sediments on polysomes, and catalyzes the selective decay of a PMR1 substrate mRNA. Importantly, the expression of human PMR1 stimulates cell motility in a manner similar to that of the Xenopus PMR1 expressed in human cells, thus providing definitive evidence linking endonuclease decay to the regulation of cell motility.

Dmd of bacteriophage T4 functions as an antitoxin against Escherichia coli LsoA and RnlA toxins.

Enterohaemorrhagic Escherichia coli O157:H7 harbours a cryptic plasmid, pOSAK1, that carries only three ORFs: mobA (involved in plasmid mobilization), ORF1 and ORF2. Predicted proteins encoded by these two ORFs were found to share a weak homology with RnlA and RnlB, respectively, a toxin­antitoxin system encoded on the E. coli K-12 chromosome. Here, we report that lsoA (ORF1) encodes a toxin and lsoB (ORF2) an antitoxin. In spite of the homologies, RnlB and LsoB functioned as antitoxins against only their cognate toxins and not interchangeably with each other. Interestingly, T4 phage Dmd suppressed the toxicities of both RnlA and LsoA by direct interaction, the first example of a phage with an antitoxin against multiple toxins.

A finite element study on the interactive effect between the damage of the cup-bone interface and the bone strain of hip implants under various fixation conditions.

Interfacial damage has a high impact on the loosening of the acetabular cup. However, monitoring this damage induced by the variations in loading conditions, such as the angle, amplitude, and frequency in vivo, is challenging. In this study, we evaluated the risk of loosening of the acetabular cup due to interfacial damages induced by the deviation in loading conditions and amplitudes. A three-dimensional model of the acetabular cup component was developed, and the interfacial crack growth between the cup and the bone was modeled using a fracture mechanics approach, which simulated the extent of interfacial damage and associated cup displacement. The interfacial delamination mechanism changed with the increasing inclination angle, wherein a fixation angle of 60° exhibited the largest area of contact loss. The compressive strain of embedding the simulated bone at the remaining bonding area accumulated as the lost contact area widened. Such interfacial damages, namely, the growth of the lost contact area and accumulated compressive strain in the simulated bone, promoted both embedding and rotational displacement of the acetabular cup. In the worst case of a fixation angle of 60°, the total displacement of the acetabular cup exceeded the limit of the modified safe zone, suggesting a quantitative risk of dislocation of the acetabular cup induced by the accumulated interfacial damage. Furthermore, nonlinear regression analyses between the degree of displacement of the acetabular cup and the extent of the two types of interfacial damage demonstrated that the interactive effect of the fixation angle with the loading amplitude showed a significant effect on increasing cup displacement. These findings suggest that proper control of the fixation angle during operation is useful in preventing the loosening of the hip joint.

Phage single-stranded DNA-binding protein or host DNA damage triggers the activation of the AbpAB phage defense system.

IMPORTANCE: Although numerous phage defense systems have recently been discovered in bacteria, how these systems defend against phage propagation or sense phage infections remains unclear. The Escherichia coli AbpAB defense system targets several lytic and lysogenic phages harboring DNA genomes. A phage-encoded single-stranded DNA-binding protein, Gp32, activates this system similar to other phage defense systems such as Retron-Eco8, Hachiman, ShosTA, Nhi, and Hna. DNA replication inhibitors or defects in DNA repair factors activate the AbpAB system, even without phage infection. This is one of the few examples of activating phage defense systems without phage infection or proteins. The AbpAB defense system may be activated by sensing specific DNA-protein complexes.

Generation of equatorial plasma bubble after the 2022 Tonga volcanic eruption.

Equatorial plasma bubbles are a phenomenon of plasma density depletion with small-scale density irregularities, normally observed in the equatorial ionosphere. This phenomenon, which impacts satellite-based communications, was observed in the Asia-Pacific region after the largest-on-record January 15, 2022 eruption of the Tonga volcano. We used satellite and ground-based ionospheric observations to demonstrate that an air pressure wave triggered by the Tonga volcanic eruption could cause the emergence of an equatorial plasma bubble. The most prominent observation result shows a sudden increase of electron density and height of the ionosphere several ten minutes to hours before the initial arrival of the air pressure wave in the lower atmosphere. The propagation speed of ionospheric electron density variations was ~ 480-540 m/s, whose speed was higher than that of a Lamb wave (~315 m/s) in the troposphere. The electron density variations started larger in the Northern Hemisphere than in the Southern Hemisphere. The fast response of the ionosphere could be caused by an instantaneous transmission of the electric field to the magnetic conjugate ionosphere along the magnetic field lines. After the ionospheric perturbations, electron density depletion appeared in the equatorial and low-latitude ionosphere and extended at least up to ±25° in geomagnetic latitude.

Utility of preoperative small-bowel endoscopy for hemorrhagic lesions in the small intestine.

PURPOSE: We evaluated a small group of patients with hemorrhagic lesions in the small intestine diagnosed preoperatively by video capsule endoscopy (VCE) and/or double-balloon endoscopy (DBE), who underwent surgery in our hospital. METHODS: The subjects were 20 patients with hemorrhagic lesions in the small intestine diagnosed preoperatively by VCE and/or DBE, who underwent surgery between April 2004 and December 2008. RESULTS: VCE, DBE, and computed tomography were performed in 12, 17, and 20 patients, respectively. Eleven patients also had a biopsy taken during DBE, resulting in a definitive diagnosis in eight. Because of the risk of hemorrhage during DBE in five patients, a biopsy was not taken and the sites of the lesion were marked in these patients. Twenty patients underwent surgery, and the diagnoses were small-intestinal cancer in eight, gastrointestinal stromal tumor in seven, arteriovenous malformation in two, and Crohn's disease, angioectasia, and leiomyoma in one each. The interval between the onset of symptoms and surgery was less than 50 days for six patients, 50-100 days for two, 100-200 days for five, and more than 200 days for seven. CONCLUSION: Preoperative small-bowel endoscopy proved useful for diagnosing the cause of hemorrhagic lesions in the small intestine.

Effects of composition of hydroxyapatite/gray titania coating fabricated by suspension plasma spraying on mechanical and antibacterial properties.

While several metallic implants with bioactive coatings have been developed thus far for treating bone deformations or deterioration, a multifunctional coating with the desired mechanical and antibacterial properties has not been demonstrated. This study aimed to reveal the effect of the composition of hydroxyapatite (HAp)/gray titania coatings on the mechanical and antibacterial properties for biomedical applications. Suspension plasma spray (SPS) aided successful deposition of HAp/gray titania coatings on the surface of titanium substrates. The microstructure of coatings with different compositions was then characterized using scanning electron microscopy, X-ray diffraction, and Raman spectroscopy to identify the crystal structure. All results consistently demonstrated that SPS could transform Ti2O3 into TiO2 with mixed Magneli phases, such as Ti4O7 and Ti3O5, which could typically demonstrate photocatalytic activity. Hardness, Young's modulus, and interfacial strength of composite coatings commonly increased with an increase in the weight percentage of TiO2. A multi-modal damage assessment combining acoustic emission (AE), infrared ray camera (IR), and digital-image-correlation (DIC) was performed to monitor the damage process of HAp composite coating, which successfully revealed initiations of microcracks and nonlinear deformation at interface until fracture. Antibacterial test performed for examining the cytotoxic effects against E. coli under LED light irradiation conditions revealed that SPS HAp/gray titania coating could significantly enhance the antibacterial properties. Enhanced antibacterial properties can be attributed to an increase in the number of Magneli phases and better bacterial adhesion was attributed to hydrophilic properties conferred by submicron-sized particles. Hence, SPS can help fabricate visible light-responsive antibacterial coating, which can be used for medical devices.

Surface modification of hydroxyapatite nanoparticles for bone regeneration by controlling their surface hydration and protein adsorption states.

Autogenous bone and metallic implant grafting has been used to repair and regenerate bone defects. However, there are still many unresolved problems. It is suggested that bioceramic nanoparticles should be developed and designed to promote effective bone regeneration. In addition, it is necessary to synthesize bioceramic nanoparticles that can support proteins related to bone repair and regeneration such as collagen and albumin. As the protein-interactive bioceramic, hydroxyapatite (HA) deserves to be mentioned and has several attractive properties that are useful in biomedical fields (e.g., biocompatibility, protein adsorption capacity and stability in the physiological environment). In order to prepare novel HA nanoparticles with high biocompatibility, it can be considered that human bones are mainly composed of HA and contain a small amount of silicate, and therefore, the design of coexistence of HA with silicate can be focused. Moreover, it is proposed that the state of the hydration layer on the nanoparticle surfaces can be controlled by introducing heteroelements and polymer chains, which have a great influence on the subsequent protein adsorption and cell adhesion. In this perspective, in order to develop novel bioceramic nanoparticles for the treatment of bone defect, the design of highly biocompatible HA nanoparticles and the control of the hydration layer and protein adsorption states on the surfaces were systematically discussed based on their surface modification techniques, which are very important for the proper understanding of the interface between cells and bioceramics, leading to the further application in biomedical fields.

Localized mesospheric ozone destruction corresponding to isolated proton aurora coming from Earth's radiation belt.

Relativistic electron precipitation (REP) from the Earth's radiation belt plays an important role in mesospheric ozone loss as a connection between space weather and the climate system. However, the rapid (tens of minutes) destruction of mesospheric ozone directly caused by REP has remained poorly understood due to the difficulty of recognizing its location and duration. Here we show a compelling rapid correspondence between localized REP and ozone destruction during a specific auroral phenomenon, the called an isolated proton aurora (IPA). The IPA from the Earth's radiation belt becomes an important spatial and temporal proxy of REP, distinct from other auroral phenomena, and allowing visualizing micro-ozone holes. We found ozone destruction of as much as 10-60% within 1.5 h of the initiation of IPA. Electromagnetic ion cyclotron waves in the oxygen ion band observed as the driver of REP likely affect through resonance with mainly ultra-relativistic (> 2 mega-electron-volts) energy electrons. The rapid REP impact demonstrates its crucial role and direct effect on regulating the atmospheric chemical balance.

The hokW-sokW Locus Encodes a Type I Toxin-Antitoxin System That Facilitates the Release of Lysogenic Sp5 Phage in Enterohemorrhagic Escherichia coli O157.

The toxin-antitoxin (TA) genetic modules control various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. They also exist in mobile genetic elements, including prophages; however, their physiological roles remain poorly understood. Here, we demonstrate that hokW-sokW, a putative TA locus encoded in Sakai prophage 5 (Sp5) in enterohemorrhagic Escherichia coli O157: H7 Sakai strain, functions as a type I TA system. Bacterial growth assays showed that the antitoxic activity of sokW RNA against HokW toxin partially requires an endoribonuclease, RNase III, and an RNA chaperone, Hfq. We also demonstrated that hokW-sokW assists Sp5-mediated lysis of E. coli cells when prophage induction is promoted by the DNA-damaging agent mitomycin C (MMC). We found that MMC treatment diminished sokW RNA and increased both the expression level and inner membrane localization of HokW in a RecA-dependent manner. Remarkably, the number of released Sp5 phages decreased by half in the absence of hokW-sokW. These results suggest that hokW-sokW plays a novel role as a TA system that facilitates the release of Sp5 phage progeny through E. coli lysis.

Higher-order topological Mott insulator on the pyrochlore lattice.

We provide the first unbiased evidence for a higher-order topological Mott insulator in three dimensions by numerically exact quantum Monte Carlo simulations. This insulating phase is adiabatically connected to a third-order topological insulator in the noninteracting limit, which features gapless modes around the corners of the pyrochlore lattice and is characterized by a [Formula: see text] spin-Berry phase. The difference between the correlated and non-correlated topological phases is that in the former phase the gapless corner modes emerge only in spin excitations being Mott-like. We also show that the topological phase transition from the third-order topological Mott insulator to the usual Mott insulator occurs when the bulk spin gap solely closes.

Damage evaluation of HAp-coated porous titanium foam in simulated body fluid based on compression fatigue behavior.

Although pure Ti is nontoxic, alloying elements may be released into the surrounding tissue when Ti alloys are used, and this can cause cytotoxicity. Therefore, this study performed the damage evaluation of hydroxyapatite (HAp)-coated porous Ti components subjected to cyclic compression in a simulated body fluid (SBF). The HAp coating layer was deposited on the surface of porous Ti by electrophoresis, and a dense and homogeneous coating morphology was observed on the surface of the porous Ti. To specify damage types of HAp coating in situ, acoustic emission (AE) measurements and microscopic observations were simultaneously conducted during compressive fatigue loading tests to detect the specific failure mode. Compression tests revealed that the interfacial strength between the HAp coating and porous Ti was higher than the yield strength of the porous body (7-9 MPa). The AE signals were detected only in the plastic deformation stage of porous Ti, which indicated that they were generated because of plastic deformation/fractures in the porous body. Compressive fatigue tests revealed that no significant HAp coating damage occurred when the applied maximum stress was within the elastic limit of porous Ti in air. In contrast, the HAp coating exhibited delamination at the initial stage of cyclic loading at all stress levels in SBF, while the fatigue limit of the coated porous substrate, 2 MPa, was not affected by the SBF medium. Though the delamination of the HAp coating in SBF occurred during the early stages of fatigue loading, the amorphous calcium phosphate layer was recovered partly through re-precipitation from SBF. The AE signals from the delamination of the HAp coating or fracture in porous Ti could be identified using the peak voltage and frequencies. As microscopic observations were limited to certain parts of the porous body, AE signals were clustered according to the types of failure. The clustered AE signals were successfully correlated with the fatigue behavior of porous Ti. Corrosion fatigue was determined to be the primary mechanism for the delamination of the HAp coating on porous Ti in SBF.

Fat replacement of the distal pancreas in a case of advanced gastric cancer.

Preoperative computed tomography in a 67-year-old man admitted with type 2 advanced gastric cancer involving the upper body of the stomach revealed marked atrophy of the left lobe of the liver and atrophy of the distal pancreas. Total gastrectomy with D2 lymph node dissection was planned; however, additional distal pancreatectomy with splenectomy was also performed during the operation because of direct invasion of the pancreas by the gastric cancer. Histopathologic examination of the resected pancreas revealed the absence of lobules, acini, conduits, and pancreatic ducts, with only islets of Langerhans found scattered in the adipose tissue. The findings revealed that the gastric cancer had directly invaded the fat, replacing the distal pancreas. In patients with fat-replaced pancreas, preoperative evaluation of direct invasion of adjacent organs/tissues by gastric cancer is difficult.