Contact-Killing Antibacterial Polystyrene Polymerized Using a Quaternized Cationic Initiator.

Contact-killing antibacterial materials are attracting attention owing to their ability for sustained antibacterial activity. However, contact-killing antibacterial polystyrene (PS) has not been extensively studied because its chemically stable structure impedes chemical modification. In this study, we developed an antibacterial PS sheet with a contact-killing surface using PS synthesized from 2,2'-azobis-[2-(1,3-dimethyl-4,5-dihydro-1H-imidazol-3-ium-2-yl)]propane triflate (ADIP) as a radical initiator with cationic moieties. The PS sheet synthesized with ADIP (ADIP-PS) exhibited antibacterial activity in contrast to PS synthesized with other azo radical initiators. Surface ζ-potential measurements revealed that only ADIP-PS had a cationic surface, which contributed to its contact-killing antibacterial activity. The ADIP-PS sheets also exhibited antibacterial activity after washing. In contrast, PS sheets containing silver, a typical leachable antibacterial agent, lost all antibacterial activity after the same washing treatment. The antibacterial ADIP-PS sheet demonstrated strong broad-spectrum activity against both Gram-positive and Gram-negative bacteria, including drug-resistant bacteria. Cytotoxicity tests using L929 cells showed that the ADIP-PS sheets were noncytotoxic. This contact-killing antibacterial PS synthesized with ADIP thus demonstrated good prospects as an easily producible antimicrobial material.

Sixth cranial nerve palsy and trigeminal neuropathic pain due to a space-occupying lesion.

Various neuropathies of the cranil nerves can accompany trigeminal neuropathic pain attributed to space-occupying lesions. In this case report, the patient presented with persistent intraoral pain and numbness on the right side of the face. Cranial nerve examination revealed dysfunctional eye movements, diplopia, and mechanical hyposensitivity in the mandibular region. The patient was diagnosed with neuropathy due to intracranial lesions and referred to the Department of Neurosurgery and Otorhinolaryngology. The patient was suspected of having malignant lymphoma and is currently undergoing neurosurgical intervention. This article discusses the importance of the examination of the cranial nerve for patients with persistent pain in the trigeminal nerve distribution.

Pathological mutations promote proteolysis of mitochondrial tRNA-specific 2-thiouridylase 1 (MTU1) via mitochondrial caseinolytic peptidase (CLPP).

MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.

Involvement of interferon gamma signaling in spinal trigeminal caudal subnucleus astrocyte in orofacial neuropathic pain in rats with infraorbital nerve injury.

Background: Trigeminal nerve injury causes orofacial pain that can interfere with activities of daily life. However, the underlying mechanism remains unknown, and the appropriate treatment has not been established yet. This study aimed to examine the involvement of interferon gamma (IFN-γ) signaling in the spinal trigeminal caudal subnucleus (Vc) in orofacial neuropathic pain. Methods: Infraorbital nerve (ION) injury (IONI) was performed in rats by partial ION ligation. The head-withdrawal reflex threshold (HWT) to mechanical stimulation of the whisker pad skin was measured in IONI or sham rats, as well as following a continuous intracisterna magna administration of IFN-γ and a mixture of IFN-γ and fluorocitrate (inhibitor of astrocytes activation) in naïve rats, or an IFN-γ antagonist in IONI rats. The IFN-γ receptor immunohistochemistry and IFN-γ Western blotting were analyzed in the Vc after IONI or sham treatment. The glial fibrillary acid protein (GFAP) immunohistochemistry and Western blotting were also analyzed after administration of IFN-γ and the mixture of IFN-γ and fluorocitrate. Moreover, the change in single neuronal activity in the Vc was examined in the IONI, sham, and IONI group administered IFN-γ antagonist. Results: The HWT decreased after IONI. The IFN-γ and IFN-γ receptor were upregulated after IONI, and the IFN-γ receptor was expressed in Vc astrocytes. IFN-γ administration decreased the HWT, whereas the mixture of IFN-γ and fluorocitrate recovered the decrement of HWT. IFN-γ administration upregulated GFAP expression, while the mixture of IFN-γ and fluorocitrate recovered the upregulation of GFAP expression. IONI significantly enhanced the neuronal activity of the mechanical-evoked responses, and administration of an IFN-γ antagonist significantly inhibited these enhancements. Conclusions: IFN-γ signaling through the receptor in astrocytes is a key mechanism underlying orofacial neuropathic pain associated with trigeminal nerve injury. These findings will aid in the development of therapeutics for orofacial neuropathic pain.

Antibiofilm Property and Biocompatibility of Siloxane-Based Polymer Coatings Applied to Biomaterials.

Biofilm infections sometimes occur on biomaterials inserted into the body because biomaterials can block the attack of immune cells such as macrophages, promoting biofilm formation by invading bacteria. Owing to their use in antifouling applications, including biofilm formation, siloxane-based polymer coatings are considered a promising method to prevent biofilm formation on the surface of biomaterials. In this study, we explored the antibiofilm property and biocompatibility of siloxane-based polymer coatings. Biofilm formation and cytotoxicity tests were performed using Escherichia coli and Staphylococcus epidermidis to quantify the biofilms while U937 cells were used to measure the time course of viable cell concentration and viability, respectively. In both the biofilm formation and cytotoxicity tests, stainless steel SUS316L plates and titanium plates coated with the siloxane-based polymer and sterilized in an autoclave were used as the biomaterials. The amount of biofilm formed on the polymer-coated titanium plate was substantially higher than that on a noncoated titanium plate in the case of S. epidermidis. The viable cell concentration and viability of U937 cultured on the polymer-coated titanium plate were lower than those of U937 cultured on the noncoated titanium plate. The same trend was observed between polymer-coated and noncoated SUS316L plates. These results indicate that the siloxane-based polymer coatings need additional treatment to achieve a satisfactory antibiofilm property and that they are sensitive to autoclave treatment, resulting in cytotoxicity.

AC Electromagnetic Field Controls the Biofilms on the Glass Surface by Escherichia coli & Staphylococcus epidermidis Inhibition Effect.

Biofilms, mainly comprised of bacteria, form on materials' surfaces due to bacterial activity. They are generally composed of water, extracellular polymeric substances (polysaccharides, proteins, nucleic acids, and lipids), and bacteria. Some bacteria that form biofilms cause periodontal disease, corrosion of the metal materials that make up drains, and slippage. Inside of a biofilm is an environment conducive to the growth and propagation of bacteria. Problems with biofilms include the inability of disinfectants and antibiotics to act on them. Therefore, we have investigated the potential application of alternating electromagnetic fields for biofilm control. We obtained exciting results using various materials' specimens and frequency conditions. Through these studies, we gradually understood that the combination of the type of bacteria, the kind of material, and the application of an electromagnetic field with various low frequencies (4 kHz-12 kHz) changes the circumstances of the onset of the biofilm suppression effect. In this study, relatively high frequencies (20 and 30 kHz) were applied to biofilms caused by Escherichia coli (E. coli) and Staphylococcus epidermidis (S. epidermidis), and quantitative evaluation was performed using staining methods. The sample surfaces were analyzed by Raman spectroscopy using a Laser Raman spectrometer to confirm the presence of biofilms on the surface.

Activation of the urotensin-II receptor by remdesivir induces cardiomyocyte dysfunction.

Remdesivir is an antiviral drug used for COVID-19 treatment worldwide. Cardiovascular side effects have been associated with remdesivir; however, the underlying molecular mechanism remains unknown. Here, we performed a large-scale G-protein-coupled receptor screening in combination with structural modeling and found that remdesivir is a selective, partial agonist for urotensin-II receptor (UTS2R) through the Gαi/o-dependent AKT/ERK axis. Functionally, remdesivir treatment induced prolonged field potential and APD90 in human induced pluripotent stem cell (iPS)-derived cardiomyocytes and impaired contractility in both neonatal and adult cardiomyocytes, all of which mirror the clinical pathology. Importantly, remdesivir-mediated cardiac malfunctions were effectively attenuated by antagonizing UTS2R signaling. Finally, we characterized the effect of 110 single-nucleotide variants in UTS2R gene reported in genome database and found four missense variants that show gain-of-function effects in the receptor sensitivity to remdesivir. Collectively, our study illuminates a previously unknown mechanism underlying remdesivir-related cardiovascular events and that genetic variations of UTS2R gene can be a potential risk factor for cardiovascular events during remdesivir treatment, which collectively paves the way for a therapeutic opportunity to prevent such events in the future.

Post-Traumatic Trigeminal Neuropathic Pain after Dental Implant Surgery and the Injustice Experience Questionnaire.

Painful post-traumatic trigeminal neuropathy (PTTN) is a known complication of dental implant therapy. Patients with PTTN develop sensory abnormalities in the orofacial region, which may be a psychosocial aspect, and dentists should assess somatosensory testing and psychosocial factors. The patients were assessed using quantitative sensory testing (QST). A 64-year-old female presented with allodynia of the left lower lip that occurred after a surgical implant procedure. Persistent pain started 4 months after the placement of two dental implants in the mandible. Sensory testing of these areas revealed warm hyposensitivity and mechanical hypersensitivity of the mandibular region. We also assessed PTTN-related perceived injustice using the Injustice Experience Questionnaire. The patient refused medication therapy such as pregabalin; therefore, autogenic training was adopted as an alternative management strategy. We conclude that for expensive dental procedures, such as implant placement, sufficient consensus should be obtained preoperatively before proceeding with surgery.

Quantitative Analyses of Biofilm by Using Crystal Violet Staining and Optical Reflection.

Biofilms have caused many problems, not only in the industrial fields, but also in our daily lives. Therefore, it is important for us to control them by evaluating them properly. There are many instrumental analytical methods available for evaluating formed biofilm qualitatively. These methods include the use of Raman spectroscopy and various microscopes (optical microscopes, confocal laser microscopes, scanning electron microscopes, transmission electron microscopes, atomic force microscopes, etc.). On the other hand, there are some biological methods, such as staining, gene analyses, etc. From the practical viewpoint, staining methods seem to be the best due to various reasons. Therefore, we focused on the staining method that used a crystal violet solution. In the previous study, we devised an evaluation process for biofilms using a color meter to analyze the various staining situations. However, this method was complicated and expensive for practical engineers. For this experiment, we investigated the process of using regular photos that were quantified without any instruments except for digitized cameras. Digitized cameras were used to compare the results. As a result, we confirmed that the absolute values were different for both cases, respectively. However, the tendency of changes was the same. Therefore, we plan to utilize the changes before and after biofilm formation as indicators for the future.

First Isolation of Neoscytalidium dimidiatum from Human Dermatomycosis in Japan.

Neoscytalidium dimidiatum is a common fungus that causes non-dermatophyte dermatomycosis in tropical regions, but there have been no reports of infection with N. dimidiatum in Japan. Here, we report the first isolation of N. dimidiatum from human dermatomycosis in Japan. A 62-year-old healthy Japanese male had been treated with oral terbinafine for tinea pedis diagnosed from a microscopic examination in 2003 with a lesion that was intractable. In 2020, re-identification by sequencing the internal transcribed spacer regions and the D1/D2 domain of the large-subunit (LSU) ribosomal RNA gene revealed that the pathogen was N. dimidiatum. Antifungal susceptibility tests showed that the minimum inhibitory concentration of the drug luliconazole (LLCZ) against the pathogen was 0.00049 µg/mL. The patient's lesions were cured by topical LLCZ. The clinical course and drug susceptibility suggest that LLCZ is a suitable first-line drug for treatment.

Proposal for Some Affordable Laboratory Biofilm Reactors and Their Critical Evaluations from Practical Viewpoints.

Biofilms are a result of bacterial activities and are found everywhere. They often form on metal surfaces and on the surfaces of polymeric compounds. Biofilms are sticky and mostly consist of water. They have a strong resistance to antimicrobial agents and can cause serious problems for modern medicine and industry. Biofilms are composed of extracellular polymeric substances (EPS) such as polysaccharides produced from bacterial cells and are dominated by water at the initial stage. In a series of experiments, using Escherichia coli, we developed three types of laboratory biofilm reactors (LBR) to simulate biofilm formation. For the first trial, we used a rotary type of biofilm reactor for stirring. For the next trial, we tried another rotary type of reactor where the circular plate holding specimens was rotated. Finally, a circular laboratory biofilm reactor was used. Biofilms were evaluated by using a crystal violet staining method and by using Raman spectroscopy. Additionally, they were compared to each other from the practical (industrial) viewpoints. The third type was the best to form biofilms in a short period. However, the first and second were better from the viewpoint of "ease of use". All of these have their own advantages and disadvantages, respectively. Therefore, they should be properly selected and used for specific and appropriate purposes in the future.

Impedance Characteristics of Monolayer and Bilayer Graphene Films with Biofilm Formation and Growth.

Biofilms are the result of bacterial activity. When the number of bacteria (attached to materials' surfaces) reaches a certain threshold value, then the bacteria simultaneously excrete organic polymers (EPS: extracellular polymeric substances). These sticky polymers encase and protect the bacteria. They are called biofilms and contain about 80% water. Other components of biofilm include polymeric carbon compounds such as polysaccharides and bacteria. It is well-known that biofilms cause various medical and hygiene problems. Therefore, it is important to have a sensor that can detect biofilms to solve such problems. Graphene is a single-atom-thick sheet in which carbon atoms are connected in a hexagonal shape like a honeycomb. Carbon compounds generally bond easily to graphene. Therefore, it is highly possible that graphene could serve as a sensor to monitor biofilm formation and growth. In our previous study, monolayer graphene was prepared on a glass substrate by the chemical vapor deposition (CVD) method. Its biofilm forming ability was compared with that of graphite. As a result, the CVD graphene film had the higher sensitivity for biofilm formation. However, the monolayer graphene has a mechanical disadvantage when used as a biofilm sensor. Therefore, for this new research project, we prepared bilayer graphene with high mechanical strength by using the CVD process on copper substrates. For these specimens, we measured the capacitance component of the specimens' impedance. In addition, we have included a discussion about the possibility of applying them as future sensors for monitoring biofilm formation and growth.

Simple Methods for Evaluating Acid Permeation and Biofilm Formation Behaviors on Polysiloxane Films.

The sulfuric acid permeation and biofilm formation behaviors of polysiloxane films have been investigated, and simple methods for evaluating the sulfuric acid permeation and biofilm formation behaviors have been proposed in this paper. The polysiloxane films used in these experiments were practically impermeable to the aqueous sulfuric acid solution, and the amount of biofilm formation varied depending on the composition of the films. Further, the amount of sulfuric acid permeation can be estimated by measuring the polarization curves of polysiloxane films with different thicknesses formed on iron electrodes. By measuring the adhesion work of pure water and simulated biofilm droplets on polysiloxane films of different compositions, we can estimate the resistance of biofilm formation on the polysiloxane films.

Protocol for preparation and measurement of intracellular and extracellular modified RNA using liquid chromatography-mass spectrometry.

About 150 modifications have been identified in RNA species. Besides their regulatory roles in the intracellular gene expression, abundant modified RNA nucleosides are catabolized from RNA and released into extracellular fluids, which can impact extracellular signaling as ligands for receptors. Here, we describe a protocol to prepare samples from biological specimens, including cultured cells, extracellular fluid, and tissues, to measure both intracellular and extracellular RNA modifications using mass spectrometry. For complete details on the use and execution of this protocol, please refer to Ogawa et al. (2021).

Potential roles of the IL-6 family in conjunctival fibrosis.

Elevated intraocular pressure (IOP) is a significant risk factor for vision loss due to glaucoma, which is a major cause of blindness worldwide. Glaucoma filtration surgery (GFS) is an important method to reduce IOP by guidance of aqueous humor into a newly built filtration bleb in the conjunctiva; management of the wound healing mechanism is essential for the success of GFS. Here, we investigated the roles of interleukin (IL)-6 family members during the wound healing process after GFS. At the surgical site, the expression levels of genes encoding IL-6, oncostatin M (OSM), their receptors, and collagen I were elevated at 3 h after GFS, whereas the levels of genes encoding transforming growth factor (TGF)-ß, α-smooth muscle actin (SMA), type IV collagen, and fibronectin were elevated at 3 days after GFS. IL-6 trans-signaling and OSM signaling suppressed TGF-ß-induced expression of α-SMA and collagen IV, as well as activation of the non-canonical TGF-ß pathway, suggesting that IL-6 and OSM may aid in controlling the phase transition from inflammation to proliferation and remodeling. The suppressive effects of OSM were accompanied by STAT3 activation, such that STAT1 function was complementary to STAT3. Taken together, these observations indicated that IL-6 family members constitute early response genes after GFS, which can suppress TGF-ß-induced expression of late response genes at the surgical site after GFS.

Orofacial Neuropathic Pain-Basic Research and Their Clinical Relevancies.

Trigeminal nerve injury is known to cause severe persistent pain in the orofacial region. This pain is difficult to diagnose and treat. Recently, many animal studies have reported that rewiring of the peripheral and central nervous systems, non-neuronal cell activation, and up- and down-regulation of various molecules in non-neuronal cells are involved in the development of this pain following trigeminal nerve injury. However, there are many unknown mechanisms underlying the persistent orofacial pain associated with trigeminal nerve injury. In this review, we address recent animal data regarding the involvement of various molecules in the communication of neuronal and non-neuronal cells and examine the possible involvement of ascending pathways in processing pathological orofacial pain. We also address the clinical observations of persistent orofacial pain associated with trigeminal nerve injury and clinical approaches to their diagnosis and treatment.

N6-methyladenosine (m6A) is an endogenous A3 adenosine receptor ligand.

About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.

Microglia-Astrocyte Communication via C1q Contributes to Orofacial Neuropathic Pain Associated with Infraorbital Nerve Injury.

Trigeminal nerve injury causes a distinct time window of glial activation in the trigeminal spinal subnucleus caudalis (Vc), which are involved in the initiation and maintenance phases of orofacial neuropathic pain. Microglia-derived factors enable the activation of astrocytes. The complement component C1q, which promotes the activation of astrocytes, is known to be synthesized in microglia. However, it is unclear whether microglia-astrocyte communication via C1q is involved in orofacial neuropathic pain. Here, we analyzed microglia-astrocyte communication in a rat model with infraorbital nerve injury (IONI). The orofacial mechanical hypersensitivity induced by IONI was significantly attenuated by preemptive treatment with minocycline. Immunohistochemical analyses revealed that minocycline inhibited the increase in c-Fos immune-reactive (IR) cells and the fluorescence intensity of both Iba1 and glial fibrillary acidic protein (GFAP) in the Vc following IONI. Intracisternal administration of C1q caused orofacial mechanical hypersensitivity and an increase in the number of c-Fos-IR cells and fluorescence intensity of GFAP. C1q-induced orofacial mechanical hypersensitivity was completely abrogated by intracisternal administration of fluorocitrate. The present findings suggest that the enhancement in the excitability of Vc nociceptive neurons is produced by astrocytic activation via the signaling of C1q released from activated microglia in the Vc following IONI, resulting in persistent orofacial neuropathic pain.

Investigation of Biofilms Formed on Steelmaking Slags in Marine Environments for Water Depuration.

Steelmaking slags are a promising resource as artificial seaweed beds for the reconstitution of marine environments. To grow seaweed well, the formation of biofilms is an essential process in biofouling. This study focused on the formation of initial biofilms on steelmaking slag samples and analyzed the resulting bacterial communities using the next-generation sequencing technique. Three types of steelmaking slag were submerged in an area of Ise Bay in Mie Prefecture, Japan, for 3 and 7 days in the summer and winter seasons to allow the formation of biofilms. The bacterial communities of these biofilms were richer in sulfur-oxidizing bacteria compared to the biofilms formed on polyurethane sponges. It was found that Helicobacteraceae dominantly grew on the biofilms formed on the slag samples. This shows that steelmaking slags have potential to be used as artificial seaweed beds and marine water purifiers.

Analgesic effect of gum chewing in patients with burning mouth syndrome.

The cause of burning mouth syndrome (BMS) is unknown. Although no effective treatment has been established, BMS patients frequently chew gum to alleviate pain. To identify the cause and new treatments for BMS, this study investigated the psychophysical and pharmacological properties of gum chewing to better understand its pain-relieving effects. In this prospective, blinded study, plasma catecholamine and serotonin levels and Profile of Mood States (POMS) scores were assessed after gum chewing or simulated chewing in 40 women (20 BMS patients and 20 age-matched controls). Visual analogue scale (VAS) scores for pain decreased significantly in BMS patients after gum chewing and simulated chewing. Moreover, resting VAS scores of BMS patients were significantly positively correlated with plasma adrenaline level. Furthermore, gum chewing was significantly correlated with lower plasma adrenaline level, VAS score, and tension-anxiety score. These results suggest that adrenaline is important in the pathogenesis of BMS pain and that the analgesic effect of gum chewing is induced through the potential effects of anxiety reduction, although this effect might not be specific to BMS. In addition, the analgesic effect of gum chewing was not induced solely by chewing motion.