Proposal of Helicobacter higonensis sp. nov. isolated from a human clinical specimen, and emended description of Helicobacter valdiviensis Collado, 2014.

We have previously isolated a gram-negative microaerophilic strain, PAGU2000T from a patient presenting with a fever in Kumamoto Prefecture, Japan. The present study aimed to comprehensively analyze the taxonomy of the isolated strain using a polyphasic approach. The 16S rRNA gene sequence analysis indicated that the strain was a member of enterohepatic Helicobacter. The strain PAGU2000T shared a 97.5% 16S rRNA gene nucleotide identity with Helicobacter valdiviensis, and this taxonomic position was confirmed by phylogenetic analysis of the GyrA amino acid sequences. The proposed strain PAGU2000T has a 1.482 Mbp chromosome with a DNA G + C content of 31.3 mol% and encodes 1520 predicted coding sequences. The average nucleotide identity between the strain PAGU2000T and type strain of H. valdiviensis was 70.3%, which was lower than the recommended threshold of 95% for species delineation. The strain PAGU2000T was a motile, non-spore-forming, and spiral-shaped bacterium, exhibiting catalase and oxidase activities but not urease and nitrate reduction. This study demonstrates that the isolate represents a novel species within enterohepatic Helicobacter, for which the name Helicobacter higonensis is proposed (type strain: PAGU2000T = GTC 16811T = LMG 33095T). In this study, we describe the phenotypic and morphological features of this strain and propose an emended description of some biochemical traits of H. valdiviensis.

Thioglucose-derived tetrasulfide, a unique polysulfide model compound.

Polysulfides have received increased interest in redox biology due to their role as the precursors of H2S and persulfides. However, the compounds that are suitable for biological investigations are limited to cysteine- and glutathione-derived polysulfides. In this work, we report the preparation and evaluation of a novel polysulfide derived from thioglucose, which represents the first carbohydrate-based polysulfide. This compound, thioglucose tetrasulfide (TGS4), showed excellent stability and water solubility. H2S and persulfide production from TGS4, as well as its associated antioxidative property were also demonstrated. Additionally, TGS4 was demonstrated to significantly induce cellular sulfane sulfur level increase, in particular for the formation of hydropersulfides/trisulfides. These results suggest that TGS4 is a useful tool for polysulfide research.

Regulation of innate immune and inflammatory responses by supersulfides.

Innate immunity plays an important role in host defense against microbial infections. It also participates in activation of acquired immunity through cytokine production and antigen presentation. Pattern recognition receptors such as Toll-like receptors and nucleotide oligomerization domain-like receptors sense invading pathogens and associated tissue injury, after which inflammatory mediators such as pro-inflammatory cytokines and nitric oxide are induced. Supersulfides are molecular species possessing catenated sulfur atoms such as persulfide and polysulfide moieties. They have recently been recognized as important regulators in cellular redox homeostasis by acting as potent antioxidants and nucleophiles. In addition, recent studies suggested that supersulfides are critically involved in the regulation of innate immune and inflammatory responses. In this review, we summarize current knowledge of the chemistry and biology of supersulfides, with particular attention to their roles in regulation of innate immune, and inflammatory responses. Studies with animal models of infection and inflammation demonstrated the potent anti-inflammatory functions of supersulfides such as blocking pro-inflammatory signaling cascades, reducing oxidative stresses, and inhibiting replication of microbial pathogens including severe acute respiratory syndrome coronavirus 2. Precise understanding of how supersulfides regulate innate immune responses is the necessary requirement for developing supersulfide-based diagnostic as well as therapeutic strategies against inflammatory disorders.

[Redox Biology Involved in the Toxicity of Enterohemorrhagic Escherichia coli Toxin SubAB].

AB5 toxins of pathogenic bacteria enter host cells and utilize the retrograde trafficking pathway to translocate to the cytoplasm and exert its pathogenesis. Cholera toxin and Shiga toxin reach the endoplasmic reticulum (ER), and the A subunit undergoes redox regulation by ER proteins to become active fragments, which pass through the ER membrane and translocate to the cytoplasm. By acting on molecular targets in the cytoplasm, the normal function of host cells are disrupted, causing diseases. ER chaperone proteins such as protein disulfide isomerase (PDI) and binding immunoglobulin protein (BiP) induce conformational changes triggered by the reduction of disulfide bonds in the A subunit. This is thought to be dependent on cysteine thiol-mediated redox regulation, but the detailed mechanism remains unclear. On the other hand, subtilase cytotoxin (SubAB), produced by enterohemorrhagic Escherichia coli (EHEC), localizes to the ER without translocating to the cytoplasm and cleaves BiP as a substrate. Therefore, it is thought that ER stress-based cytotoxicity and intestinal bleeding occur without translocating to the cytoplasm. We reported that PDI is involved in BiP cleavage through SubAB localization to the ER. Like other AB5 toxins, this indicates the involvement of redox regulation via chaperone proteins in the ER, but also suggests that SubAB does not translocate to the cytoplasm because it cleaves BiP. Although there are few reports on the redox state of ER protein thiols, it is suggested that polysulfidation, which is discussed in this symposium, may be involved.

Acute Kidney Injury Caused by Rhabdomyolysis Is Ameliorated by Serum Albumin-Based Supersulfide Donors through Antioxidative Pathways.

Oxidative stress is responsible for the onset and progression of various kinds of diseases including rhabdomyolysis-induced acute kidney injury (AKI). Antioxidants are, therefore, thought to aid in the recovery of illnesses linked to oxidative stress. Supersulfide species have been shown to have substantial antioxidative activity; however, due to their limited bioavailability, few supersulfide donors have had their actions evaluated in vivo. In this study, human serum albumin (HSA) and N-acetyl-L-cysteine polysulfides (NACSn), which have polysulfides in an oxidized form, were conjugated to create a supersulfide donor. HSA is chosen to be a carrier of NACSn because of its extended blood circulation and high level of biocompatibility. In contrast to a supersulfide donor containing reduced polysulfide in HSA, the NACSn-conjugated HSAs exhibited stronger antioxidant activity than HSA and free NACSn without being uptaken by the cells in vitro. The supersulfide donor reduced the levels of blood urea nitrogen and serum creatinine significantly in a mouse model of rhabdomyolysis-induced AKI. Supersulfide donors significantly reduced the expression of oxidative stress markers in the kidney. These results indicate that the developed supersulfide donor has the therapeutic effect on rhabdomyolysis-induced AKI.

Differential Ability of Spike Protein of SARS-CoV-2 Variants to Downregulate ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19) and employs angiotensin-converting enzyme 2 (ACE2) as the receptor. Although the expression of ACE2 is crucial for cellular entry, we found that the interaction between ACE2 and the Spike (S) protein in the same cells led to its downregulation through degradation in the lysosomal compartment via the endocytic pathway. Interestingly, the ability of the S protein from previous variants of concern (VOCs) to downregulate ACE2 was variant-dependent and correlated with disease severity. The S protein from the Omicron variant, associated with milder disease, exhibited a lower capacity to downregulate ACE2 than that of the Delta variant, which is linked to a higher risk of hospitalization. Chimeric studies between the S proteins from the Delta and Omicron variants revealed that both the receptor-binding domain (RBD) and the S2 subunit played crucial roles in the reduced ACE2 downregulation activity observed in the Omicron variant. In contrast, three mutations (L452R/P681R/D950N) located in the RBD, S1/S2 cleavage site, and HR1 domain were identified as essential for the higher ACE2 downregulation activity observed in the Delta variant compared to that in the other VOCs. Our results suggested that dysregulation of the renin-angiotensin system due to the ACE2 downregulation activity of the S protein of SARS-CoV-2 may play a key role in the pathogenesis of COVID-19.

Redox Regulation of Xenobiotics by Reactive Sulfur and Supersulfide Species.

Significance: Routine exposure to xenobiotics is unavoidable during our lifetimes. Certain xenobiotics are hazardous to human health, and are metabolized in the body to render them less toxic. During this process, several detoxification enzymes cooperatively metabolize xenobiotics. Glutathione (GSH) conjugation plays an important role in the metabolism of electrophilic xenobiotics. Recent Advances: Recent advances in reactive sulfur and supersulfide (RSS) analyses showed that persulfides and polysulfides bound to low-molecular-weight thiols, such as GSH, and to protein thiols are abundant in both eukaryotes and prokaryotes. The highly nucleophilic nature of hydropersulfides and hydropolysulfides contributes to cell protection against oxidative stress and electrophilic stress. Critical Issues: In contrast to GSH conjugation to electrophiles that is aided by glutathione S-transferase (GST), persulfides and polysulfides can directly form conjugates with electrophiles without the catalytic actions of GST. The polysulfur bonds in the conjugates are further reduced by perthioanions and polythioanions derived from RSS to form sulfhydrated metabolites that are no longer electrophilic but rather nucleophilic, and differ from metabolites that are formed via GSH conjugation. Future Directions: In view of the abundance of RSS in cells and tissues, metabolism of xenobiotics that is mediated by RSS warrants additional investigations, such as studies of the impact of microbiota-derived RSS on xenobiotic metabolism. Metabolites formed from reactions between electrophiles and RSS may be potential biomarkers for monitoring exposure to electrophiles and for studying their metabolism by RSS. Antioxid. Redox Signal. 40, 679-690.

Understanding impacts of COVID-19 restrictions on glycemic control for patients with diabetes in Japan.

Objective: This study evaluated the changes in the status of glycemic control and lipid management in patients with diabetes under COVID-19 containment restrictions, in order to better understand the impacts of events causing lifestyle restrictions. Patient characteristics with worsened glycemic control were also assessed. Methods: We conducted a retrospective and observational cohort study using the electronic health records of 5,169 patients with diabetes seeking medical care in two healthcare centers. Laboratory test results including glycemic and lipid goal attainment rates were compared between pre-COVID-19 (January to December 2019) and the first wave of COVID-19 (February to June 2020). Multiple regression models were used to evaluate the association between glycated hemoglobin (HbA1c) at baseline and during the first wave with covariates such as concomitant medications and comorbidities. Results: The HbA1c goal achievement rate improved significantly from 39.0% to 43.1% (p < 0.0001) overall, and more patients reached their glycemic target during COVID-19 restrictions. No significant changes were observed in lipid control. An indexed change in HbA1c level showed that glycemic control improved in 2,230 patients and worsened in 1,619 patients. Administration of insulin, GLP-1, and sulfonylureas were each identified as factors correlated with elevated HbA1c, during the first wave of COVID-19. Conclusion: Although the glycemic control in patients with diabetes improved overall under COVID-19 restrictions, those on insulin, GLP-1, or sulfonylureas worsened. These findings suggest the need to better understand what drives differences in glycemic control to better support people with diabetes for future epidemiological outbreaks. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-023-01302-5.

Turkish Plants, Including Quercetin and Oenothein B, Inhibit the HIV-1 Release and Accelerate Cell Apoptosis.

The introduction of combined anti-retroviral therapy (cART) in 1996, along with a continual breakthrough in anti-human immunodeficiency virus-1 (HIV-1) drugs, has improved the life expectancies of HIV-1-infected individuals. However, the incidence of drug-resistant viruses between individuals undergoing cART and treatment-naïve individuals is a common challenge. Therefore, there is a requirement to explore potential drug targets by considering various stages of the viral life cycle. For instance, the late stage, or viral release stage, remains uninvestigated extensively in antiviral drug discovery. In this study, we prepared a natural plant library and selected candidate plant extracts that inhibited HIV-1 release based on our laboratory-established screening system. The plant extracts from Epilobium hirsutum L. and Chamerion angustifolium (L.) Holub, belonging to the family Onagraceae, decreased HIV-1 release and accelerated the apoptosis in HIV-1-infected T cells but not uninfected T cells. A flavonol glycoside quercetin with oenothein B in Onagraceae reduced HIV-1 release in HIV-1-infected T cells. Moreover, extracts from Chamerion angustifolium (L.) Holub and Senna alexandrina Mill. inhibited the infectivity of progeny viruses. Together, these results suggest that C. angustifolium (L.) Holub contains quercetin with oenothein B that synergistically blocks viral replication and kills infected cells via an apoptotic pathway. Consequently, the plant extracts from the plant library of Turkey might be suitable candidates for developing novel anti-retroviral drugs that target the late phase of the HIV-1 life cycle.

Interaction of TSG101 with the PTAP Motif in Distinct Locations of Gag Determines the Incorporation of HTLV-1 Env into the Retroviral Virion.

Human T-cell tropic virus type 1 (HTLV-1) is known to be mainly transmitted by cell-to-cell contact due to the lower infectivity of the cell-free virion. However, the reasons why cell-free HTLV-1 infection is poor remain unknown. In this study, we found that the retrovirus pseudotyped with HTLV-1 viral envelope glycoprotein (Env) was infectious when human immunodeficiency virus type 1 (HIV-1) was used to produce the virus. We found that the incorporation of HTLV-1 Env into virus-like particles (VLPs) was low when HTLV-1 Gag was used to produce VLPs, whereas VLPs produced using HIV-1 Gag efficiently incorporated HTLV-1 Env. The production of VLPs using Gag chimeras between HTLV-1 and HIV-1 Gag and deletion mutants of HIV-1 Gag showed that the p6 domain of HIV-1 Gag was responsible for the efficient incorporation of HTLV-1 Env into the VLPs. Further mutagenic analyses of the p6 domain of HIV-1 Gag revealed that the PTAP motif in the p6 domain of HIV-1 Gag facilitates the incorporation of HTLV-1 Env into VLPs. Since the PTAP motif is known to interact with tumor susceptibility gene 101 (TSG101) during the budding process, we evaluated the effect of TSG101 knockdown on the incorporation of HTLV-1 Env into VLPs. We found that TSG101 knockdown suppressed the incorporation of HTLV-1 Env into VLPs and decreased the infectivity of cell-free HIV-1 pseudotyped with HTLV-1 Env. Our results suggest that the interaction of TSG101 with the PTAP motif of the retroviral L domain is involved not only in the budding process but also in the efficient incorporation of HTLV-1 Env into the cell-free virus.

Alkyl gallates inhibit serine O-acetyltransferase in bacteria and enhance susceptibility of drug-resistant Gram-negative bacteria to antibiotics.

A principal concept in developing antibacterial agents with selective toxicity is blocking metabolic pathways that are critical for bacterial growth but that mammalian cells lack. Serine O-acetyltransferase (CysE) is an enzyme in many bacteria that catalyzes the first step in l-cysteine biosynthesis by transferring an acetyl group from acetyl coenzyme A (acetyl-CoA) to l-serine to form O-acetylserine. Because mammalian cells lack this l-cysteine biosynthesis pathway, developing an inhibitor of CysE has been thought to be a way to establish a new class of antibacterial agents. Here, we demonstrated that alkyl gallates such as octyl gallate (OGA) could act as potent CysE inhibitors in vitro and in bacteria. Mass spectrometry analyses indicated that OGA treatment markedly reduced intrabacterial levels of l-cysteine and its metabolites including glutathione and glutathione persulfide in Escherichia coli to a level similar to that found in E. coli lacking the cysE gene. Consistent with the reduction of those antioxidant molecules in bacteria, E. coli became vulnerable to hydrogen peroxide-mediated bacterial killing in the presence of OGA. More important, OGA treatment intensified susceptibilities of metallo-ß-lactamase-expressing Gram-negative bacteria (E. coli and Klebsiella pneumoniae) to carbapenem. Structural analyses showed that alkyl gallate bound to the binding site for acetyl-CoA that limits access of acetyl-CoA to the active site. Our data thus suggest that CysE inhibitors may be used to treat infectious diseases caused by drug-resistant Gram-negative bacteria not only via direct antibacterial activity but also by enhancing therapeutic potentials of existing antibiotics.

Supersulfide biology and translational medicine for disease control.

For decades, the major focus of redox biology has been oxygen, the most abundant element on Earth. Molecular oxygen functions as the final electron acceptor in the mitochondrial respiratory chain, contributing to energy production in aerobic organisms. In addition, oxygen-derived reactive oxygen species including hydrogen peroxide and nitrogen free radicals, such as superoxide, hydroxyl radical and nitric oxide radical, undergo a complicated sequence of electron transfer reactions with other biomolecules, which lead to their modified physiological functions and diverse biological and pathophysiological consequences (e.g. oxidative stress). What is now evident is that oxygen accounts for only a small number of redox reactions in organisms and knowledge of biological redox reactions is still quite limited. This article reviews a new aspects of redox biology which is governed by redox-active sulfur-containing molecules-supersulfides. We define the term 'supersulfides' as sulfur species with catenated sulfur atoms. Supersulfides were determined to be abundant in all organisms, but their redox biological properties have remained largely unexplored. In fact, the unique chemical properties of supersulfides permit them to be readily ionized or radicalized, thereby allowing supersulfides to actively participate in redox reactions and antioxidant responses in cells. Accumulating evidence has demonstrated that supersulfides are indispensable for fundamental biological processes such as energy production, nucleic acid metabolism, protein translation and others. Moreover, manipulation of supersulfide levels was beneficial for pathogenesis of various diseases. Thus, supersulfide biology has opened a new era of disease control that includes potential applications to clinical diagnosis, prevention and therapeutics of diseases.

Sulfur metabolic response in macrophage limits excessive inflammatory response by creating a negative feedback loop.

The excessive inflammatory response of macrophages plays a vital role in the pathogenesis of various diseases. The dynamic metabolic alterations in macrophages, including amino acid metabolism, are known to orchestrate their inflammatory phenotype. To explore a new metabolic pathway that regulates the inflammatory response, we examined metabolome changes in mouse peritoneal macrophages (PMs) in response to lipopolysaccharide (LPS) and found a coordinated increase of cysteine and its related metabolites, suggesting an enhanced demand for cysteine during the inflammatory response. Because Slc7a11, which encodes a cystine transporter xCT, was remarkably upregulated upon the pro-inflammatory challenge and found to serve as a major channel of cysteine supply, we examined the inflammatory behavior of Slc7a11 knockout PMs (xCT-KO PMs) to clarify an impact of the increased cysteine demand on inflammation. The xCT-KO PMs exhibited a prolonged upregulation of pro-inflammatory genes, which was recapitulated by cystine depletion in the culture media of wild-type PMs, suggesting that cysteine facilitates the resolution of inflammation. Detailed analysis of the sulfur metabolome revealed that supersulfides, such as cysteine persulfide, were increased in PMs in response to LPS, which was abolished in xCT-KO PMs. Supplementation of N-acetylcysteine tetrasulfide (NAC-S2), a supersulfide donor, attenuated the pro-inflammatory gene expression in xCT-KO PMs. Thus, activated macrophages increase cystine uptake via xCT and produce supersulfides, creating a negative feedback loop to limit excessive inflammation. Our study highlights the finely tuned regulation of macrophage inflammatory response by sulfur metabolism.

Supersulphides provide airway protection in viral and chronic lung diseases.

Supersulphides are inorganic and organic sulphides with sulphur catenation with diverse physiological functions. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulphide synthase (CPERS). Here, we identify protective functions of supersulphides in viral airway infections (influenza and COVID-19), in aged lungs and in chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF). We develop a method for breath supersulphur-omics and demonstrate that levels of exhaled supersulphides increase in people with COVID-19 infection and in a hamster model of SARS-CoV-2 infection. Lung damage and subsequent lethality that result from oxidative stress and inflammation in mouse models of COPD, IPF, and ageing were mitigated by endogenous supersulphides production by CARS2/CPERS or exogenous administration of the supersulphide donor glutathione trisulphide. We revealed a protective role of supersulphides in airways with various viral or chronic insults and demonstrated the potential of targeting supersulphides in lung disease.

Cystathionine γ-Lyase Self-Inactivates by Polysulfidation during Cystine Metabolism.

Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. It also has ß-lyase activity toward cystine, generating cysteine persulfide (Cys-SSH). The chemical reactivity of Cys-SSH is thought to be involved in the catalytic activity of particular proteins via protein polysulfidation, the formation of -S-(S)n-H on their reactive cysteine residues. The Cys136/171 residues of CSE have been proposed to be redox-sensitive residues. Herein, we investigated whether CSE polysulfidation occurs at Cys136/171 during cystine metabolism. Transfection of wild-type CSE into COS-7 cells resulted in increased intracellular Cys-SSH production, which was significantly increased when Cys136Val or Cys136/171Val CSE mutants were transfected, instead of the wild-type enzyme. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CSE polysulfidation occurs at Cys136 during cystine metabolism. In vitro incubation of CSE with CSE-enzymatically synthesized Cys-SSH resulted in the inhibition of Cys-SSH production. In contrast, the mutant CSEs (Cys136Val and Cys136/171Val) proved resistant to inhibition. The Cys-SSH-producing CSE activity of Cys136/171Val CSE was higher than that of the wild-type enzyme. Meanwhile, the cysteine-producing CSE activity of this mutant was equivalent to that of the wild-type enzyme. It is assumed that Cys-SSH-producing CSE activity could be auto-inactivated via the polysulfidation of the enzyme during cystine metabolism. Thus, the polysulfidation of CSE at the Cys136 residue may be an integral feature of cystine metabolism, which functions to down-regulate Cys-SSH synthesis by the enzyme.

Modification of Silver Nanoplates with Cell-Binding Subunit of Bacterial Toxin and Their Antimicrobial Activity against Intracellular Bacteria.

Intracellular bacteria are able to survive and grow in host cells and often cause serious infectious diseases. The B subunit of the subtilase cytotoxin (SubB) found in enterohemorrhagic Escherichia coli O113:H21 recognizes sialoglycans on cell surfaces and triggers the uptake of cytotoxin by the cells, meaning that Sub B is a ligand molecule that is expected to be useful for drug delivery into cells. In this study, we conjugated SubB to silver nanoplates (AgNPLs) for use as an antibacterial drug and examined their antimicrobial activity against intracellularly infecting Salmonella typhimurium (S. typhimurium). The modification of AgNPLs with SubB improved their dispersion stability and antibacterial activity against planktonic S. typhimurium. The SubB modification enhanced the cellular uptake of AgNPLs, and intracellularly infecting S. typhimurium were killed at low concentrations of AgNPLs. Interestingly, larger amounts of SubB-modified AgNPLs were taken up by infected cells compared with uninfected cells. These results suggest that the S. typhimurium infection activated the uptake of the nanoparticles into the cells. SubB-modified AgNPLs are expected to be useful bactericidal systems for intracellularly infecting bacteria.

Helicobacter kumamotonensis sp. nov., isolated from human clinical specimens.

A Gram-stain-negative, spiral bacterium (PAGU 1991T) was isolated from the blood of a patient with diffuse large B-cell lymphoma. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate was very closely related to Helicobacter equorum LMG 23362T (99.1 % similarity), originally isolated from a faecal sample from a healthy horse. PAGU 1991T was also very closely related to PAGU 1750 in our strain library (=CCUG 41437) with 99.7 % similarity. Additional phylogenetic analyses based on the 23S rRNA gene sequence and GyrA amino acid sequence further supported the close relationship between the two human isolates (PAGU 1991T and PAGU 1750) and the horse strain. However, a phylogenetic analysis based on 16S rRNA showed that the two human isolates formed a lineage that was distinct from the horse strain (less than 99.2 % similarity). In silico whole-genome comparisons based on digital DNA-DNA hybridization, average nucleotide identity based on blast and orthologous average nucleotide identity using usearch between the two human isolates and the type strain of H. equorum showed values of less than 52.40, 93.47, and 93.50 %, respectively, whereas those between the two human isolates were 75.8, 97.2, and 97.2 %, respectively. These data clearly demonstrated that the two human isolates formed a single species, distinct from H. equorum. Morphologically, the human isolates could be distinguished by the type of flagella; the human isolates showed a bipolar sheathed flagellum, whereas that of H. equorum was monopolar. Biochemically, the human isolate was characterized by growth at 42 °C under microaerobic conditions and nitrate reduction unability. We conclude that the two human isolates, obtained from geographically and temporally distinct sources, were a novel species, for which we propose the name Helicobacter kumamotonensis sp. nov., with the type strain PAGU 1991T (=GTC 16810T=CCUG 75774T).

Enhanced Permeability and Retention Effect as a Ubiquitous and Epoch-Making Phenomenon for the Selective Drug Targeting of Solid Tumors.

In 1979, development of the first polymer drug SMANCS [styrene-co-maleic acid (SMA) copolymer conjugated to neocarzinostatin (NCS)] by Maeda and colleagues was a breakthrough in the cancer field. When SMANCS was administered to mice, drug accumulation in tumors was markedly increased compared with accumulation of the parental drug NCS. This momentous result led to discovery of the enhanced permeability and retention effect (EPR effect) in 1986. Later, the EPR effect became known worldwide, especially in nanomedicine, and is still believed to be a universal mechanism for tumor-selective accumulation of nanomedicines. Some research groups recently characterized the EPR effect as a controversial concept and stated that it has not been fully demonstrated in clinical settings, but this erroneous belief is due to non-standard drug design and use of inappropriate tumor models in investigations. Many research groups recently provided solid evidence of the EPR effect in human cancers (e.g., renal and breast), with significant diversity and heterogeneity in various patients. In this review, we focus on the dynamics of the EPR effect and restoring tumor blood flow by using EPR effect enhancers. We also discuss new applications of EPR-based nanomedicine in boron neutron capture therapy and photodynamic therapy for solid tumors.

Structural Determination of the Nanocomplex of Borate with Styrene-Maleic Acid Copolymer-Conjugated Glucosamine Used as a Multifunctional Anticancer Drug.

The development of effective anticancer drugs is essential for chemotherapy that specifically targets cancer tissues. We recently synthesized a multifunctional water-soluble anticancer polymer drug consisting of styrene-maleic acid copolymer (SMA) conjugated with glucosamine and boric acid (BA) (SGB complex). It demonstrated about 10 times higher tumor-selective accumulation compared with accumulation in normal tissues because of the enhanced permeability and retention effect, and it inhibited tumor growth via glycolysis inhibition, mitochondrial damage, and thermal neutron irradiation. Gaining insight into the anticancer effects of this SGB complex requires a determination of its structure. We therefore investigated the chemical structure of the SGB complex by means of nuclear magnetic resonance, infrared (IR) spectroscopy, and liquid chromatography-mass spectrometry. To establish the chemical structure of the SGB complex, we synthesized a simple model compound─maleic acid-glucosamine (MAG) conjugate─by using a maleic anhydride (MA) monomer unit instead of the SMA polymer. We obtained two MAG-BA complexes (MAGB) with molecular weights of 325 and 343 after the MAG reaction with BA. We confirmed, by using IR spectroscopy, that MAGB formed a stable complex via an amide bond between MA and glucosamine and that BA bound to glucosamine via a diol bond. As a result of this chemical design, identified via analysis of MAGB, the SGB complex can release BA and demonstrate toxicity to cancer cells through inhibition of lactate secretion in mild hypoxia that mimics the tumor microenvironment. For clinical application of the SGB complex, we confirmed that this complex is stable in the presence of serum. These findings confirm that our design of the SGB complex has various advantages in targeting solid cancers and exerting therapeutic effects when combined with neutron irradiation.

Non-canonical inflammasome activation analysis in a mouse model of Citrobacter rodentium infection.

Infection of mice with Citrobacter rodentium is a useful model for studying the pathogenicity of enteropathogenic and enterohemorrhagic Escherichia coli, pathogens that have a close association with humans. Here, we provide a protocol detailing the approaches for non-canonical inflammasome analysis in a mouse model of C. rodentium infection, including preparation of bacteria, oral administration of bacteria to mice, counting colony-forming units to quantify bacterial colonization, and analysis of expression and activation of inflammasome-related factors. For complete details on the use and execution of this protocol, please refer to Tsutsuki et al. (2022).