[A Case of Advanced Gastric Cancer in Which Tumor Relapse Was Observed after Discontinuation of Nivolumab Due to Complete Response, and for Which Re-Administration Was Initiated].

A 61-year-old male was diagnosed with unresectable advanced gastric cancer(cT4b[SI; panc], N+, M0, cStage ⅣA). However he was administered S-1 plus oxaliplatin as a primary treatment and ramucirumab plus paclitaxel as a secondary treatment, the primary tumor and lymph nodes were enlarged. We judged PD and switched to the third-line treatment with nivolumab. After starting nivolumab, both the primary tumor and the lymph nodes shrank, and the PET-CT scan after 24 courses showed no FDG accumulation in the primary tumor or lymph nodes, so we judged the response as CR. The patient requested discontinuation of nivolumab, and nivolumab administration was stopped. Twenty months later after nivolumab administration was discontinued, CT scan showed re-growth of the primary tumor, and nivolumab administration was resumed. After resumption, he received 22 courses of nivolumab for 10 months with maintenance of SD.

Occurrence of different fucoidanase genes in Flavobacterium sp. SW and enzyme characterization.

Fucoidans are hetero-sulfated polysaccharides that are widely distributed in brown algae and have been extensively studied for their various biological activities. The structure-function relationship of fucoidans remains unclear but can be studied using fucoidan-degrading enzymes (fucoidanases). Here, we isolated and identified Flavobacterium sp. SW as a microbial strain that can grow on fucoidan from Cladosiphon okamuranus as the sole carbon source. Genomic analysis of this strain revealed the presence of two genes, swfct and swfcn2, that are homologous to fct114 from Luteolibacter algae H18 and fcnA from Psychromonas sp. SW5A, respectively. The gene products were produced in Escherichia coli and showed significantly different specificities for fucoidan. Swfct catalyzed the degradation of deacetylated fucoidan from C. okamuranus, and Swfcn2 degraded fucoidans from Saccharina sculpera and Macrocystis pyrifera. The general properties of Swfct were examined by measuring the amounts of reducing ends produced by the enzymatic reaction, and the enzyme properties of Swfcn2 were evaluated by carbohydrate-polyacrylamide gel electrophoresis. Our findings indicate that one microbial strain can harbor genes encoding two different types of fucoidanases.

[A Resected Case of the Pancreatic Tail Cancer with Colonic Obstruction].

A 71-year-old man complained of abdominal pain. He showed fecal occult blood positive and he was referred to our hospital for further examination and treatment. During examinations, he developed colonic obstruction. As a result of examinations, he was diagnosed with pancreatic tail cancer invading to the colon. We underwent distal pancreatectomy, partial colectomy, partial gastrectomy, and left adrenalectomy. Although chylous fistula was observed, he was discharged from hospital 35 days after surgery. He has received adjuvant chemotherapy using S-1, and no recurrence has been observed 4 months after operation.

Volatile Anesthetic Sevoflurane Precursor 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP) Exerts an Anti-Prion Activity in Prion-Infected Culture Cells.

Prion disease is a neurodegenerative disorder with progressive neurologic symptoms and accelerated cognitive decline. The causative protein of prion disease is the prion protein (PrP), and structural transition of PrP from the normal helix rich form (PrPC) to the abnormal ß-sheet rich form (PrPSc) occurs in prion disease. While so far numerous therapeutic agents for prion diseases have been developed, none of them are still useful. A fluorinated alcohol, hexafluoro isopropanol (HFIP), is a precursor to the inhalational anesthetic sevoflurane and its metabolites. HFIP is also known as a robust α-helix inducer and is widely used as a solvent for highly aggregated peptides. Here we show that the α-helix-inducing activity of HFIP caused the conformational transformation of the fibrous structure of PrP into amorphous aggregates in vitro. HFIP added to the ScN2a cell medium, which continuously expresses PrPSc, reduced PrPSc protease resistance after 24-h incubation. It was also clarified that ScN2a cells are more susceptible to HFIP than any of the cells being compared. Based on these findings, HFIP is expected to develop as a therapeutic agent for prion disease.

Limited spread of pathology within the brainstem of α-synuclein BAC transgenic mice inoculated with preformed fibrils into the gastrointestinal tract.

Parkinson's disease (PD) is pathologically characterized by intraneuronal α-synuclein (α-Syn) aggregates called Lewy bodies (LBs) as well as the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). On the basis of autopsy studies, Braak et al. hypothesized that Lewy pathology initially occurs in the enteric nervous system, subsequently spreading to the dorsal motor nucleus of the vagus nerve (dmX) and then ascending in the brainstem to the SNpc. However, this hypothetical progression lacks adequate experimental evidence. We previously reported that inoculation of α-Syn preformed fibrils (PFFs) into the gastric wall of wild-type (WT) mice induced LB-like α-Syn aggregates in the dmX via the vagus nerve. However, α-Syn pathology did not spread beyond the dmX up to 12 months postinoculation. In the present study, we inoculated α-Syn PFFs into the gastric wall of bacterial artificial chromosome (BAC) transgenic mice harboring the human α-Syn gene with an A53 T mutation and analyzed the pathology. The transgenic mice had ∼1.5-fold overexpression of α-Syn in the brains and ∼6-fold overexpression of α-Syn in the stomach compared with WT mice. After inoculation of α-Syn PFFs, the transgenic mice developed a higher number of phosphorylated α-Syn (p-α-Syn)-positive neurons in the dmX compared with similarly inoculated WT mice. However, the number of p-α-Syn-positive neurons in the dmX decreased over time, and α-Syn pathology was not observed in other brain regions except in the ambiguous nucleus up to 8 months postinoculation. Taken together, BAC transgenic expression of α-Syn facilitated induction of α-Syn pathology in the brainstem, but not subsequent caudo-rostral spread in accordance with Braak's hypothesis.

Correction to: Inoculation of α-synuclein preformed fibrils into the mouse gastrointestinal tract induces Lewy body-like aggregates in the brainstem via the vagus nerve.

The original article [1] mistakenly omitted essential information regarding Fig. 1c; thus, the authors would like to note that Fig. 1c describes transmission electron microscopy of α-Syn PFFs before sonication.

Differences in biochemical properties of two 5'-nucleotidases from deep- and shallow-sea Shewanella species under various harsh conditions.

Deep-sea Shewanella violacea 5'-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions. Abbreviations: NTase: 5'-nucleotidase; SANTase: Shewanella amazonensis 5'-nucleotidase; SVNTase: Shewanella violacea 5'-nucleotidase; CD: circular dichroism.

Identification and characterization of the fucoidanase gene from Luteolibacter algae H18.

Fucoidan is a hetero-sulfated polysaccharide found in brown algae and has received much attention as an ingredient in functional and health foods. The marine bacterial strain Luteolibacter algae H18 degrades fucoidan from Cladosiphon okamuranus. We purified the fucoidanase from a cell-free extract of L. algae H18, used it to decrease the molecular weight of deacetylated-fucoidan, determined the N-terminal amino acid sequence of the enzyme, and identified the gene involved in the degradation of fucoidan, fct114, in a draft genome sequence of strain H18. The gene product was heterologously produced in Escherichia coli and demonstrated to catalyze the degradation of deacetylated-fucoidan into lower molecular weight fragments. The mass of the gene product Fct114 is 112 kDa (1026 amino acid residues). The general properties of the enzyme were investigated by measuring the amount of reducing ends produced from deacetylated-fucoidan during the reaction. The enzyme was inactive toward fucoidans from other brown seaweed species or toward polysaccharides such as alginic acid, carrageenan, hyaluronic acid, and chondroitin sulfate. The amino acid sequence of Fct114 shared less than 25% identity and had no conserved motifs when compared with previously identified fucoidanases from other marine bacterial strains. These data suggest that Fct114 is a novel polysaccharide-degrading enzyme.

Inoculation of α-synuclein preformed fibrils into the mouse gastrointestinal tract induces Lewy body-like aggregates in the brainstem via the vagus nerve.

BACKGROUND: Intraneuronal α-synuclein (α-Syn) aggregates known as Lewy bodies (LBs) and the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are the pathological hallmarks of Parkinson's disease (PD). Braak's hypothesis based on autopsy studies suggests that Lewy pathology initially occurs in the enteric nervous system (ENS) and then travels retrogradely to the dorsal motor nucleus of the vagus nerve (dmX), proceeding from there in a caudo-rostral direction. Recent evidence that α-Syn aggregates propagate between interconnected neurons supports this hypothesis. However, there is no direct evidence demonstrating this transmission from the ENS to the dmX and then to the SNpc. METHODS: We inoculated α-Syn preformed fibrils (PFFs) or phosphate-buffered saline (PBS) into the mouse gastric wall and analyzed the progression of the pathology. RESULTS: The mice inoculated with α-Syn PFFs, but not with PBS, developed phosphorylated α-Syn (p-α-Syn)-positive LB-like aggregates in the dmX at 45 days postinoculation. This aggregate formation was completely abolished when vagotomy was performed prior to inoculation of α-Syn PFFs, suggesting that the aggregates in the dmX were retrogradely induced via the vagus nerve. Unexpectedly, the number of neurons containing p-α-Syn-positive aggregates in the dmX decreased over time, and no further caudo-rostral propagation beyond the dmX was observed up to 12 months postinoculation. P-α-Syn-positive aggregates were also present in the myenteric plexus at 12 months postinoculation. However, unlike in patients with PD, there was no cell-type specificity in neurons containing those aggregates in this model. CONCLUSIONS: These results indicate that α-Syn PFF inoculation into the mouse gastrointestinal tract can induce α-Syn pathology resembling that of very early PD, but other factors are apparently required if further progression of PD pathology is to be replicated in this animal model.

Microbial and genomic characterization of Geobacillus thermodenitrificans OS27, a marine thermophile that degrades diverse raw seaweeds.

Seaweeds are a nonlignocellulosic biomass, but they are often abundant in unique polysaccharides that common microbes can hardly utilize; therefore, polysaccharide degradation is key for the full utilization of seaweed biomass. Here, we isolated 13 thermophiles from seaweed homogenates that had been incubated at high temperature. All of the isolates were Gram-positive and preferentially grew at 60-70 °C. Most formed endospores and were tolerant to seawater salinity. Despite different sources, all isolates were identical regarding 16S rRNA gene sequences and were categorized as Geobacillus thermodenitrificans. Their growth occurred on seaweed polysaccharides with different profiles but required amino acids and/or vitamins, implying that they existed as proliferative cells by utilizing nutrients on seaweed viscous surfaces. Among 13 isolates, strain OS27 was further characterized to show that it can utilize a diverse range of seaweed polysaccharides and hemicelluloses. Notably, strain OS27 degraded raw seaweeds while releasing soluble saccharides. The degradation seemed to depend on enzymes that were extracellularly produced in an inducible manner. The strain could be genetically modified to produce heterologous endoglucanase, providing a transformant that degrades more diverse seaweeds with higher efficiency. The draft sequences of the OS27 genome contained 3766 coding sequences, which included intact genes for 28 glycoside hydrolases and many hypothetical proteins unusual among G. thermodenitrificans. These results suggest that G. thermodenitrificans OS27 serves as a genetic resource for thermostable enzymes to degrade seaweeds and potentially as a microbial platform for high temperature seaweed biorefinery via genetic modification.

Characterization of a novel endo-type alginate lyase derived from Shewanella sp. YH1.

Alginate, which is an anionic polysaccharide, is widely distributed in the cell wall of brown algae. Alginate and the products of its degradation (oligosaccharides) are used in stabilizers, thickeners and gelling agents, especially in the food industry. The degradation of alginate generally involves a combination of several alginate lyases (exo-type, endo-type and oligoalginate lyase). Enhancing the efficiency of the production of alginate degradation products may require the identification of novel alginate lyases with unique characteristics. In this study, we isolated an alginate-utilizing bacterium, Shewanella sp. YH1, from seawater collected off the coast of Tottori prefecture, Japan. The detected novel alginate lyase was named AlgSI-PL7, and was classified in polysaccharide lyase family 7. The enzyme was purified from Shewanella sp. YH1 and a recombinant AlgSI-PL7 was produced in Escherichia coli. The optimal temperature and pH for enzyme activity were around 45°C and 8, respectively. Interestingly, we observed that AlgSI-PL7 was not thermotolerant, but could refold to its active form following an almost complete denaturation at approximately 60°C. Moreover, the degradation of alginate by AlgSI-PL7 produced two to five oligosaccharides, implying this enzyme was an endo-type lyase. Our findings suggest that AlgSI-PL7 may be useful as an industrial enzyme.

Characterization of a Long-Lived Alginate Lyase Derived from Shewanella Species YH1.

Polysaccharides from seaweeds are widely used in various fields, including the food, biomedical material, cosmetic, and biofuel industries. Alginate, which is a major polysaccharide in brown algae, and the products of its degradation (oligosaccharides) have been used in stabilizers, thickeners, and gelling agents, especially in the food industry. Discovering novel alginate lyases with unique characteristics for the efficient production of oligosaccharides may be relevant for the food and pharmaceutical fields. In this study, we identified a unique alginate lyase derived from an alginate-utilizing bacterium, Shewanella species YH1. The recombinant enzyme (rAlgSV1-PL7) was produced in an Escherichia coli system and it was classified in the Polysaccharide Lyase family 7. The optimal temperature and pH for rAlgSV1-PL7 activity were around 45 °C and 8, respectively. Interestingly, we observed that rAlgSV1-PL7 retained over 80% of its enzyme activity after incubation at 30 °C for at least 20 days, indicating that rAlgSV1-PL7 is a long-lived enzyme. Moreover, the degradation of alginate by rAlgSV1-PL7 produced one to four sugars because of the broad substrate specificity of this enzyme. Our findings suggest that rAlgSV1-PL7 may represent a new commercially useful enzyme.

Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp. A11-2, reveal potential for expanding its substrate selectivity.

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO2) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate-FMN-enzyme complex remains unknown. Herein, we report the crystal structures of the DBT-FMN-TdsC and DBTO-FMN-TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications.

Gene identification and characterization of fucoidan deacetylase for potential application to fucoidan degradation and diversification.

Fucoidan is an α-l-fucopyranosyl polymer found in seaweeds with forms that have acetyl and sulfuric modifications and derivatives that are lower and/or diversified, with modifications that have attracted interest as potential bioactive substances. We identified the gene for a fucoidan deacetylase that cleaves acetyl moieties from fucoidan and thereby contributes to fucoidan utilization in the marine bacterium Luteolibacter algae H18. Fucoidan deacetylase was purified to homogeneity from a cell-free extract of L. algae H18, and used to determine the internal amino acid sequence and identify the gene, fud, in a draft genome sequence of the H18 strain. The gene product was heterologously produced in Escherichia coli and was demonstrated to catalyze fucoidan deacetylation, but not desulfation, and degradation into lower forms. In addition to fucoidan deacetylation, the enzyme catalyzed the hydrolysis of p-nitrophenyl esters with organic acids, and p-nitrophenyl acetate was the best substrate among those tested. The present study provides a new tool for fucoidan degradation, potentially expanding investigations on fucoidan derivatives.

Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae.

The application of marine resources, instead of fossil fuels, for biomass production is important for building a sustainable society. Seaweed is valuable as a source of marine biomass for producing biofuels such as ethanol, and can be used in various fields. Alginate is an anionic polysaccharide that forms the main component of brown algae. Various alginate lyases (e.g. exo- and endo-types and oligoalginate lyase) are generally used to degrade alginate. We herein describe a novel alginate lyase, AlgC-PL7, which belongs to the polysaccharide lyase 7 family. AlgC-PL7 was isolated from the halophilic Gram-negative bacterium Cobetia sp. NAP1 collected from the brown algae Padina arborescens Holmes. The optimal temperature and pH for AlgC-PL7 activity were 45 °C and 8, respectively. Additionally, AlgC-PL7 was thermostable and salt-tolerant, exhibited broad substrate specificity, and degraded alginate into monosaccharides. Therefore, AlgC-PL7 is a promising enzyme for the production of biofuels.

A small-angle X-ray scattering study of alpha-synuclein from human red blood cells.

α-synuclein (α-syn) is the main component of Lewy bodies, which are neuropathological hallmarks of patients with Parkinson's disease. As it has been controversial whether human α-syn from erythrocytes exists as a tetramer under physiological conditions, we tried solving this issue by the small-angle X-ray solution scattering method. Under two different conditions (high ionic strength with a Tris buffer and low ionic strength with an ammonium acetate buffer), no evidence was found for the presence of tetramer. When comparing erythrocyte and recombinant α-syn molecules, we found no significant difference of the molecular weight and the secondary structure although the buffer conditions strongly affect the radius of gyration of the protein. The results indicate that, even though a stable tetramer may not be formed, conformation of α-syn depends much on its environment, which may be the reason for its tendency to aggregate in cells.

Dynamical Behavior of Human α-Synuclein Studied by Quasielastic Neutron Scattering.

α-synuclein (αSyn) is a protein consisting of 140 amino acid residues and is abundant in the presynaptic nerve terminals in the brain. Although its precise function is unknown, the filamentous aggregates (amyloid fibrils) of αSyn have been shown to be involved in the pathogenesis of Parkinson's disease, which is a progressive neurodegenerative disorder. To understand the pathogenesis mechanism of this disease, the mechanism of the amyloid fibril formation of αSyn must be elucidated. Purified αSyn from bacterial expression is monomeric but intrinsically disordered in solution and forms amyloid fibrils under various conditions. As a first step toward elucidating the mechanism of the fibril formation of αSyn, we investigated dynamical behavior of the purified αSyn in the monomeric state and the fibril state using quasielastic neutron scattering (QENS). We prepared the solution sample of 9.5 mg/ml purified αSyn, and that of 46 mg/ml αSyn in the fibril state, both at pD 7.4 in D2O. The QENS experiments on these samples were performed using the near-backscattering spectrometer, BL02 (DNA), at the Materials and Life Science Facility at the Japan Accelerator Research Complex, Japan. Analysis of the QENS spectra obtained shows that diffusive global motions are observed in the monomeric state but largely suppressed in the fibril state. However, the amplitude of the side chain motion is shown to be larger in the fibril state than in the monomeric state. This implies that significant solvent space exists within the fibrils, which is attributed to the αSyn molecules within the fibrils having a distribution of conformations. The larger amplitude of the side chain motion in the fibril state than in the monomeric state implies that the fibril state is entropically favorable.

Nucleus factory on cavitation bubble for amyloid ß fibril.

Structural evolution from monomer to fibril of amyloid ß peptide is related to pathogenic mechanism of Alzheimer disease, and its acceleration is a long-running problem in drug development. This study reveals that ultrasonic cavitation bubbles behave as catalysts for nucleation of the peptide: The nucleation reaction is highly dependent on frequency and pressure of acoustic wave, and we discover an optimum acoustical condition, at which the reaction-rate constant for nucleation is increased by three-orders-of magnitudes. A theoretical model is proposed for explaining highly frequency and pressure dependent nucleation reaction, where monomers are captured on the bubble surface during its growth and highly condensed by subsequent bubble collapse, so that they are transiently exposed to high temperatures. Thus, the dual effects of local condensation and local heating contribute to dramatically enhance the nucleation reaction. Our model consistently reproduces the frequency and pressure dependences, supporting its essential applicability.

A Stable Mutant Predisposes Antibody Domains to Amyloid Formation through Specific Non-Native Interactions.

The aggregation of mostly antibody light chain variable (VL) domains into amyloid fibrils in various tissues is the main cause of death in systemic amyloid light chain amyloidosis. Point mutations within the domain are important to shift the VL into the fibrillar pathway, but why and how only some site-specific mutations achieve this still remains elusive. We show here that both destabilizing and surprisingly stable mutants readily predispose an amyloid-resistant VL domain to amyloid formation. The decreased thermodynamic stability of the destabilizing mutant results in the accumulation of non-native intermediates that readily populate the amyloid state. Interestingly, the stable mutants establish site-specific non-native interactions with especially nearby serine/threonine residues that unexpectedly do not affect the folding behavior of the VL domain but rather readily induce and stabilize the fibril structure, a previously unrecognized mechanism. These findings provide a new concept for the molecular mechanism of amyloid fibril formation.

Synchrotron FTIR micro-spectroscopy for structural analysis of Lewy bodies in the brain of Parkinson's disease patients.

Lewy bodies (LBs), which mainly consist of α-synuclein (α-syn), are neuropathological hallmarks of patients with Parkinson's disease (PD). The fine structure of LBs is unknown, and LBs cannot be made artificially. Nevertheless, many studies have described fibrillisation using recombinant α-syn purified from E. coli. An extremely fundamental problem is whether the structure of LBs is the same as that of recombinant amyloid fibrils. Thus, we used synchrotron Fourier transform infrared micro-spectroscopy (FTIRM) to analyse the fine structure of LBs in the brain of PD patients. Our results showed a shift in the infrared spectrum that indicates abundance of a ß-sheet-rich structure in LBs. Also, 2D infrared mapping of LBs revealed that the content of the ß-sheet structure is higher in the halo than in the core, and the core contains a large amount of proteins and lipids.