Enantioselective Total Synthesis of (-)-Hunterine A Enabled by a Desymmetrization/Rearrangement Strategy.

The first enantioselective total synthesis of (-)-hunterine A is disclosed. Our strategy employs a catalytic asymmetric desymmetrization of a symmetrical diketone and subsequent Beckmann rearrangement to construct a 5,6-α-aminoketone. A convergent 1,2-addition joins a vinyl dianion nucleophile and the enantioenriched ketone. The endgame of the synthesis features an aza-Cope/Mannich reaction and azide-olefin dipolar cycloaddition to complete the pentacyclic ring system. The synthesis is completed through a regioselective aziridine ring opening.

Ultrafast Halogen Dance Reactions of Bromoarenes Enabled by Catalytic Potassium Hexamethyldisilazide.

Lochmann-Schlosser base, a stoichiometric combination of nBuLi and KOtBu, is commonly used as a superbase for deprotonating a wide range of organic compounds. In the present study, we report that catalytic potassium hexamethyldisilazide (KHMDS) exhibits higher catalytic activity than KOtBu for successive bromine-metal exchanges. Accordingly, 1-10 mol% of KHMDS dramatically enhances halogen dance reactions to introduce various electrophiles to bromopyridine, bromoimidazole, bromothiophene, bromofuran, and bromobenzene derivatives with the bromo group translocated from the original position. A dual catalytic cycle is proposed to explain the ultrafast bromine transfer.

Formal Halogen Transfer of Bromoarenes via Stepwise Reactions.

A two-step halogen transfer of bromoarenes is reported. Mono-, di-, and tribromoaryllithiums generated through deprotonative lithiation were converted into organozinc species by in situ zincation, which were then subjected to bromination to provide the corresponding di-, tri-, and tetrabromoarenes, respectively, in 41-95% yields. Regioselective bromine-magnesium exchange with ethylmagnesium chloride followed by electrophilic trapping afforded benzene, pyridine, quinoline, pyrimidine, and thiazole derivatives with the bromo group translocated from the original position in 28-86% yields.

Performance of stacked microbial fuel cells with barley-shochu waste.

The treatment of barley-shochu waste combined with electricity generation was examined using stacked microbial fuel cells (SMFCs). The maximum chemical oxygen demand (CODCr) removal efficiency and maximum power density were achieved at 36.7 ± 1.1% and 4.3 ± 0.2 W m⁻³ (15.7 ± 0.9 mW m-2). The acetic acid concentration in effluent increased, whereas the citric acid, ethanol and sugar concentrations decreased during the operation. Microbial community analysis of the anode cell suspension and raw barley-shochu waste revealed that Clostridiaceae, Acetobacteraceae, and Enterobacteriaceae became predominant after the operation, implying that microorganisms belonging to these families might be involved in organic waste decomposition and electricity generation in the SMFCs.

Unexpected genomic features of high current density-producing Geobacter sulfurreducens strain YM18.

Geobacter sulfurreducens produces high current densities and it has been used as a model organism for extracellular electron transfer studies. Nine G. sulfurreducens strains were isolated from biofilms formed on an anode poised at -0.2 V (vs SHE) in a bioelectrochemical system in which river sediment was used as an inoculum. The maximum current density of an isolate, strain YM18 (9.29 A/m2), was higher than that of the strain PCA (5.72 A/m2), the type strain of G. sulfurreducens, and comparable to strain KN400 (8.38 A/m2), which is another high current-producing strain of G. sulfurreducens. Genomic comparison of strains PCA, KN400 and YM18 revealed that omcB, xapD, spc and ompJ, which are known to be important genes for iron reduction and current production in PCA, were not present in YM18. In the PCA and KN400 genomes, two and one region(s) encoding CRISPR/Cas systems were identified, respectively, but they were missing in the YM18 genome. These results indicate that there is genetic variation in the key components involved in extracellular electron transfer among G. sulfurreducens strains.

Complete Genome Sequence of High Current-Producing Geobacter sulfurreducens Strain YM35, Isolated from River Sediment in Japan.

Here, we report the complete genome sequence of Geobacter sulfurreducens strain YM35, which was isolated from biofilms formed on an anode in a bioelectrochemical system where river sediment was used as an inoculum. The chromosome is 3,745,223 bp with a G+C content of 60.9%. The chromosome contains 3,324 protein-coding genes.

"Snapshot" Trapping of Multiple Transient Azolyllithiums in Batch.

Invited for the cover of this issue is Kentaro Okano and co-workers at Kobe University. The image depicts that the 'dancing' transient organolithiums in the 'halogen dance' are successfully trapped in a batch reactor as if their individual snapshots were taken. Read the full text of the article at 10.1002/chem.202101256.

Crystal structure of the ferredoxin reductase component of carbazole 1,9a-dioxygenase from Janthinobacterium sp. J3.

Carbazole 1,9a-dioxygenase (CARDO), which consists of an oxygenase component and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), is a Rieske nonheme iron oxygenase (RO). ROs are classified into five subclasses (IA, IB, IIA, IIB and III) based on their number of constituents and the nature of their redox centres. In this study, two types of crystal structure (type I and type II) were resolved of the class III CARDO-R from Janthinobacterium sp. J3 (CARDO-RJ3). Superimposition of the type I and type II structures revealed the absence of flavin adenine dinucleotide (FAD) in the type II structure along with significant conformational changes to the FAD-binding domain and the C-terminus, including movements to fill the space in which FAD had been located. Docking simulation of NADH into the FAD-bound form of CARDO-RJ3 suggested that shifts of the residues at the C-terminus caused the nicotinamide moiety to approach the N5 atom of FAD, which might facilitate electron transfer between the redox centres. Differences in domain arrangement were found compared with RO reductases from the ferredoxin-NADP reductase family, suggesting that these differences correspond to differences in the structures of their redox partners ferredoxin and terminal oxygenase. The results of docking simulations with the redox partner class III CARDO-F from Pseudomonas resinovorans CA10 suggested that complex formation suitable for efficient electron transfer is stabilized by electrostatic attraction and complementary shapes of the interacting regions.

"Snapshot" Trapping of Multiple Transient Azolyllithiums in Batch.

Recent developments in flow microreactor technology have allowed the use of transient organolithium compounds that cannot be realized in a batch reactor. However, trapping the transient aryllithiums in a "halogen dance" is still challenging. Herein is reported the trapping of such short-lived azolyllithiums in a batch reactor by developing a finely tuned in situ zincation using zinc halide diamine complexes. The reaction rate is controlled by the appropriate choice of diamine ligand. The reaction is operationally simple and can be performed at 0 °C with high reproducibility on a multigram scale. This method was applicable to a wide range of brominated azoles allowing deprotonative functionalization, which was used for the concise divergent syntheses of both constitutional isomers of biologically active azoles.

Proteolytic Maturation of the Outer Membrane c-Type Cytochrome OmcZ by a Subtilisin-Like Serine Protease Is Essential for Optimal Current Production by Geobacter sulfurreducens.

An outer membrane c-type cytochrome (OmcZ) in Geobacter sulfurreducens is essential for optimal current production in microbial fuel cells. OmcZ exists in two forms, small and large, designated OmcZS and OmcZL, respectively. However, it is still not known how these two structures are formed. A mutant with a disruption of the GSU2075 gene encoding a subtilisin-like serine protease (designated ozpA for the OmcZprotease), which is located downstream of omcZ, produced low currents at a level similar to that of the omcZ-deficient mutant strain. Biochemical analyses revealed that the ozpA mutant accumulated OmcZL and did not produce OmcZS, which is thought to be a mature form that is essential for the extracellular electron transfer to the electrode. A heterologous expression system cell lysate from an Escherichia coli strain producing OzpA cleaved OmcZL and generated OmcZS as the proteolytic product. Among the culture supernatant, loosely bound outer surface, and intracellular protein fractions from wild-type G. sulfurreducens, only the culture supernatant protein fraction showed OmcZL cleavage activity, indicating that the mature form of OmcZ, OmcZS, can be produced outside the cells. These results indicate that OzpA is an essential protease for current production via the maturation of OmcZ, and OmcZS is the key to the extracellular electron transfer to electrodes. This proteolytic maturation of OmcZ is a unique regulation among known c-type cytochromes in G. sulfurreducens. IMPORTANCE Microbial fuel cells are a promising technology for energy generation from various waste types. However, the molecular mechanisms of microbial extracellular electron transfer to the electrode need to be elucidated. G. sulfurreducens is a common key player in electricity generation in mixed-culture microbial fuel cell systems and a model microorganism for the study of extracellular electron transfer. Outer membrane c-type cytochrome OmcZ is essential for an optimal current production by G. sulfurreducens. OmcZ proteolytic cleavage occurs during maturation, but the underlying mechanism is unknown. This study identifies a subtilisin-like protease, OzpA, which plays a role in cleaving OmcZ and generating the mature form of OmcZ (OmcZS). OzpA is essential for current production and, thus, the proteolytic maturation of OmcZ. This is a novel regulation of the c-type cytochrome for G. sulfurreducens extracellular electron transfer. This study also provides new insights into the design strategy and development of microbial extracellular electron transfer for an efficient energy conversion from chemical energy to electricity.

Determination of Plasmid pSN1216-29 Host Range and the Similarity in Oligonucleotide Composition Between Plasmid and Host Chromosomes.

Plasmids are extrachromosomal DNA that can be horizontally transferred between different bacterial cells by conjugation. Horizontal gene transfer of plasmids can promote rapid evolution and adaptation of bacteria by imparting various traits involved in antibiotic resistance, virulence, and metabolism to their hosts. The host range of plasmids is an important feature for understanding how they spread in environmental microbial communities. Earlier bioinformatics studies have demonstrated that plasmids are likely to have similar oligonucleotide (k-mer) compositions to their host chromosomes and that evolutionary host ranges of plasmids could be predicted from this similarity. However, there are no complementary studies to assess the consistency between the predicted evolutionary host range and experimentally determined replication/transfer host range of a plasmid. In the present study, the replication/transfer host range of a model plasmid, pSN1216-29, exogenously isolated from cow manure as a newly discovered self-transmissible plasmid, was experimentally determined within microbial communities extracted from soil and cow manure. In silico prediction of evolutionary host range was performed with the pSN1216-29 using its oligonucleotide compositions independently. The results showed that oligonucleotide compositions of the plasmid pSN1216-29 had more similarities to those of hosts (transconjugants genera) than those of non-hosts (other genera). These findings can contribute to the understanding of how plasmids behave in microbial communities, and aid in the designing of appropriate plasmid vectors for different bacteria.

Dysfunction of Poly (ADP-Ribose) Glycohydrolase Induces a Synthetic Lethal Effect in Dual Specificity Phosphatase 22-Deficient Lung Cancer Cells.

Poly (ADP-ribose) glycohydrolase (PARG) is the main enzyme responsible for catabolism of poly (ADP-ribose) (PAR), synthesized by PARP. PARG dysfunction sensitizes certain cancer cells to alkylating agents and cisplatin by perturbing the DNA damage response. The gene mutations that sensitize cancer cells to PARG dysfunction-induced death remain to be identified. Here, we performed a comprehensive analysis of synthetic lethal genes using inducible PARG knockdown cells and identified dual specificity phosphatase 22 (DUSP22) as a novel synthetic lethal gene related to PARG dysfunction. DUSP22 is considered a tumor suppressor and its mutation has been frequently reported in lung, colon, and other tumors. In the absence of DNA damage, dual depletion of PARG and DUSP22 in HeLa and lung cancer A549 cells reduced survival compared with single-knockdown counterparts. Dual depletion of PARG and DUSP22 increased the apoptotic sub-G1 fraction and upregulated PUMA in lung cancer A549, PC14, and SBC5 cells, and inhibited the PI3K/AKT/mTOR pathway in A549 cells, suggesting that dual depletion of PARG and DUSP22 induced apoptosis by upregulating PUMA and suppressing the PI3K/AKT/mTOR pathway. Consistently, the growth of tumors derived from double knockdown A549 cells was slower compared with those derived from control siRNA-transfected cells. Taken together, these results indicate that DUSP22 deficiency exerts a synthetic lethal effect when combined with PARG dysfunction, suggesting that DUSP22 dysfunction could be a useful biomarker for cancer therapy using PARG inhibitors. SIGNIFICANCE: This study identified DUSP22 as a novel synthetic lethal gene under the condition of PARG dysfunction and elucidated the mechanism of synthetic lethality in lung cancer cells.

Electricity generation from sweet potato-shochu waste using microbial fuel cells.

Electricity generation and treatment of sweet potato-shochu waste, acidic and organic-rich slurry, was examined using cassette-electrode microbial fuel cells (CE-MFCs). Among CE-MFCs with raw (73 g-chemical oxygen demand chromium CODCr/L) and different concentration of diluted sweet potato-shochu waste (0.5, 1, 5, 10, and 20 g-CODCr/L) without pH control, the maximum power density (1.2 W/m3) and CODCr removal efficiency (67.4 ± 1.8%) were observed in the CE-MFCs with 10 g-CODCr/L shochu waste. The concentration of organic acid was decreased to below the quantification limits during the 9-day operation in the CE-MFC with 10 g-CODCr/L shochu waste. During the same period, the electrolyte pH was increased from 4.2 to 6.6. Microbial community analysis revealed that the genus Clostridium (75.4%) was predominant in the CE-MFCs with raw shochu waste, whereas Bacteroides (65.3%) and Clostridium (12.1%) were predominant in the CE-MFCs with 10 g-CODCr/L shochu waste.

Stability of the ABCD1 Protein with a Missense Mutation: A Novel Approach to Finding Therapeutic Compounds for X-Linked Adrenoleukodystrophy.

Mutations in the ABCD1 gene that encodes peroxisomal ABCD1 protein cause X-linked adrenoleukodystrophy (X-ALD), a rare neurodegenerative disorder. More than 70% of the patient fibroblasts with this missense mutation display either a lack or reduction of the ABCD1 protein because of posttranslational degradation. In this study, we analyzed the stability of the missense mutant ABCD1 proteins (p.A616T, p.R617H, and p.R660W) in X-ALD fibroblasts and found that the mutant ABCD1 protein p.A616T has the capacity to recover its function by incubating at low temperature. In the case of such a mutation, chemical compounds that stabilize mutant ABCD1 proteins could be therapeutic candidates. Here, we prepared CHO cell lines stably expressing ABCD1 proteins with a missense mutation in fusion with green fluorescent protein (GFP) at the C-terminal. The stability of each mutant ABCD1-GFP in CHO cells was similar to the corresponding mutant ABCD1 protein in X-ALD fibroblasts. Furthermore, it is of interest that the GFP at the C-terminal was degraded together with the mutant ABCD1 protein. These findings prompted us to use CHO cells expressing mutant ABCD1-GFP for a screening of chemical compounds that can stabilize the mutant ABCD1 protein. We established a fluorescence-based assay method for the screening of chemical libraries in an effort to find compounds that stabilize mutant ABCD1 proteins. The work presented here provides a novel approach to finding therapeutic compounds for X-ALD patients with missense mutations.

Electricity Generation from Rice Bran by a Microbial Fuel Cell and the Influence of Hydrodynamic Cavitation Pretreatment.

Single-chamber microbial fuel cells (MFCs) were constructed using rice bran (carbon source) and pond bottom mud (microbial source). The total electric charge obtained in the MFC combining rice bran with pond bottom mud was four times higher than that in MFC using only rice bran. Phylogenetic analyses revealed dominant growth of fermentative bacteria such as Bacteroides and Clostridium species, and exoelectrogenic Geobacter species in the anode biofilms, suggesting that mutualism of these bacteria is a key factor for effective electricity generation in the MFC. Furthermore, rice bran, consisting of persistent polysaccharide, was pretreated by the hydrodynamic cavitation system to improve the digestibility and enhance the efficiency in MFC, resulting in 26% increase in the total production of electricity.

Novel Self-Transmissible and Broad-Host-Range Plasmids Exogenously Captured From Anaerobic Granules or Cow Manure.

Novel self-transmissible plasmids were exogenously captured from environmental samples by triparental matings with pBBR1MCS-2 as a mobilizable plasmid and Pseudomonas resinovorans as a recipient. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. The whole nucleotide sequences of six plasmids were determined, including one IncP-1 plasmid (pSN1104-59), four PromA-like plasmids (pSN1104-11, pSN1104-34, pSN0729-62, and pSN0729-70), and one novel plasmid (pSN1216-29), whose incompatibility group has not been previously identified. No previously known antibiotic resistance genes were found in these plasmids. In-depth phylogenetic analyses showed that the PromA-like plasmids belong to subgroups of PromA (designated as PromAγ and PromAδ) different from previously proposed subgroups PromAα and PromAß. Twenty-four genes were identified as backbone genes by comparisons with other PromA plasmids. The nucleotide sequences of pSN1216-29 share high identity with those found in clinical isolates. A minireplicon of pSN1216-29 was successfully constructed from repA encoding a replication initiation protein and oriV. All the captured plasmids were found to have a broad host range and could be transferred to and replicated in different classes of Proteobacteria. Notably, repA and oriV of pSN1216-29 showed high similarity with one of two replication systems of pSRC119-A/C, known as a plasmid with multidrug resistance genes found in Salmonella enterica serovar Senftenberg. Our findings suggest that these "cryptic" but broad-host-range plasmids may be important for spreading several genes as "vehicles" in a wider range of bacteria in natural environments.

Complete Genome Sequence of Geobacter sulfurreducens Strain YM18, Isolated from River Sediment in Japan.

Geobacter sulfurreducens is known to be a dominant species in the anode biofilms of microbial fuel cells. Here, we report the complete genome sequence of G. sulfurreducens strain YM18. Strain YM18 was isolated from a biofilm formed on an anode poised at -400 mV (versus an Ag/AgCl electrode) in a bioelectrochemical system.

Synergistic degradation of pyrene by five culturable bacteria in a mangrove sediment-derived bacterial consortium.

A pyrene-degrading microbial consortium was obtained after enrichment with mangrove sediment collected from Thailand. Five cultivable bacteria (Mycobacterium spp. PO1 and PO2, Novosphingobium pentaromativorans PY1, Ochrobactrum sp. PW1, and Bacillus sp. FW1) were successfully isolated from the consortium. Draft genomes of them showed that two different morphotypes of Mycobacterium (PO1 and PO2), possessed a complete gene set for pyrene degradation. PY1 contained genes for phthalate assimilation via protocatechuate, a central intermediate, by meta-cleavage pathway, and PW1 possessed genes for protocatechuate degradation via ortho-cleavage pathway. The occurrence of biosurfactant-producing genes in FW1 suggests the involvement in enhancing the pyrene bioavailability. Biotransformation experiments revealed that Mycobacterium completely degraded 100mgL-1 pyrene within six days, whereas no significant degradation was observed with the others. Notably, PY1 and PW1 exhibited higher activity for protocatechuate degradation than the others. The artificially reconstructed consortia containing Mycobacterium with the other three strains (PY1, PW1 and FW1) showed three-fold higher degradation rate for pyrene than the individual Mycobacterium. The enhanced pyrene biodegradation achieved in the consortium was due to the cooperative interaction of bacterial mixture. Our findings showing that synergistic degradation of pyrene in the consortium will facilitate the application of the defined bacterial consortium in bioremediation.

Establishment of disseminated intravascular coagulation (DIC) model by a single iv administration of Escherichia coli-derived lipopolysaccharide (LPS) to cynomolgus monkeys and evaluation of its pathophysiological status.

We prepared a DIC model by administrating LPS to cynomolgus monkeys, and investigated its potential for evaluations of new medicines for DIC therapy. Peripheral blood mononuclear cells (PBMC) collected from cynomolgus monkeys were incubated with LPS (8 types), and TNF-α levels in the media were measured. LPS from Escherichia coli (K-235) was most appropriate in terms of larger increases and smaller variation in TNF-α levels. PBMC from rats, cynomolgus monkeys or humans were incubated with LPS (K-235), and the TNF-α response to LPS was investigated. The response was comparable between cynomolgus monkeys and humans but small in rats. In an in vivo experiment, LPS (K-235) was administered once intravenously to cynomolgus monkeys with or without recombinant human thrombomodulin (rhTM) to investigate any changes in coagulation and fibrinolysis biomarkers and the suppressive effect of rhTM. The liver, kidney, and lung were examined histopathologically. Almost all of the changes resembled the pathophysiological status of human DIC and were suppressed by co-administration of rhTM. The DIC model resembling human DIC was established by LPS (K-235) treatment in cynomolgus monkeys, and therapeutic effect of rhTM was noted, suggesting that this model is useful in evaluations of the efficacy of new medicines for DIC therapy.

PARG Inhibitors and Functional PARG Inhibition Models.

Poly(ADP-ribose) polymerases (PARPs) family proteins catalyze poly(ADP-ribosylation) (PARylation) by conjugating ADP-ribose residues repeatedly on amino acid residues using nicotinamide adenine dinucleotide as a substrate. The inhibitors of PARP widely block DNA repair processes and are currently examined in clinical trials of cancer therapy. Poly(ADP-ribose) glycohydrolase (PARG) is the main nuclear enzyme, which digests poly(ADP-ribose) into ADP-ribose. PARG inhibitor could also be considered as a chemotherapeutic agent for cancer, because of its involvement in DNA repair. Various PARG inhibitors with IC50 value of micromolar to submicromolar range have been reported. However, for most of these chemicals, the specificity of inhibition has not been fully evaluated. PARG functional inhibition models in various organisms have been developed. Here, inducible PARG knockdown system was developed in HeLa cells and the cell line will be useful for identifying the synthetic lethal genes or affecting genes for PARG inhibitor treatment and also for functional elucidation of PARP superfamily molecules.