Clustering evaluation of water quality for various classes of in-flow rivers in connected brackish lakes.

Brackish waters and estuaries at the lower reaches of rivers accumulate organic matter and nutrients from various sources in the watershed. Sufficient light and shallow water depth stimulate phytoplankton growth, resulting in a more diversified ecosystem with higher trophic levels. For effective watershed management, it is crucial to characterize the water quality of all rivers, including small and medium-sized ones. Our field survey assessed water quality parameters in 26 inflow rivers surrounding Lakes Shinji and Nakaumi, two consolidated brackish lakes in Japan. The parameters included water temperature, salinity, chlorophyll-a, and nutrients. The study used hierarchical clustering. The Silhouette Index was used to assess clustering outcomes and identify any difficulties in dispersion across clusters. The 26 rivers surrounding Lakes Shinji and Nakaumi were classified into six groups based on their water quality characteristics. This classification distinguishes itself from earlier subjective methods that relied on geographical factors. The new approach identifies a need for improved management of river water quality. The results of the cluster analysis provide valuable insights for future management initiatives. It is important to consider these findings alongside established watershed criteria.

[A Case of Intrahepatic Cholangiocarcinoma in Contact with the Gastroduodenal Anastomosis in Which Proton Beam Therapy Was Performed after Placement of an Absorbable Spacer].

Chemotherapy is the standard therapy for unresectable intrahepatic cholangiocarcinoma(ICC), but chemotherapy is not efficacious. Proton beam therapy(PBT)has been covered by Japanese health insurance for ICC since 2022, and the number of cases is expected to increase. In some cases, irradiation is difficult due to the close proximity of the gastrointestinal tract to the tumor. We report our management of a patient with ICC close to the gastrointestinal tract. The patient was a 69-year- old woman with a history of distal gastrectomy and Billroth-Ⅰ reconstruction for gastric cancer. A CT scan showed a tumor in liver S3; a biopsy revealed ICC. Because the tumor was in contact with the gastroduodenal anastomosis, we placed an absorbable spacer and performed PBT. After the treatment, the tumor shrank slightly. Although the liver is anatomically adjacent to the digestive tract, the placement of absorbable spacers facilitates performing PBT without adverse events, and is thus considered a useful treatment.

Phylogenetic Analysis and Characterization of Odorous Compound-Producing Actinomycetes in Sediments in the Sanbe Reservoir, A Drinking Water Reservoir in Japan.

Odor caused by the presence of geosmin and 2-methylisoborneol (2-MIB) in aquatic ecosystems leads to considerable economic loss worldwide. The odorous compounds are primarily produced by cyanobacteria and actinomycetes. While the contribution of odorous compounds-producing cyanobacteria has been thoroughly investigated, the production of geosmin and 2-MIB by actinomycetes in aquatic ecosystems is poorly understood. In this study, we isolated geosmin and/or 2-MIB-producing actinomycetes in sediments collected from the Sanbe Reservoir, Japan, identified the biosynthetic gene of geosmin and 2-MIB, and investigated the production of the odorous compounds by the isolated strains. Partial sequence of 16S rRNA and the biosynthetic genes was determined to analyze the phylogenetic relationship among the strains. The geosmin and 2-MIB concentrations in the culture of the isolated strains were measured using gas chromatography mass spectrometry. Fifty-four strains of odorous compounds-producing and non-geosmin-producing actinomycetes were isolated from sediments from the Sanbe Reservoir. Diverse actinomycetes were identified and many of them produced geosmin and/or 2-MIB. Many odorous compounds-producing actinomycetes were phylogenetically different from previously reported producing actinomycetes. The producing ability of the odorous compounds of the isolated strains in this study was not significantly related with the phylogenetic groups of 16S rRNA and the biosynthetic genes. The findings suggest that the odorous compounds-producing actinomycetes in the sediments are diverse and different from previously reported strains.

Spectral imaging of pharmaceutical materials with a compact terahertz difference-frequency generation semiconductor source.

Spectral imaging of pharmaceutical material using a compact ultra-broadband (1-4 THz) terahertz semiconductor source was demonstrated. False-color RGB images could be obtained using a simple procedure (calibration free). The ability to distinguish the polymorphism of carbamazepine (CBZ), the hydrate forms of D-(+)-glucose and caffeine, and the crystallinity of nifedipine was demonstrated using the THz DFG source. Crystal forms of pharmaceutical materials can be distinguished using this method.

Multiple Gene Clusters and Their Role in the Degradation of Chlorophenoxyacetic Acids in Bradyrhizobium sp. RD5-C2 Isolated from Non-Contaminated Soil.

Bradyrhizobium sp. RD5-C2, isolated from soil that is not contaminated with 2,4-dichlorophenoxyacetic acid (2,4-D), degrades the herbicides 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). It possesses tfdAα and cadA (designated as cadA1), which encode 2,4-D dioxygenase and the oxygenase large subunit, respectively. In the present study, the genome of Bradyrhizobium sp. RD5-C2 was sequenced and a second cadA gene (designated as cadA2) was identified. The two cadA genes belonged to distinct clusters comprising the cadR1A1B1K1C1 and cadR2A2B2C2K2S genes. The proteins encoded by the cad1 cluster exhibited high amino acid sequence similarities to those of other 2,4-D degraders, while Cad2 proteins were more similar to those of non-2,4-D degraders. Both cad clusters were capable of degrading 2,4-D and 2,4,5-T when expressed in non-2,4-D-degrading Bradyrhizobium elkanii USDA94. To examine the contribution of each degradation gene cluster to the degradation activity of Bradyrhizobium sp. RD5-C2, cadA1, cadA2, and tfdAα deletion mutants were constructed. The cadA1 deletion resulted in a more significant decrease in the ability to degrade chlorophenoxy compounds than the cadA2 and tfdAα deletions, indicating that degradation activity was primarily governed by the cad1 cluster. The results of a quantitative reverse transcription-PCR analysis suggested that exposure to 2,4-D and 2,4,5-T markedly up-regulated cadA1 expression. Collectively, these results indicate that the cad1 cluster plays an important role in the degradation of Bradyrhizobium sp. RD5-C2 due to its high expression.

Recent advances in functional analysis of polyunsaturated fatty acid synthases.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid, eicosapentaenoic acid, and arachidonic acid are essential fatty acids for humans. PUFAs are biosynthesized by either desaturases/elongases from oleic acid or PUFA synthases from acetyl units. PUFA synthases are composed of three or four subunits, and each creates a specific PUFA even though the multiple catalytic domains in each subunit are very similar. We recently dissected these PUFA synthases by in vivo and in vitro experiments and elucidated how the enzymes control PUFA profiles. Moreover, for the first time, we converted a practical microalgal docosahexaenoic acid synthase into an eicosapentaenoic acid synthase based on the results.

Off-Loading Mechanism of Products in Polyunsaturated Fatty Acid Synthases.

Marine microorganisms de novo biosynthesize polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid by polyunsaturated fatty acid (PUFA) synthases composed of three or four polypeptides in a manner similar to fatty acid synthases (FASs). FASs usually possess thioesterase (TE) domains to release free fatty acids from acyl carrier protein (ACP)-tethered intermediates. Here, we investigated the off-loading mechanism with microalgal and bacterial PUFA synthases through in vivo and in vitro experiments. The in vitro experiments with acyltransferase (AT)-like domains and acyl-ACP substrates clearly demonstrated that the AT-like domains catalyzed the hydrolysis of acyl-ACPs to yield free fatty acids.

Metagenomic study of endophytic bacterial community of sweet potato (Ipomoea batatas) cultivated in different soil and climatic conditions.

The aim of this study was to clarify effects of soil and climatic conditions on community structure of sweet potato bacterial endophytes by applying locked nucleic acid oligonucleotide-PCR clamping technique and metagenomic analysis. For this purpose, the soil samples in three locations were transferred each other and sweet potato nursery plants from the same farm were cultivated for ca. 3 months. After removal of plastid, mitochondria and undefined sequences, the averaged numbers of retained sequences and operational taxonomic units per sample were 20,891 and 846, respectively. Proteobacteria (85.0%), Bacteroidetes (6.6%) and Actinobacteria (6.3%) were the three most dominant phyla, accounting for 97.9% of the reads, and γ-Proteobacteria (66.3%) being the most abundant. Top 10 genera represented 81.2% of the overall reads in which Pseudomonas (31.9-45.0%) being the most predominant. The overall endophytic bacterial communities were similar among the samples which indicated that the soil and the climatic conditions did not considerably affect the entire endophytic community. The original endophytic bacterial community might be kept during the cultivation period.

Subtle Control of Carbon Chain Length in Polyunsaturated Fatty Acid Synthases.

Some marine bacteria synthesize docosahexaenoic acid (DHA; C22) and eicosapentaenoic acid (EPA; C20) by enzyme complexes composed of four subunits (A-D). We recently revealed that ß-ketoacyl synthase (KSC)/chain length factor (CLF)-like domains in the "C" subunit of DHA synthase catalyzed the last elongation step (C20 to C22) even though their amino acid sequences are very similar to those of EPA synthase. To investigate the amino acid residues controlling the product chain length, conserved residues in the KSC/CLF-like domains in DHA synthase were replaced with corresponding EPA synthase residues. Among 12 mutants, two CLF-like domain-mutated genes completely lost DHA productivity and produced trace amounts of EPA when coexpressed with dha-ABD in Escherichia coli, whereas when coexpressed with epa-ABD, they produced the same amounts of EPA as epa-ABCD. These results suggest that the product profiles were subtly controlled by several amino acid residues.

Identification of geosmin biosynthetic gene in geosmin-producing colonial cyanobacteria Coelosphaerium sp. and isolation of geosmin non-producing Coelosphaerium sp. from brackish Lake Shinji in Japan.

Geosmin is an earthy-muddy smelling compound produced in aquatic ecosystems by microorganisms including cyanobacteria. An increase in geosmin levels affecting the local fishery occurred in May 2007 in Lake Shinji, Japan, and geosmin-producing colonial cyanobacterium, Coelosphaerium sp. G2, was isolated from a water sample from the lake and identified. Cyanobacteria Coelosphaerium sp. is commonly found in Lake Shinji; however, prior to 2007, earthy-muddy odors were not a frequent issue. Further, there was no information regarding the geosmin biosynthetic gene in colonial cyanobacteria. Here, the geosmin biosynthetic gene (geoA) in strain G2 was identified and its nucleotide sequence was determined. It was found that geoA had 79% and 78% identity with geoA from filamentous geosmin-producing cyanobacteria Fischerella sp. PCC 9431 and geoA2 from Phormidium sp. P2r, respectively. The deduced amino acid sequence of GeoA consisted of two domains that were annotated as terpene cyclase. In 2015, geosmin non-producing Coelosphaerium sp. S3C5 was isolated from Lake Shinji and identified by morphological and genetic analyses. There was no difference in morphology or nucleotide sequences of 16S rRNA and 16S-23S internal transcribed spacer (ITS) between geosmin-producing and non-producing strains, which are therefore closely related and can exist in Lake Shinji. Distinguishing the two strains by observation under a microscope and sequencing of 16S rRNA and 16S-23S ITS have proven difficult. Inconsistency between the appearance of Coelosphaerium cells and the detection of the odor in water samples could therefore be attributed to dominance by the geosmin-producing strain or the non-producing strain. The increase in earthy smell is assumed to be caused by an increase in the geosmin-producing strain in Lake Shinji. Genetic analysis of geoA in Coelosphaerium sp. and the relative abundances of geosmin-producing and non-producing Coelosphaerium strains in Lake Shinji can be used to mitigate the economic damages caused by geosmin. Development of a molecular method to monitor the geosmin-producing strain in water ecosystems is equally important to alleviate the earthy smell caused by this particular strain.

Regional differences in facial skin blood flow responses to thermal stimulation.

PURPOSE: The facial skin blood flow (SkBF) shows regional differences in the responses to a given stimulation. The facial SkBFs, especially in the eyelid and nose exhibit unique response to physiological and psychological stimuli, but the mechanisms inducing those regional differences remain unclear. To investigate whether the regional differences in the local control of vasomotion in facial vessels correspond to the regional differences in facial SkBF response, we monitored the relative change of facial SkBF to regional thermal stimulation. We hypothesized that heat stimulation dilates the cutaneous vessels in the eyelid, while cold stimulation constricts those in the nose, which was based on previous findings METHODS: A thermal stimulator was used to apply temperature increase (from 20 to 40 °C at 2 °C/min) and decrease (from 40 to 20 °C at 2°C/min) in a randomized order to the right eyelid, nose, right cheek, and forehead of 14 healthy young males. The facial SkBF was measured for 10 s using laser-speckle flowgraphy when temperatures of 20 °C, 30 °C, and 40 °C had been applied for 30 s in both trials. RESULTS: The SkBF in the eyelid did not change significantly during any thermal stimulation, and the nasal SkBF did not decrease significantly during cold stimulation. The SkBFs in the cheek and forehead increased significantly with the applied temperature. CONCLUSIONS: These findings indicate that a large regional variation exists in facial skin blood flow response to local heating or cooling and that the regional variation did not correspond to the unique SkBF responses in the previous studies.

Control Mechanism for Carbon-Chain Length in Polyunsaturated Fatty-Acid Synthases.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are essential fatty acids. PUFA synthases are composed of three to four subunits and each create a specific PUFA without undesirable byproducts. However, detailed biosynthetic mechanisms for controlling final product profiles have been obscure. Here, the bacterial DHA and EPA synthases were carefully dissected by in vivo and in vitro experiments. In vitro analysis with two KS domains (KSA and KSC ) and acyl-acyl carrier protein (ACP) substrates showed that KSA accepted short- to medium-chain substrates while KSC accepted medium- to long-chain substrates. Unexpectedly, condensation from C18 to C20 , the last elongation step in EPA biosynthesis, was catalyzed by KSA domains in both EPA and DHA synthases. Conversely, condensation from C20 to C22 , the last elongation step for DHA biosynthesis, was catalyzed by the KSC domain in DHA synthase. KSC domains therefore determine the chain lengths.

Control Mechanism for cis Double-Bond Formation by Polyunsaturated Fatty-Acid Synthases.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans. Some microorganisms biosynthesize these PUFAs through PUFA synthases composed of four subunits with multiple catalytic domains. These PUFA synthases each create a specific PUFA without undesirable byproducts, even though the multiple catalytic domains in each large subunit are very similar. However, the detailed biosynthetic pathways and mechanisms for controlling final-product profiles are still obscure. In this study, the FabA-type dehydratase domain (DHFabA ) in the C-subunit and the polyketide synthase-type dehydratase domain (DHPKS ) in the B-subunit of ARA synthase were revealed to be essential for ARA biosynthesis by in vivo gene exchange assays. Furthermore, in vitro analysis with truncated recombinant enzymes and C4 - to C8 -acyl ACP substrates showed that ARA and EPA synthases utilized two types of DH domains, DHPKS and DHFabA , depending on the carbon-chain length, to introduce either saturation or cis double bonds to growing acyl chains.

Longitudinal changes and body composition assessment using bioelectrical impedance in elderly patients with mild disequilibrium and different care needs.

[Purpose] This study involved performing longitudinal measurements of muscle mass in elderly patients with mild disequilibrium using a body composition meter. The rate of change and characteristics were determined according to the level of care needed. [Participants and Methods] Bioelectrical impedance was used to measure body composition in 20 elderly females in Care Needs Category 1 (n=10) and 2 (n=8); body composition was measured every 3 months for 1 year. [Results] Compared to Category 1, the muscle mass at each body site was lower in Category 2 and the muscle mass of the whole body and thighs in Category 2 decreased throughout the year. [Conclusion] Muscle mass in elderly patients needing assistance depended on the level of care, as suggested by the decrease in muscle mass in the whole body and thighs in Category 2 over time. In addition, effective rehabilitation intervention for the trunk is important.

Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo.

Goadsporin (GS) is a member of ribosomally synthesized and post-translationally modified peptides (RiPPs), containing an N-terminal acetyl moiety, six azoles and two dehydroalanines in the peptidic main chain. Although the enzymes involved in GS biosynthesis have been defined, the principle of how the respective enzymes control the specific modifications remains elusive. Here we report a one-pot synthesis of GS using the enzymes reconstituted in the 'flexible' in vitro translation system, referred to as the FIT-GS system. This system allows us to readily prepare not only the precursor peptide from its synthetic DNA template but also 52 mutants, enabling us to dissect the modification determinants of GodA for each enzyme. The in vitro knowledge has also led us to successfully produce designer GS analogues in vivo. The methodology demonstrated in this work is also applicable to other RiPP biosynthesis, allowing us to rapidly investigate the principle of modification events with great ease.

Enhanced production of polyunsaturated fatty acids by enzyme engineering of tandem acyl carrier proteins.

In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A. These ACPs were previously shown to be important for PUFA productivity. In this study, we examined their function in more detail. PUFA productivities increased depending on the number of ACPs without profile changes in each subunit A of eukaryotic and prokaryotic PUFA synthases. We also constructed derivative enzymes from subunit A with 5 × ACPs. Enzymes possessing one inactive ACP at any position produced ~30% PUFAs compared with the parental enzyme but unexpectedly had ~250% productivity compared with subunit A with 4 × ACPs. Enzymes constructed by replacing the 3rd ACP with an inactive ACP from another subunit A or ACP-unrelated sequences produced ~100% and ~3% PUFAs compared with the parental 3rd ACP-inactive enzyme, respectively. These results suggest that both the structure and number of ACP domains are important for PUFA productivity.

2,4-Dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading gene cluster in the soybean root-nodulating bacterium Bradyrhizobium elkanii USDA94.

Herbicides 2,4-dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading Bradyrhizobium strains possess tfdAα and/or cadABC as degrading genes. It has been reported that root-nodulating bacteria belonging to Bradyrhizobium elkanii also have tfdAα and cadA like genes but lack the ability to degrade these herbicides and that the cadA genes in 2,4-D-degrading and non-degrading Bradyrhizobium are phylogenetically different. In this study, we identified cadRABCK in the genome of a type strain of soybean root-nodulating B. elkanii USDA94 and demonstrated that the strain could degrade the herbicides when cadABCK was forcibly expressed. cadABCK-cloned Escherichia coli also showed the degrading ability. Because co-spiked phenoxyacetic acid (PAA) could induce the degradation of 2,4-D in B. elkanii USDA94, the lack of degrading ability in this strain was supposed to be due to the low inducing potential of the herbicides for the degrading gene cluster. On the other hand, tfdAα from B. elkanii USDA94 showed little potential to degrade the herbicides, but it did for 4-chlorophenoxyacetic acid and PAA. The 2,4-D-degrading ability of the cad cluster and the inducing ability of PAA were confirmed by preparing cadA deletion mutant. This is the first study to demonstrate that the cad cluster in the typical root-nodulating bacterium indeed have the potential to degrade the herbicides, suggesting that degrading genes for anthropogenic compounds could be found in ordinary non-degrading bacteria.

Insights into the Biosynthesis of Dehydroalanines in Goadsporin.

Dehydroalanines in goadsporin are proposed to be formed by GodF and GodG, which show slight homology to the N-terminal glutamylation and C-terminal elimination domains, respectively, of LanB, a class I lanthipeptide dehydratase. Although similar, separated-type LanBs are conserved among thiopeptides and indispensable for their biosynthesis and biological activities, these enzymes had not yet been characterized. Here, we identified goadsporin B, which has unmodified Ser4 and Ser14, from both godF and godG disruptants. The godG disruptant also produced goadsporin C, a glutamylated-Ser4 variant of goadsporin B. These results suggested that dehydroalanines are formed by glutamylation and glutamate elimination. NMR analysis revealed for the first time that the glutamyl group was attached to a serine via an ester bond, by the catalysis of LanB-type enzymes. Our findings provide insights into the function of separated-type LanBs involved in the biosynthesis of goadsporin and thiopeptides.

Genes of Bacillus subtilis 168 that Support Growth of the Cyanobacterium, Synechococcus leopoliensis CCAP1405/1 on Agar Media.

Synechococcus leopoliensis CCAP1405/1 cannot grow on common solid media; however, the strain can grow when co-cultured with Bacillus subtilis 168. Gene-disruptant strains of B. subtilis 168 by pMUTINs at the following sites lost the ability to support the growth of S. leopoliensis CCAP1405/1 on agar media: yxeO and yufO (transporter), yxdK (histidine kinase), sdhC (succinate dehydrogenase), yvgQR (sulfite reductase), acoB (acetoin dehydrogenase), yusE (thioredoxin), yrdA (function unknown). Involvement of the assimilatory sulfate reduction pathway was the suggested reason for loss of the function.