Annual phenology and migration routes to breeding grounds in western-central North Pacific sei whales.

The sei whale (Balaenoptera borealis) is an important species among baleen whales in the North Pacific and plays a significant role in the ecosystem. Despite the importance of this species, information regarding its migration patterns and breeding locations remains limited. To enhance the understanding of the phenology of North Pacific sei whales, we deployed satellite-monitored tags on these whales in the western and central North Pacific from 2017 to 2023. We fitted 55 sei whale tracks to a state-space model to describe the whales' seasonal movements at feeding grounds and their migratory behavior. The whales typically leave their feeding grounds between November and December, with migration pathways extending from off Japan to the west of the Hawaiian Islands. These southward transits converge in the waters of the Marshall Islands and north of Micronesia between 20° N and 7° N, which appear to be breeding grounds. After a brief stay at these breeding grounds, the whales migrate northward from January to February, reaching their feeding grounds around 30°N by March. To the best of our knowledge, this is the first study to present the phenology of feeding and breeding seasons and the migration pattern of North Pacific sei whales.

Successful excision of an inflammatory endobronchial polyp using biopsy forceps with improvement in FEV1 in a patient with allergic bronchopulmonary aspergillosis: A case report.

Inflammatory endobronchial polyps (IEPs) are rare, benign bronchial tumors posing diagnostic and therapeutic challenges owing to limited data. A 55-year-old man, receiving treatment for allergic bronchopulmonary aspergillosis, presented with a one-week history of fever and purulent sputum. Diagnosed with pneumonia, he received antimicrobial treatment. However, because of persistent symptoms, an endobronchial tumor was suspected on computed tomography. IEP was confirmed through flexible bronchoscopy with forceps biopsy, and polyp removal improved symptoms, lung function, and imaging.

Pretreatment asthma control test score as a predictive score for clinical remission after bronchial thermoplasty in younger patients with severe asthma and preserved lung function.

OBJECTIVE: Bronchial thermoplasty (BT) decreases the incidence of asthma exacerbations, emergency room visits, and hospitalizations among patients with severe asthma. Predictors of BT effectiveness remain unclear as its mechanism of action and invasiveness remain obscure. This study aimed to identify factors that could predict BT outcomes. METHODS: Two respiratory physicians treated 20 consecutive patients with severe asthma using BT. The patients were assigned to groups based on clinical remission following an expert consensus proposed in 2020. Predictors of clinical remission were analyzed using asthma control test (ACT) score, pulmonary function and blood tests, and fractional exhaled nitric oxide. RESULTS: At baseline, the median age was 44 years (interquartile range [IQR], 31.0-52.8), and pre-bronchodilator (pre-BD) percent predicted forced expiratory volume in one second (%FEV1) was 85.9% (IQR, 74.8-100.5). Six (30%) patients achieved clinical remission. Among the patients treated with biologics, 20% had clinical remission, and 20% discontinued biologic therapy. The pre-BT ACT score was significantly lower in the group with than without remission (11.0 [IQR, 8.0-14.5] vs. 15.0 [IQR, 11.0-17.3], p = .016). Adverse events did not significantly differ between the groups. CONCLUSIONS: To the best of our knowledge, this is the first study to use clinical remission as a criterion for evaluating BT efficacy. The pre-BT ACT score might a the predict response to BT in younger adult patients with severe asthma and pre-BD %FEV1 ≥ 70%.

Disruption of SMC-related genes promotes recombinant cholesterol esterase production in Burkholderia stabilis.

Burkholderia stabilis strain FERMP-21014 secretes cholesterol esterase (BsChe), which is used in clinical settings to determine serum cholesterol levels. Previously, we constructed an expression plasmid with an endogenous constitutive promoter to enable the production of recombinant BsChe. In this study, we obtained one mutant strain with 13.1-fold higher BsChe activity than the wild type, using N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen. DNA-sequencing analysis revealed that the strain had lost chromosome 3 (∆Chr3), suggesting that the genes hindering BsChe production may be encoded on Chr3. We also identified common mutations in the functionally unknown BSFP_068720/30 genes in the top 10 active strains generated during transposon mutagenesis. As BSFP_068720/30/40 comprised an operon on Chr3, we created the BSFP_068720/30/40 disruption mutant and confirmed that each disruption mutant containing the expression plasmid exhibited ~ 16.1-fold higher BsChe activity than the wild type. Quantitative PCR showed that each disruption mutant and ΔChr3 had a ~ 9.4-fold higher plasmid copy number than the wild type. Structural prediction models indicate that BSFP_068730/40 is structurally homologous to the structural maintenance of chromosomes (SMC) protein MukBE, which is responsible for chromosome segregation during cell division. Conversely, BSFP_068720/30/40 disruption did not lead to a Chr3 drop-out. These results imply that BSFP_068720/30/40 is not a SMC protein but is involved in destabilizing foreign plasmids to prevent the influx of genetic information from the environment. In conclusion, the disruption of BSFP_068720/30/40 improved plasmid stability and copy number, resulting in exceptionally high BsChe production. KEY POINTS: • Disruption of BSFP_068720/30/40 enabled mass production of Burkholderia Che/Lip. • BSFP_068730/40 is an SMC protein homolog not involved in chromosome retention. • BSFP_068720/30/40 is likely responsible for the exclusion of exogenous plasmids.

Enzymatic measurement of short-chain fatty acids and application in periodontal disease diagnosis.

Periodontal disease is a chronic inflammatory condition caused by periodontal pathogens in the gingival sulcus. Short-chain fatty acids (SCFAs) produced by causal bacteria are closely related to the onset and progression of periodontal disease and have been reported to proliferate in the periodontal sulcus of patients experiencing this pathology. In such patients, propionic acid (C3), butyric acid (C4), isobutyric acid (IC4), valeric acid (C5), isovaleric acid (IC5), and caproic acid (C6), henceforth referred to as [C3-C6], has been reported to have a detrimental effect, while acetic acid (C2) exhibits no detrimental effect. In this study, we established an inexpensive and simple enzymatic assay that can fractionate and measure these acids. The possibility of applying this technique to determine the severity of periodontal disease by adapting it to specimens collected from humans has been explored. We established an enzyme system using acetate kinase and butyrate kinase capable of measuring SCFAs in two fractions, C2 and [C3-C6]. The gingival crevicular fluid (GCF) and saliva of 10 healthy participants and 10 participants with mild and severe periodontal disease were measured using the established enzymatic method and conventional gas chromatography-mass spectrometry (GC-MS). The quantification of C2 and [C3-C6] in human GCF and saliva was well correlated when using the GC-MS method. Furthermore, both C2 and [C3-C6] in the GCF increased with disease severity. However, while no significant difference was observed between healthy participants and periodontal patients when using saliva, [C3-C6] significantly differed between mild and severe periodontal disease. The enzymatic method was able to measure C2 and [C3-C6] separately as well as using the GC-MS method. Furthermore, the C2 and [C3-C6] fractions of GCF correlated with disease severity, suggesting that this method can be applied clinically. In contrast, the quantification of C2 and [C3-C6] in saliva did not differ significantly between healthy participants and patients with periodontal disease. Future studies should focus on inflammation rather than on tissue destruction.

Bacterial triacylglycerol lipase is a potential cholesterol esterase: Identification of a key determinant for sterol-binding specificity.

Cholesterol esterase (Che) from Burkholderia stabilis (BsChe) is a homolog of well-characterized and industrially relevant bacterial triacylglycerol lipases (Lips). BsChe is a rare bacterial Lip enzyme that exhibits practical Che activity and is currently used in clinical applications to determine total serum cholesterol levels. To investigate the sterol specificity of BsChe, we determined the X-ray structure of BsChe. We discovered a local structural change in the active-site cleft, which might be related to substrate binding and product release. We also performed molecular docking studies by using the X-ray models of BsChe and cholesterol linoleate (CLL), the most favorable substrate for BsChe. The results showed that the sterol moieties of reasonable CLL docking poses localized to a specific active-site cleft surface formed by Leu266 and Ile287, which are unconserved among Burkholderia Lip homologs. Site-directed mutagenesis identified these residues as essential for the Che activity of BsChe, and Leu or Ile substitution conferred marked Che activity to Burkholderia Lips. In particular, Burkholderia cepacia and Burkholderia ubonensis Lips with the V266L/L287I double mutation exhibited ~50-fold and 500-fold higher Che activities than those of the wild-type enzymes, respectively. These results provide new insights into the substrate-binding mechanisms and selectivities of bacterial Lips.

Regulation of Sporangium Formation, Spore Dormancy, and Sporangium Dehiscence by a Hybrid Sensor Histidine Kinase in Actinoplanes missouriensis: Relationship with the Global Transcriptional Regulator TcrA.

The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores. In response to water, the sporangia open and release the spores into external environments. The orphan response regulator TcrA functions as a global transcriptional activator during sporangium formation and dehiscence. Here, we report the characterization of an orphan hybrid histidine kinase, HhkA. Sporangia of an hhkA deletion mutant contained many distorted or ectopically germinated spores and scarcely opened to release the spores under sporangium dehiscence-inducing conditions. These phenotypic changes are quite similar to those observed in a tcrA deletion mutant. Comparative RNA sequencing analysis showed that genes controlled by HhkA mostly overlap TcrA-regulated genes. The direct interaction between HhkA and TcrA was suggested by a bacterial two-hybrid assay, but this was not conclusive. The phosphorylation of TcrA using acetyl phosphate as a phosphate donor markedly enhanced its affinity for the TcrA box sequences in the electrophoretic mobility shift assay. Taking these observations together with other results, we proposed that HhkA and TcrA compose a cognate two-component regulatory system, which controls the transcription of the genes involved in many aspects of morphological development, including sporangium formation, spore dormancy, and sporangium dehiscence in A. missouriensisIMPORTANCEActinoplanes missouriensis goes through complex morphological differentiation, including formation of flagellated spore-containing sporangia, sporangium dehiscence, swimming of zoospores, and germination of zoospores to filamentous growth. Although the orphan response regulator TcrA globally activates many genes required for sporangium formation, spore dormancy, and sporangium dehiscence, its partner histidine kinase remained unknown. Here, we analyzed the function of an orphan hybrid histidine kinase, HhkA, and proposed that HhkA constitutes a cognate two-component regulatory system with TcrA. That HhkA and TcrA homologues are highly conserved among the genus Actinoplanes and several closely related rare actinomycetes indicates that this possible two-component regulatory system is employed for complex morphological development in sporangium- and/or zoospore-forming rare actinomycetes.

Production of recombinant extracellular cholesterol esterase using consistently active promoters in Burkholderia stabilis.

Burkholderia stabilis FERMP-21014 produces highly active cholesterol esterase in the presence of fatty acids. To develop an overexpression system for cholesterol esterase production, we carried out RNA sequencing analyses to screen strongly active promoters in FERMP-21014. Based on gene expression consistency analysis, we selected nine genes that were consistently expressed at high levels, following which we constructed expression vectors using their promoter sequences and achieved overproduction of extracellular cholesterol esterase under fatty acid-free conditions. Of the tested promoters, the promoter of BSFP_0720, which encodes the alkyl hydroperoxide reductase subunit AhpC, resulted in the highest cholesterol esterase activity (24.3 U mL-1). This activity level was 243-fold higher than that of the wild-type strain under fatty acid-free conditions. We confirmed that cholesterol esterase was secreted without excessive accumulation within the cells. The gene expression consistency analysis will be useful to screen promoters applicable to the overexpression of other industrially important enzymes.

Regulation of sporangium formation by the orphan response regulator TcrA in the rare actinomycete Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores, which can swim in aquatic environments after release from sporangium. However, gene regulation for its characteristic morphological development is largely unknown. Here, we report the functional analysis of an orphan response regulator, TcrA, which is encoded next to the chemotaxis-flagellar gene cluster. The tcrA null (ΔtcrA) mutant formed sporangium, in which sporulation proceeded. However, many distorted spores were produced and some spores ectopically germinated in the mutant sporangia. In addition, spores were hardly released from the mutant sporangia. A comparative RNA-Seq analysis between the wild-type and ΔtcrA strains showed that TcrA upregulated the transcription of more than 263 genes, which were integrated into 185 transcriptional units. In silico searches identified a 21-bp direct repeat sequence, 5'-nnGCA(A/C)CCG-n4 -GCA(A/C)CCGn-3', as the TcrA box, which was confirmed by electrophoretic mobility shift assays. Finally, we identified 34 transcriptional units as the TcrA regulon. TcrA seems to regulate a few hundred genes through the transcriptional activation of three FliA-family sigma factor genes besides its own regulon. We concluded that TcrA is a global transcriptional activator that controls many aspects of sporangium formation, including flagellar biogenesis, spore dormancy and sporangium dehiscence.

Characterization and application of a novel nicotinamide mononucleotide adenylyltransferase from Thermus thermophilus HB8.

Herein, we describe a novel enzymatic cycling method to measure nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN), which are precursors of NAD biosynthesis. A gene encoding an NMN adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified in Thermus thermophilus HB8. The gene from T. thermophilus (TtNMNAT) was engineered for expression in Escherichia coli and the recombinant enzyme found to be stable, retaining full activity after incubation for 45 min at 70°C. The Km values for NMN and ATP were calculated to be 0.263 and 1.27 mM, respectively, with a Vmax value of 60.3 µmoL/min/mg. TtNMNAT was successfully applied to the colorimetric NMN or NaMN assays, which employed (i) adenylation of NMN to NAD by TtNMNAT or adenylation of NaMN to deamido-NAD (NaAD) by TtNMNAT followed by amidation of NaAD to NAD by NAD synthetase (NADS, EC 6.3.1.5) and (ii) an NAD cycling reaction using 12α-hydroxysteroid dehydrogenase (12α-HSD, EC 1.1.1.176) and diaphorase (DI, EC 1.6.99.3) to accumulate reduced WST-8. This enzymatic cycling method enabled detection of 0.5 µM (12.2 nM in the reaction mixture) NMN or NaMN in an automatic clinical analyzer.

Complete Genome Sequence of Burkholderia stabilis FERMP-21014.

Cholesterol esterase (EC 3.1.1.13) was identified in a bacterium, Burkholderia stabilis strain FERMP-21014. Here, we report the complete genome sequence of B. stabilis FERMP-21014, which has been used in the commercial production of cholesterol esterase. The genome sequence information may be useful for improving production levels of cholesterol esterase.

Regulation of Sporangium Formation by BldD in the Rare Actinomycete Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms sporangia, including hundreds of flagellated spores that start swimming as zoospores after their release. Under conditions suitable for vegetative growth, zoospores stop swimming and germinate. A comparative proteome analysis between zoospores and germinating cells identified 15 proteins that were produced in larger amounts in germinating cells. They include an orthologue of BldD (herein named AmBldD [BldD of A. missouriensis]), which is a transcriptional regulator involved in morphological development and secondary metabolism in Streptomyces AmBldD was detected in mycelia during vegetative growth but was barely detected in mycelia during the sporangium-forming phase, in spite of the constant transcription of AmbldD throughout growth. An AmbldD mutant started to form sporangia much earlier than the wild-type strain, and the resulting sporangia were morphologically abnormal. Recombinant AmBldD bound a palindromic sequence, the AmBldD box, located upstream from AmbldD 3',5'-Cyclic di-GMP significantly enhanced the in vitro DNA-binding ability of AmBldD. A chromatin immunoprecipitation-sequencing analysis and an in silico search for AmBldD boxes revealed that AmBldD bound 346 genomic loci that contained the 19-bp inverted repeat 5'-NN(G/A)TNACN(C/G)N(G/C)NGTNA(C/T)NN-3' as the consensus AmBldD-binding sequence. The transcriptional analysis of 27 selected AmBldD target gene candidates indicated that AmBldD should repress 12 of the 27 genes, including bldM, ssgB, whiD, ddbA, and wblA orthologues. These genes are involved in morphological development in Streptomyces coelicolor A3(2). Thus, AmBldD is a global transcriptional regulator that seems to repress the transcription of tens of genes during vegetative growth, some of which are likely to be required for sporangium formation.IMPORTANCE The rare actinomycete Actinoplanes missouriensis undergoes complex morphological differentiation, including sporangium formation. However, almost no molecular biological studies have been conducted on this bacterium. BldD is a key global regulator involved in the morphological development of streptomycetes. BldD orthologues are highly conserved among sporulating actinomycetes, but no BldD orthologues, except one in Saccharopolyspora erythraea, have been studied outside the streptomycetes. Here, it was revealed that the BldD orthologue AmBldD is essential for normal developmental processes in A. missouriensis The AmBldD regulon seems to be different from the BldD regulon in Streptomyces coelicolor A3(2), but they share four genes that are involved in morphological differentiation in S. coelicolor A3(2).

Preliminary evaluation of an improved enzymatic assay method for measuring potassium concentrations in serum.

BACKGROUND: K(+) has important physiological functions. K(+) concentrations in serum are generally determined using ion-selective electrodes (ISEs), though measurement using reagents in aqueous medium is also useful. METHODS: K(+) concentrations were measured using recombinant inosine 5'-monophosphate dehydrogenase (IMPDH), which was activated only by K(+) and NH4(+). Exogenous NH4(+) and endogenous NH4(+) were eliminated using glutamine synthase. RESULTS: Regression analysis of the enzymatic assay (y) vs. the ISE method (x) gave the following relation: y=1.03x+0.09 (n=54, Sy,x=0.06 mmol/l). The linear range was up to 12 mmol/l when 1 U/ml IMPDH was used. CONCLUSION: Advantages of the proposed assay method are: (i) the measured range is wider than that of existing enzymatic methods; (ii) the conditions for K(+) determination can be maintained constant, regardless of the amount of NH4(+) in the analyte and reagents; and (iii) the elimination system is simpler because the recombinant IMPDH is stimulated by only K(+) and NH4(+) and is unaffected by biological materials.

[An autopsy case of tension pneumothorax due to the rupture of intrapulmonary cavity by mucormycosis during treatment with a ventilator].

 A 50-year-old man with chronic renal failure (hemodialysis treatment) and interstitial pneumonia (IP) was referred to our hospital for exacerbation of IP. We immediately administered a mechanical ventilation, broad spectrum antibiotics, steroid pulse therapy, and endoxan pulse therapy in the intensive care unit, but alveolar opacities became worse. Subsequently, an intrapulmonary cavity appeared in the left middle lung field on the chest X-ray and we also administered amphotericin B. However he died of tension pneumothorax on the tenth day of hospitalization. In an autopsy the rupture of the intrapulmonary cavity of the left S3 region was detected and we diagnosed as invasive pulmonary mucormycosis by Grocott stain of the cavitary lesion. We report a rare case that complicated by fatal tension pneumothorax during treatment with a ventilator in invasive pulmonary mucormycosis and review the literature.

[Pulmonary hypertension in a patient on chronic hemodialysis].

A 52-year-old woman with end-stage renal disease and long-term hemodialysis complained of worsening exertional dyspnea. A chest X-ray showed cardiomegaly. She was admitted to our hospital because an echocardiogram suggested pulmonary hypertension. Right heart catheterization revealed pulmonary hypertension, but pulmonary perfusion scintigraphy with Tc-99m-MAA showed no evidence of pulmonary thromboembolism. We gave her a diagnosis of pulmonary arterial hypertension associated with Sjögren syndrome on the basis of a positive serological test (SS-A. SS-B) and the findings of lip biopsy. After four months of therapy with bosentan, her 6-minute walk distance, estimated pulmonary arterial pressure and brain natriuretic peptide (BNP) improved. Bosentan is mainly cleared by hepatic elimination and its dialysis clearance is low. Bosentan for the treatment of pulmonary hypertension was safe as well as effective in this patient with end-stage renal disease and hemodialysis. In consideration of the relationship between pulmonary hypertension and end-stage renal disease, and hemodialysis, bosentan was considered to be a reasonable and effective treatment.

Epidemiological survey of Babesia gibsoni infection in dogs in Japan by enzyme-linked immunosorbent assay using B. gibsoni thrombospondin-related adhesive protein antigen.

A nationwide epidemiological survey of Babesia gibsoni infection in non-fighting dogs was conducted using an improved ELISA with recombinant B. gibsoni thrombospondin-related adhesive protein (BgTRAP). A total of 1206 dogs from 27 prefectures were examined and 128 (10.6%) tested positive. In the eastern part of Japan, 39 dogs out of the 559 (7.0%) examined were positive, while 89 dogs out of 647 (13.8%) tested positive in the western part of Japan. Although the percentage of dogs that tested positive was significantly (p=0.0001) lower in the eastern part compared to the western part of Japan, overall these results indicate that B. gibsoni infection of dogs has a widespread geographic distribution throughout the country. A history of tick infestation was identified as a significant risk factor for B. gibsoni infection (p=0.0091), while sex (p=0.9411), age (p=0.0920) and breed (p=0.0549) of dogs were not statistically significant risk factors. These results indicate that tick infestation is the most dominant risk factor for B. gibsoni infection of non-fighting dogs in Japan and suggest that other B. gibsoni transmission routes, such as fighting and transplacental transmission, may be less important.

Eosinophilic cellulitis as a cutaneous manifestation of idiopathic hypereosinophilic syndrome.

Three patients with eosinophilic cellulitis associated with sustained peripheral blood eosinophilia of unidentified cause are reported. They also presented with diversities of extracutaneous symptoms such as bronchospasm, sensory polyneuropathies, epigastralgia, and gangrenous eosinophilic enteritis. These cases suggest that eosinophilic cellulitis can develop as a cutaneous manifestation of idiopathic hypereosinophilic syndrome.

Relapsing coalescent papular dermatosis of elderly patients: a precursor or an abortive lesion of Ofuji papuloerythroderma.

We describe a group of elderly patients (average: 71.1 year-old, 37 male and 6 female patients) with relapsing, coalescent, papular dermatosis. The skin rash started as intensely pruritic, discrete, solid papules developing in crops in a fairly localized area. The primary papular lesions tended to coalesce into flat-topped, polygonal papules or plaques that often showed a cobble stone-like appearance. The disease took a protracted course of repeated relapses without known precipitating factors. Both papular and coalescent plaque lesions showed compact perivascular infiltration of mononuclear cells containing varying populations of eosinophils in the upper dermis. Approximately half of the patients had mild hypereosinophilia and elevated serum IgE levels without histories of atopic diathesis. These clinicopathological features were consistent with those of the papular lesions that Ofuji originally defined as the primary skin rash of papuloerythroderma. We propose that our cases represent a precursor or an abortive lesion of Ofuji papuloerythroderma.