Greedy reduction of Bacillus subtilis genome yields emergent phenotypes of high resistance to a DNA damaging agent and low evolvability.

Genome-scale engineering enables rational removal of dispensable genes in chassis genomes. Deviating from this approach, we applied greedy accumulation of deletions of large dispensable regions in the Bacillus subtilis genome, yielding a library of 298 strains with genomes reduced up to 1.48 Mb in size. High-throughput physiological phenotyping of these strains confirmed that genome reduction is associated with substantial loss of cell fitness and accumulation of synthetic-sick interactions. Transcriptome analysis indicated that <15% of the genes conserved in our genome-reduced strains exhibited a twofold or higher differential expression and revealed a thiol-oxidative stress response. Most transcriptional changes can be explained by loss of known functions and by aberrant transcription at deletion boundaries. Genome-reduced strains exhibited striking new phenotypes relative to wild type, including a very high resistance (increased >300-fold) to the DNA-damaging agent mitomycin C and a very low spontaneous mutagenesis (reduced 100-fold). Adaptive laboratory evolution failed to restore cell fitness, except when coupled with a synthetic increase of the mutation rate, confirming low evolvability. Although mechanisms underlying this emergent phenotype are not understood, we propose that low evolvability can be leveraged in an engineering strategy coupling reductive cycles with evolutive cycles under induced mutagenesis.

An unusual presentation of dermatofibrosarcoma protuberans: A case of fibrosarcomatous dermatofibrosarcoma protuberans with pleomorphic angiectatic tumor-like changes.

Dermatofibrosarcoma protuberans (DFSP) is a locally aggressive superficial mesenchymal neoplasm characterized by monomorphic spindle-cell proliferation with a storiform pattern. It can demonstrate pigmentation, myxoid changes, myoid differentiation, plaque-like growth, and fibrosarcomatous features; its varied presentation often complicates diagnosis. We report an extremely rare case of fibrosarcomatous DFSP with features reminiscent of a pleomorphic hyalinizing angiectatic tumor (PHAT) in a 73-year-old male. The diagnosis was confirmed using a reverse transcription polymerase chain reaction. To the best of our knowledge, PHAT-like changes in DFPS have not been described so far. Therefore, this report provides a novel variant of DFSP and expands the differential diagnosis of DFSP and PHAT.

Genes encoding a novel thermostable bacteriocin in the thermophilic bacterium Aeribacillus pallidus PI8.

AIM: Aeribacillus pallidus PI8 is a Gram-positive thermophilic bacterium that produces thermostable antimicrobial substances against several bacterial species, including Geobacillus kaustophilus HTA426. In the present study, we sought to identify genes of PI8 with antibacterial activity. METHODS AND RESULTS: We isolated, cloned, and characterized a thermostable bacteriocin from A. pallidus PI8 and named it pallidocyclin. Mass spectrometric analyses of pallidocyclin revealed that it had a circular peptide structure, and its precursor was encoded by pcynA in the PI8 genome. pcynA is the second gene within the pcynBACDEF operon. Expression of the full-length pcynBACDEF operon in Bacillus subtilis produced intact pallidocyclin, whereas expression of pcynF in G. kaustophilus HTA426 conferred resistance to pallidocyclin. CONCLUSION: Aeribacillus pallidus PI8 possesses the pcynBACDEF operon to produce pallidocyclin. pcynA encodes the pallidocyclin precursor, and pcynF acts as an antagonist of pallidocyclin.

Constitutive glucose dehydrogenase elevates intracellular NADPH levels and luciferase luminescence in Bacillus subtilis.

BACKGROUND: Genetic modifications in Bacillus subtilis have allowed the conversion of myo-inositol into scyllo-inositol, which is proposed as a therapeutic agent for Alzheimer's disease. This conversion comprises two reactions catalyzed by two distinct inositol dehydrogenases, IolG and IolW. The IolW-mediated reaction requires the intracellular regeneration of NADPH, and there appears to be a limit to the endogenous supply of NADPH, which may be one of the rate-determining factors for the conversion of inositol. The primary mechanism of NADPH regeneration in this bacterium remains unclear. RESULTS: The gdh gene of B. subtilis encodes a sporulation-specific glucose dehydrogenase that can use NADP+ as a cofactor. When gdh was modified to be constitutively expressed, the intracellular NADPH level was elevated, increasing the conversion of inositol. In addition, the bacterial luciferase derived from Photorhabdus luminescens became more luminescent in cells in liquid culture and colonies on culture plates. CONCLUSION: The results indicated that the luminescence of luciferase was representative of intracellular NADPH levels. Luciferase can therefore be employed to screen for mutations in genes involved in NADPH regeneration in B. subtilis, and artificial manipulation to enhance NADPH regeneration can promote the production of substances such as scyllo-inositol.

A novel method for transforming Geobacillus kaustophilus with a chromosomal segment of Bacillus subtilis transferred via pLS20-dependent conjugation.

BACKGROUND: Geobacillus kaustophilus is a thermophilic Gram-positive bacterium. Methods for its transformation are still under development. Earlier studies have demonstrated that pLS20catΔoriT mobilized the resident mobile plasmids from Bacillus subtilis to G. kaustophilus and transferred long segments of chromosome from one cell to another between B. subtilis. RESULTS: In this study, we applied mobilization of the B. subtilis chromosome mediated by pLS20catΔoriT to transform G. kaustophilus. We constructed a gene cassette to be integrated into G. kaustophilus and designed it within the B. subtilis chromosome. The pLS20catΔoriT-mediated conjugation successfully transferred the gene cassette from the B. subtilis chromosome into the G. kaustophilus allowing for the desired genetic transformation. CONCLUSIONS: This transformation approach described here will provide a new tool to facilitate the flexible genetic manipulation of G. kaustophilus.

Identification of a repressor for the two iol operons required for inositol catabolism in Geobacillus kaustophilus.

Geobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, feeds on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster of G. kaustophilus comprises two tandem operons induced in the presence of inositol; however, the mechanism underlying this induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding scyllo-inositol dehydrogenase, and its homologue in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and was termed iolQ in G. kaustophilus. When iolQ was inactivated in G. kaustophilus, not only cellular myo-inositol dehydrogenase activity due to gk1899 expression but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal histidine (His)-tagged fusion protein in Escherichia coli and subjected to an in vitro gel electrophoresis mobility shift assay to examine its DNA-binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions and that DNA binding was antagonized by myo-inositol. Moreover, DNase I footprinting analyses identified two tandem binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to form a palindrome of 5'-RGWAAGCGCTTSCY-3' (where R=A or G, W=A or T, S=G or C, and Y=C or T). IolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.

Skeletal EWSR1-NFATC2 sarcoma previously diagnosed as Ewing-like adamantinoma: A case report and literature review emphasizing its unique radiological features.

Ewing-like adamantinoma (EAD) is a rare bone tumor. It remains unclear whether EAD belongs to adamantinoma, Ewing sarcoma (ES), or an independent category. Herein, we present a case of femoral sarcoma previously diagnosed as EAD in a 26-year-old woman. We observed amplified EWSR1 and NFATC2 fusion signals using fluorescence in situ hybridization. Prompted by its unique radiological features, we reviewed the current literature on skeletal EWSR1-NFATC2 sarcoma (ENS) and EAD. In addition to the similar histological features, we found that both ENS and EAD displayed similar characteristic radiological features, such as the tendency to occur in the diaphysis of long bones, cortical expansion and buttressing-type thickening, and bone surface involvement with saucer-like erosion without cortical destruction. We believe that these unique radiological features were related to its indolent behavior. Altogether, it is possible that previously reported EAD cases may be neither ES nor the classic adamantinoma but ENS. Further studies are needed to clarify the relationship between EAD and ENS.

Intermittent hypoxia induces turbinate mucosal hypertrophy via upregulating the gene expression related to inflammation and EMT in rats.

PURPOSE: Chronic intermittent hypoxia (IH) plays a pivotal role in the consequences of obstructive sleep apnea (OSA). It has been demonstrated that IH impairs nasomaxillary complex growth to reduce nasal airway cavity size in rodent models. Although turbinate dysfunction with inflammatory mucosal hypertrophy is related to OSA, the role of IH in turbinate hypertrophy with inflammation-driven fibrosis is unknown. Here, we aimed to clarify the pathogenesis of inflammatory mucosal hypertrophy and epithelial-mesenchymal transition (EMT) in the nasal turbinate under IH. METHODS: Seven-week-old male Sprague-Dawley rats were exposed to IH (4% O2 to 21% O2 with 0% CO2) at a rate of 20 cycles/h. RESULTS: Hypertrophy of the turbinate mucosa occurred after 3 weeks, with the turbinate mucosa of the experimental group becoming significantly thicker than in the control group. Immunostaining showed that IH increased the expression of TGFß and N-cadherin and decreased E-cadherin expression in the turbinate mucosa. Quantitative PCR analysis demonstrated that IH enhanced the expression of not only the inflammatory markers Tnf-a, Il-1b, and Nos2 but also the EMT markers Tgf-b1, Col1a1, and Postn. CONCLUSIONS: Collectively, these results suggest that IH induced turbinate hypertrophy via upregulation of gene expression related to inflammation and EMT in the nasal mucosa.

Intermittent hypoxia retards mandibular growth and alters RANKL expression in adolescent and juvenile rats.

OBJECTIVES: Chronic intermittent hypoxia (IH), a common state experienced in obstructive sleep apnoea (OSA), retards mandibular growth in adolescent rats. The aim of this study was to elucidate the differential effects of IH on mandibular growth in different growth stages. MATERIALS AND METHODS: Three-week-old (juvenile stage) and 7-week-old (adolescent stage) male Sprague-Dawley rats underwent IH for 3 weeks. Age-matched control rats were exposed to room air. Mandibular growth was evaluated by radiograph analysis, micro-computed tomography, real-time polymerase chain reaction and immunohistology. Tibial growth was evaluated as an index of systemic skeletal growth. RESULTS: IH had no significant impact on the general growth of either the juvenile or adolescent rats. However, it significantly decreased the total mandibular length and the posterior corpus length of the mandible in the adolescent rats and the anterior corpus length in the juvenile rats. IH also increased bone mineral density (BMD) of the condylar head in adolescent rats but did not affect the BMD of the tibia. Immunohistological analysis showed that the expression level of receptor activation of nuclear factor-κB ligand significantly decreased (in contrast to its messenger ribonucleicacid level) in the condylar head of adolescent rats with IH, while the number of osteoprotegerin-positive cells was comparable in the mandibles of adolescent IH rats and control rats. LIMITATIONS: The animal model could not simulate the pathological conditions of OSA completely and there were differences in bone growth between humans and rodents. CONCLUSIONS: These results suggest that the susceptibility of mandibular growth retardation to IH depends on the growth stage of the rats.

Absence of H3F3A mutation in a subset of malignant giant cell tumor of bone.

Giant cell tumor of bone typically involves the epiphysis of the long bones of skeletally mature patients. It is genetically characterized by highly recurrent and specific mutations of the H3F3A gene, which encodes histone H3.3. The most common mutation H3F3A G34W can readily be detected by a recently developed mutation-specific antibody. Giant cell tumor of bone rarely transforms to a sarcoma (malignant giant cell tumor of bone), which has not been genetically characterized in detail. We studied seven clinicopathologically defined malignant giant cell tumors, as well as two H3F3A-mutant bone sarcomas without giant cell tumor histology using a combination of clinicopathological, immunohistochemical, and molecular methods (Sanger sequencing + pyrosequencing or next generation sequencing). The cases included five men and four women, with a median age at initial diagnosis of 27 years. The two H3F3A G34W-positive sarcomas without giant cell tumor histology involved the subarticular epiphyseal sites, suggesting relatedness with giant cell tumor of bone. In two of the seven clinicopathologically defined malignant giant cell tumor cases, the sarcoma tissue showed the H3F3A G34W mutation. However, in the remaining five cases, in contrast to their associated H3F3A G34W-mutant giant cell tumor, the sarcoma lacked the H3F3A G34W mutation, either entirely or sub-clonally in the samples tested. This discordant mutation status was confirmed in all instances by immunohistochemistry and sequencing. A FISH analysis suggested that the absence of the H3F3A G34W mutation may be related to deletion of the H3F3A gene. Therefore, we have demonstrated that H3F3A G34W mutation, a critical driver in giant cell tumor, is absent in a subset of malignant giant cell tumor of bone. This novel recurrent phenomenon has potential biological and diagnostic implications, and further study is required to better characterize this progression pathway and understand its mechanism.

Discovery of Novel 7-[(1R,5S)-1-Amino-5-fluoro-3-azabicyclo[3.3.0]octan-3-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropane]-8-(methoxy or methyl)quinolones.

A series of 8-methoxy or 8-methylquinolones bearing novel 3-aminooctahydrocyclopenta[c]pyrrole derivatives at the C-7 position was synthesized, and the pharmacological, physicochemical, and toxicological properties of the individual compounds were evaluated. Novel 8-methylquinolone 7, which includes a 3-amino-7-fluorooctahydrocyclopenta[c]pyrrole moiety at the C-7 position, showed potent antibacterial activity against both Gram-positive and negative pathogens. Compound 7 also demonstrated favorable pharmacokinetic and pharmacodynamic properties and an acceptably safe toxicological profile. Consequently, compound 7 was selected as a clinical candidate.

Estrogen replacement enhances insulin-induced AS160 activation and improves insulin sensitivity in ovariectomized rats.

Menopause predisposes women to impaired glucose metabolism, but the role of estrogen remains unclear. In this study, we examined the effects of chronic estrogen replacement on whole body insulin sensitivity and insulin signaling in ovariectomized rats. Female Wistar rats aged 9 wk were ovariectomized under anesthesia. After 4 wk, pellets containing either 17ß-estradiol (E2) or placebo (Pla) were subcutaneously implanted in the rats. After 4 wk of treatment, the intra-abdominal fat accumulation was greater in the Pla group than that in the E2 group. Hyperinsulinemic-euglycemic clamp analysis and intravenous glucose tolerance test revealed that insulin sensitivity was significantly lower in the Pla group than in the E2 group. In addition, Western blotting showed that in vivo insulin stimulation increased protein kinase B (Akt) phosphorylation to a similar degree in the gastrocnemius and liver of both groups, but phosphorylated Akt2 Ser474 was enhanced in the muscle of the E2 group compared with the Pla group. Moreover, insulin-stimulated phosphorylation of Akt substrate of 160 kDa (AS160) Thr642 was observed only in the E2 group, resulting in the difference between the two groups. Additionally, AS160 protein and mRNA levels were higher in muscle of the E2 group than the Pla group. In contrast, E2 replacement had no effect on glucose transporter 4 protein levels in muscle and glycogen synthase kinase-3ß in muscle and liver. These results suggest that estrogen replacement improves insulin sensitivity by activating the Akt2/AS160 pathway in the insulin-stimulated muscle of ovariectomized rats.

Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation.

BACKGROUND: Bradyrhizobium diazoefficiens USDA110 nodulates soybeans for nitrogen fixation. It accumulates poly-3-hydroxybutyrate (PHB), which is of physiological importance as a carbon/energy source for survival during starvation, infection, and nitrogen fixation conditions. PHB accumulation is orchestrated by not only the enzymes for PHB synthesis but also PHB-binding phasin proteins (PhaPs) stabilizing the PHB granules. The transcription factor PhaR controls the phaP genes. RESULTS: Inactivation of phaR led to decreases in PHB accumulation, less cell yield, increases in exopolysaccharide (EPS) production, some improvement in heat stress tolerance, and slightly better growth under microaerobic conditions. Changes in the transcriptome upon phaR inactivation were analyzed. PhaR appeared to be involved in the repression of various target genes, including some PHB-degrading enzymes and others involved in EPS production. Furthermore, in vitro gel shift analysis demonstrated that PhaR bound to the promoter regions of representative targets. For the phaP1 and phaP4 promoter regions, PhaR-binding sites were determined by DNase I footprinting, allowing us to deduce a consensus sequence for PhaR-binding as TGCRNYGCASMA (R: A or G, Y: C or T, S: C or G, M: A or C). We searched for additional genes associated with a PhaR-binding sequence and found that some genes involved in central carbon metabolism, such as pdhA for pyruvate dehydrogenase and pckA for phosphoenolpyruvate carboxykinase, may be regulated positively and directly by PhaR. CONCLUSIONS: These results suggest that PhaR could regulate various genes not only negatively but also positively to coordinate metabolism holistically in response to PHB accumulation.

A novel method for transforming the thermophilic bacterium Geobacillus kaustophilus.

BACKGROUND: Bacterial strains of the genus Geobacillus grow at high temperatures of 50-75 °C and could thus be useful for biotechnological applications. However, genetic manipulation of these species is difficult because the current techniques for transforming Geobacillus species are not efficient. In this study, we developed an easy and efficient method for transforming Geobacillus kaustophilus using the conjugative plasmid pLS20cat. RESULTS: We constructed a transformation system comprising (i) a mobilizable Bacillus subtilis-G. kaustophilus shuttle plasmid named pGK1 that carries the elements for selection and replication in Geobacillus, and (ii) a pLS20cat-harboring B. subtilis donor strain expressing the dam methylase gene of Escherichia coli and the conjugation-stimulating rapLS20 gene of pLS20cat. This system can be used to efficiently introduce pGK1 into G. kaustophilus by mobilization in a pLS20cat-dependent way. Whereas the thermostable kanamycin marker and Geobacillus replication origin of pGK1 as well as expression of dam methylase in the donor were indispensable for mobilization, ectopic expression of rapLS20 increased its efficiency. In addition, the conditions of the recipient influenced mobilization efficiency: the highest mobilization efficiencies were obtained using recipient cells that were in the exponential growth phase. Furthermore, elimination of the origin of transfer from pLS20cat enhanced the mobilization. CONCLUSIONS: We describe a novel method of plasmid mobilization into G. kaustophilus recipient from B. subtilis donor depending on the helper function of pLS20cat, which enables simple, rapid, and easy transformation of the thermophilic Gram-positive bacterium.

Rapid conjugative mobilization of a 100 kb segment of Bacillus subtilis chromosomal DNA is mediated by a helper plasmid with no ability for self-transfer.

BACKGROUND: The conjugative plasmid, pLS20, isolated from Bacillus subtilis natto, has an outstanding capacity for rapid self-transfer. In addition, it can function as a helper plasmid, mediating the mobilization of an independently replicating co-resident plasmid. RESULTS: In this study, the oriT sequence of pLS20cat (oriTLS20) was eliminated to obtain the plasmid, pLS20catΔoriT. This resulted in the complete loss of the conjugative transfer of the plasmid but still allowed it to mobilize a co-resident mobilizable plasmid. Moreover, pLS20catΔoriT was able to mobilize longer DNA segments, up to 113 kb of chromosomal DNA containing oriTLS20, after mixing the liquid cultures of the donor and recipient for only 15 min. CONCLUSIONS: The chromosomal DNA mobilization mediated by pLS20catΔoriT will allow us to develop a novel genetic tool for the rapid, easy, and repetitive mobilization of longer DNA segments into a recipient chromosome.

Evaluation of Inhibitory Activities of UK-2A, an Antimycin-Type Antibiotic, and Its Synthetic Analogues against the Production of Anti-inflammatory Cytokine IL-4.

The inhibitory activities of the antimycin-class antibiotics UK-2A, antimycin A, and splenocin B against the production of anti-inflammatory cytokine IL-4, which is related to IgE-mediated allergic responses in rat basophilic leukemia (RBL-2H3) cells, were evaluated. Although antimycin A and splenocin B showed cytotoxicity at concentrations at which IL-4 release from the cells was restricted, UK-2A was found to restrict IL-4 release without cytotoxicity. Three UK-2A analogues (4-6) were then synthesized and assessed. Compound 5 restricted IL-4 release dose-dependently without cytotoxicity, and its effect was more potent than that of UK-2A.

A rare case of ectopic papillary thyroid carcinoma transformed into squamous cell carcinoma.

A rare case of a metastatic ectopic papillary thyroid carcinoma (PTC) of the lung that transformed into a squamous cell carcinoma (SCC) that resembles pulmonary SCC is reported. A subcutaneous ectopic PTC in the left anterior neck area, together with a normal thyroid gland, were excised. The ectopic PTC showed thyroglobulin, TTF-1 and PAX-8 immunoreactivity and a BRAF V600E mutation. During the post-operative follow-up period, a rapidly growing 2 cm nodular lesion in the lower left lobe of the lung was detected. The lung tumor consisted of solid sheets and nests of squamous cells but without the nuclear features of PTC. Neither papillary nor follicular structures of cancer cells were identified. Carcinoma cells were positive for TTF-1, PAX-8, p40, CK14, and p63, while showing a high Ki-67 labeling index and a BRAF V600E mutation. These results support our interpretation of a PTC that originated from ectopic thyroid tissue in the left anterior neck and that developed a lung metastasis showing squamous cell differentiation.