Bacillus subtilis grown in a "breathing" vessel without sparger aeration.

Here we present a "breathing" vessel consisting of expanded polytetrafluoroethylene, which allows gas exchange but no liquid permeation. The bacterial culture inside needs only agitation to promote air supply. Using this setup, a Bacillus subtilis cell factory for scyllo-inositol production grew to produce scyllo-inositol efficiently. The results indicate that our approach represents a sustainable "greener" approach for the cell factory.

Successful pazopanib treatment of undifferentiated pleomorphic sarcoma with coamplification of PDGFRA, VEGFR2 and KIT: A case report.

Undifferentiated pleomorphic sarcoma (UPS) is a high-grade, aggressive soft tissue sarcoma (STS) with a poor prognosis, and no definitive or effective treatment is currently available for it. Pazopanib, an orally available multiple tyrosine kinase inhibitor, has been approved for the treatment of advanced STS. The present study documents the case of a 51-year-old man with advanced UPS with coamplification of platelet-derived growth factor receptor A (PDGFRA), vascular endothelial growth factor receptor 2 (VEGFR2) and stem cell factor receptor (KIT) genes. The patient exhibited a marked and sustained response to pazopanib. The patient presented with a retroperitoneal tumour with pancreatic head lymph node metastasis, and bone metastases in the second/fifth thoracic vertebrae and left femur. Based on the histological analysis of the retroperitoneal tumour and femoral mass, the patient was diagnosed with UPS. Palliative radiation therapy was administered to the left femur and second/fifth thoracic vertebrae to prevent fractures. After radiation therapy, the patient achieved a partial response after eight courses of doxorubicin. A comprehensive genomic profiling analysis (FoundationOne® CDx) revealed coamplification of PDGFRA, VEGFR2 and KIT genes. Hence, pazopanib was initiated as a second-line treatment. Notably, the retroperitoneal tumour shrank, and no new lesions developed for 3 years after the initiation of pazopanib treatment. This response suggests that the coamplification of PDGFRA, VEGFR2 and KIT may predict favourable outcomes in response to pazopanib.

High-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered Bacillus subtilis on blood agar.

Biosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis. Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity.

Efficient pathway-driven scyllo-inositol production from myo-inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control.

Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli-Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo-inositol into scyllo-inositol at 30°C. In a scaled-up reaction, 10 g of myo-inositol was converted to 1.8 g of scyllo-inositol, which was further purified to yield 970 mg of pure powder. Notably, myo-inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification. IMPORTANCE: Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo-inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo-inositol, holding potential pharmaceutical significance as scyllo-inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.

Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production.

The Gram-positive bacterium Bacillus subtilis is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced B. subtilis strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production. Different from previously characterized genome-reduced B. subtilis strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD+ and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.

Sarcoma of the Lung and Mediastinum.

Primary sarcoma of the lung and mediastinum is rare. The diagnosis requires careful exclusion of sarcomatoid carcinoma, sarcomatoid mesothelioma, and metastases from extra-thoracic sites. This review summarizes the key morphologic, immunohistochemical, and molecular characteristics of sarcomas that are encountered in the lung and mediastinum. The tumor types discussed are synovial sarcoma, well-differentiated/dedifferentiated liposarcoma, myxoid pleomorphic liposarcoma, intimal sarcoma of the pulmonary artery, inflammatory myofibroblastic tumor, epithelioid hemangioendothelioma, primary pulmonary myxoid sarcoma, malignant peripheral nerve sheath tumor, Ewing sarcoma, and CIC-rearranged sarcoma. Relevant differential diagnoses are also addressed.

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.

A case of pelvic EWSR1-PATZ1 fusion sarcoma treated with carbon ion radiotherapy.

EWSR1-PATZ1 fusion sarcoma is a type of round-cell sarcoma with EWSR1-non-EST fusion that was newly categorized in the 2020 World Health Organization classification of soft tissue and bone tumors. In general, local disease is managed via surgical resection; however, at present, there is no standard therapy for locally advanced or metastatic disease. Here, we report our experience with a middle-aged male patient with pelvic EWSR1-PATZ1 fusion sarcoma who was treated with carbon ion radiotherapy and maintained stable disease for 13 months. The patient's clinical course suggests that carbon ion radiotherapy may be effective in patients with locally advanced EWSR1-PATZ1 fusion sarcoma.

Microbial synthesis of health-promoting inositols.

D-chiro-inositol and scyllo-inositol are known for their health-promoting properties and promising as ingredients for functional foods. Strains of Bacillus subtilis and Corynebacterium glutamicum were created by metabolic engineering capable of inexpensive production of these two rare inositols from myo-inositol, which is the most common inositol in nature. In addition, further modifications have enabled the synthesis of the two rare inositols from the much-cheaper carbon sources, glucose or sucrose.

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.

Association of atmospheric temperature with out-of-hospital natural deaths occurrence before and during the COVID-19 pandemic in Osaka, Japan.

In this study, we aimed to investigate the relationship between out-of-hospital natural death (OHND) and ambient temperature and examine the seriousness of the impact of the coronavirus disease-2019 (COVID-19) pandemic on this relationship. We used data from the Osaka Prefectural Office of Medical Examiners between 2018 and 2022 and performed a retrospective observational study. A Poisson regression model was applied to examine the relationship between OHND and temperature in Osaka City. The relative risk of OHND at 5 °C and 32 °C compared to the minimum mortality temperature increased from 1.81 in the pre-COVID-19 period to 2.03 in the post-COVID-19 period at 5 °C and from 1.29 in the pre-COVID-19 period to 1.60 in the post-COVID-19 period at 32 °C. The increase in relative risk per 1 °C increase from the pre- to post-COVID-19 period was 1.0551 (rate ratio [RR], p = 0.003) in the hot environment and 1.0233 (RR, p = 0.013) in the cold environment, which was larger than that in the hot environment. Although the risk of OHND increased at both temperatures, the change in OHND risk during post-COVID-19 was larger in the hot environment than in the cold environment, implicating the effect of pandemics in the current scenario of global warming.

Bacillus subtilis NDmed, a model strain for biofilm genetic studies.

The Bacillus subtilis strain NDmed was isolated from an endoscope washer-disinfector in a medical environment. NDmed can form complex macrocolonies with highly wrinkled architectural structures on solid medium. In static liquid culture, it produces thick pellicles at the interface with air as well as remarkable highly protruding ''beanstalk-like'' submerged biofilm structures at the solid surface. Since these mucoid submerged structures are hyper-resistant to biocides, NDmed has the ability to protect pathogens embedded in mixed-species biofilms by sheltering them from the action of these agents. Additionally, this non-domesticated and highly biofilm forming strain has the propensity of being genetically manipulated. Due to all these properties, the NDmed strain becomes a valuable model for the study of B. subtilis biofilms. This review focuses on several studies performed with NDmed that have highlighted the sophisticated genetic dynamics at play during B. subtilis biofilm formation. Further studies in project using modern molecular tools of advanced technologies with this strain, will allow to deepen our knowledge on the emerging properties of multicellular bacterial life.

Genetic characterization of a novel organoid from human malignant giant-cell tumor.

Malignant giant-cell tumors are extremely rare bone sarcomas that transform from conventional giant-cell tumors during long periods of treatment. Owing to their rarity, no further analysis of their molecular pathogenesis exists, and thus, no standard treatment has been established. Recently, organoid culture methods have been highlighted for recapturing the tumor microenvironment, and we have applied the culture methods and succeeded in establishing patient-derived organoids (PDO) of rare sarcomas. This study aimed to investigate the genomic characteristics of our established novel organoids from human malignant giant-cell tumors. At our institute, we treated a patient with malignant giant-cell tumor. The remaining sarcoma specimens after surgical resection were cultured according to the air-liquid interface organoid-culture method. Organoids were xenografted into NOD-scid IL2Rgnull mice. The developed tumors were histologically and genomically analyzed to compare their characteristics with those of the original tumors. Genetic changes over time throughout treatment were analyzed, and the genomic status of the established organoid was confirmed. Organoids from malignant giant-cell tumors could be serially maintained using air-liquid interface organoid-culture methods. The tumors developed in xenografted NOD-scid IL2Rgnull mice. After several repetitions of the process, a patient-derived organoid line from the malignant giant-cell tumor was established. Immunohistochemical analyses and next-generation sequencing revealed that the established organoids lacked the H3-3A G34W mutation. The xenografted organoids of the malignant giant-cell tumor had phenotypes histologically and genetically similar to those of the original tumor. The established organoids were confirmed to be derived from human malignant giant-cell tumors. In summary, the present study demonstrated a novel organoid model of a malignant giant-cell tumor that was genetically confirmed to be a malignant transformed tumor. Our organoid model could be used to elucidate the molecular pathogenesis of a malignant giant-cell tumor and develop novel treatment modalities.

Bacillus velezensis S141, a soybean growth-promoting bacterium, hydrolyzes isoflavone glycosides into aglycones.

Bacillus velezensis S141, a plant growth-promoting rhizobacteria (PGPR), was isolated from a soybean field in Thailand. Previous studies demonstrated that S141 enhanced soybean growth, stimulating nodulation for symbiotic nitrogen fixation with soybean root nodule bacteria, including Bradyrhizobium diazoefficience USDA110. Isoflavone glycosides are produced in soybean roots and hydrolyzed into their aglycones, triggering nodulation. This study revealed that S141 efficiently hydrolyzed two isoflavone glycosides in soybean roots (daidzin and genistin) to their aglycones (daidzein and genistein, respectively). However, S141, Bacillus subtilis 168, NCIB3610, and B. velezensis FZB42 hydrolyzed isoflavone glucosides into aglycones. A BLASTp search suggested that S141 and the other three strains shared four genes encoding ß-glucosidases corresponding to bglA, bglC, bglH, and gmuD in B. subtilis 168. The gene inactivation analysis of B. subtilis 168 revealed that bglC encoded the major ß-glucosidase, contributing about half of the total activity to hydrolyze isoflavone glycosides and that bglA, bglH, and gmuD, all barely committed to the hydrolysis of isoflavone glycosides. Thus, an unknown ß-glucosidase exists, and our genetic knowledge of ß-glucosidases was insufficient to evaluate the ability to hydrolyze isoflavone glycosides. Nevertheless, S141 could predominate in the soybean rhizosphere, releasing isoflavone aglycones to enhance soybean nodulation.

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.

Postmorterm Computed Tomography and Autopsy to Confirm Sudden Death Due to Tracheal Compression by Mediastinal Fat Tissue in a Young Man With Obesity.

A man in his early twenties with obesity was found dead in his apartment. The deceased was found naked and surrounded by empty bottles of electrolytes. An autopsy performed approximately 6 days postmortem and gross inspection revealed the absence of injury and no apparent extrinsic cause of death. It was decided to dissect to investigate the cause of death. The deceased had become morbidly obese (weight, 98 kg; height, 160 cm; body mass index, 38.3). Shortly before his death, he presented at a clinic complaining of gastric discomfort and heartburn, but other than hypertension (155/91 mmHg) no specific abnormality was found. He was normothermic (36.6℃), and his blood oxygen saturation was normal (97%). Postmortem computed tomography of the thorax revealed a mediastinal mass obstructing the trachea, an upper-airway obstruction, and a narrowed thoracic cavity due to upward compression by an enlarged fatty liver. Autopsy confirmed that the tracheal mass was fatty tissue within the thymus and that upward pressure from an enlarged fatty liver had compressed the thoracic cavity. The deceased likely developed nocturnal chronic hypoxia because of compression by the mediastinal fat mass as well as intermittent hypoxia because of obstructive sleep apnea when lying supine. Chronic and intermittent hypoxia, diabetes, and obesity activate the sympathetic nervous system, increasing the risk of hypertension, heart failure, and arrhythmias. Histological findings showed pulmonary congestion and edema, reflecting heart failure as well as myocardial fragmentation and waving, showing hyper-contraction and hyper-relaxation, respectively. Hypertension, feeling overheated, and myocardial hyper-contraction can be explained as sympathetic nerve over-activation. Intra-cardiac coagulation and a renal cortical pallor suggested subacute death from cardiogenic shock due to heart failure. Postmortem computed tomography before autopsy detected airway obstruction and revealed the cause and pathophysiology of unexpected death in a young man with morbid obesity. Therefore, this could be a potentially useful clinical practice for determining the cause of death postmortem.

Metabolic dysregulation and decreased capillarization in skeletal muscles of male adolescent offspring rats exposed to gestational intermittent hypoxia.

Gestational intermittent hypoxia (IH) is a hallmark of obstructive sleep apnea that occurs frequently during pregnancy, and effects caused by this environmental change during pregnancy may be transmitted to the offspring. In this study, we aimed to clarify the effects of IH in pregnant rats on the skeletal muscle of adolescent offspring rats. Mother rats underwent IH from gestation day 7-21, and their 5-weeks-old male offspring were analyzed. All male offspring rats were born and raised under normoxia conditions. Although no general growth retardation was observed, we found that exposure to gestational IH reduces endurance running capacity of adolescent offspring rats. Both a respiratory muscle (diaphragm; DIA) and a limb muscle (tibialis anterior; TA) showed no histological abnormalities, including fiber size and fiber type distribution. To identify the possible mechanism underlying the reduced running capacity, regulatory factors associated with energy metabolism were analyzed in different parts of skeletal muscles. Compared with rats born under conditions of gestational normoxia, gestational IH offspring rats showed significantly lower expression of genes associated with glucose and lipid metabolism, and lower protein levels of phosphorylated AMPK and AKT. Furthermore, gene expression of adiponectin receptors one and two was significantly decreased in the DIA and TA muscles. In addition, the DIA muscle from adolescent rats had significantly decreased capillary density as a result of gestational IH. However, these changes were not observed in a sucking muscle (geniohyoid) and a masticating muscle (masseter) of these rats. These results suggest that respiratory and limb muscles are vulnerable to gestational IH, which induces altered energy metabolism with decreased aerobic motor function. These changes were partially owing to the decreased expression of adiponectin receptors and decreased capillary density in adolescent offspring rats.

Rapid and reliable collection of tumor tissue for successful gene panel in a patient with advanced stage lung cancer: A case report.

Rapid and reliable identification of targetable driver mutations in patients with advanced stage lung cancer is essential. Adequate amount of tumor tissue biopsies (i.e., genomic biopsies) are required to successfully analyze the gene panel. In the present case, we performed three pleural fluid investigations, including transbronchial biopsy of the primary tumor, transesophageal endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of lymph node metastasis, and thoracoscopic biopsy of the pleural seeding sites. Among the three investigations, thoracoscopic biopsy alone successfully obtained a sufficient amount of tissue. Thus, it is important to determine the technique and site of biopsy, as multiple biopsies are not only burdensome to the patient, but also lead to significant delays in therapy induction.