Time-programmable drug dosing allows the manipulation, suppression and reversal of antibiotic drug resistance in vitro

Multi-drug strategies have been attempted to prolong the efficacy of existing antibiotics, but with limited success. Here we show that the evolution of multi-drug-resistant Escherichia coli can be manipulated in vitro by administering pairs of antibiotics and switching between them in ON/OFF manner. Using a multiplexed cell culture system, we find that switching between certain combinations of antibiotics completely suppresses the development of resistance to one of the antibiotics. Using this data, we develop a simple deterministic model, which allows us to predict the fate of multi-drug evolution in this system. Furthermore, we are able to reverse established drug resistance based on the model prediction by modulating antibiotic selection stresses. Our results support the idea that the development of antibiotic resistance may be potentially controlled via continuous switching of drugs

Regulation of pH attenuates toxicity of a byproduct produced by an ethanologenic strain of Sphingomonas sp. A1 during ethanol fermentation from alginate

Marine macroalgae is a promising carbon source that contains alginate and mannitol as major carbohydrates. A bioengineered ethanologenic strain of the bacterium Sphingomonas sp. A1 can produce ethanol from alginate, but not mannitol, whereas the yeast Saccharomyces paradoxus NBRC 0259–3 can produce ethanol from mannitol, but not alginate. Thus, one practical approach for converting both alginate and mannitol into ethanol would involve two-step fermentation, in which the ethanologenic bacterium initially converts alginate into ethanol, and then the yeast produces ethanol from mannitol. In this study, we found that, during fermentation from alginate, the ethanologenic bacterium lost viability and secreted toxic byproducts into the medium. These toxic byproducts inhibited bacterial growth and killed bacterial cells and also inhibited growth of S. paradoxus NBRC 0259–3. We discovered that adjusting the pH of the culture supernatant or the culture medium containing the toxic byproducts to 6.0 attenuated the toxicity toward both bacteria and yeast, and also extended the period of viability of the bacterium. Although continuous adjustment of pH to 6.0 failed to improve the ethanol productivity of this ethanologenic bacterium, this pH adjustment worked very well in the two-step fermentation due to the attenuation of toxicity toward S. paradoxus NBRC 0259–3. These findings provide information critical for establishment of a practical system for ethanol production from brown macroalgae

Factors associated with the severity of childhood rhinoconjunctivitis

AbstractBackgroundAllergic rhinitis is one of the most common chronic diseases in children. Although it has a large impact on the patient's quality of life, little is known about the factors associated with its severity. The aim of this study was to assess the factors associated with the severity of rhinoconjunctivitis among children in the general population.MethodsA survey was conducted using an online research panel in 2012. Parents were asked to answer an International Study of Asthma and Allergies in Childhood-based questionnaire to identify children with current rhinoconjunctivitis and evaluate factors associated with the severity of its symptoms. Severity was rated according to the degree of impairment caused by the symptoms in the patient's daily life.ResultsAmong 26,725 children aged 6–12 years old, rhinoconjunctivitis was defined in 5175 (19.4%), and of these, 688 children (13.3% of children with current rhinoconjunctivitis) presented severe symptoms. Living in areas with a high cedar and cypress pollen count and having concurrent eczema were associated with severe rhinoconjunctivitis [adjusted OR (95% CI): 1.21 (1.00–1.46) and 1.45 (1.20–1.75), respectively]. Further, a maternal history of asthma and allergic rhinitis was a significant risk factor for severe rhinoconjunctivitis [1.34 (1.04–1.74) and 1.30 (1.10–1.53), respectively]. However, living with fur-bearing animals (pets) before 1 year of age proved to be a protective factor against severe rhinoconjunctivitis [0.70 (0.52–0.94)].ConclusionsEnvironmental factors such as pets and pollen, together with comorbidities and a maternal history of allergic diseases, play an important role in determining the severity of rhinoconjunctivitis

Tephra-stratigraphical study of the 1988-1989 eruption of Tokachi-dake Volcano, central Hokkaido

Twenty-three small-scale eruptions took place at Tokachi-dake from December 16, 1988 to March 5, 1989. The pyroclastic fall deposits, ballistic fragments, and pyroclastic surge and flow deposits were dispersed over the flank and leeward areas of the volcano. Because the pyroclasts of each eruption were well-preserved in snow during the winter, the stratigraphy and distribution of these deposits could be studied in detail. The volume of the pyroclastic fall deposits are nearly equal to those of the pyroclastic surge and flow deposits. The total volume of these pyroclasts is estimated to be 7.4×105 m3. Judging from the sequential changes of the volume and composition of the pyroclasts, the characteristic features of the eruption can be summarized as follows: At first, a vent was opened by ejection of altered rock fragments in December, 1988. Then, essential fragments were ejected in January, 1989. Finally the activity level of magma declined and the altered rock fragments content increased again in February to March, 1989

Multicentric Epithelioid Hemangioendothelioma of the Bone: Histologic and Radiographic Features

A multicentric epithelioid hemangioendothelioma (EHE) of the bone is reported which affected the right femur, the right patella and the right fibula of a 56-year-old man. Plain radiographs demonstrated purely lytic multicentric lesions with well-defined sclerotic borders. Computed tomography (CT) scans showed multiple osteolytic lesions in a honeycomb pattern at the epiphyseal lesion of the right femur; the other lesions of the right femoral shaft, the right patella and the right fibula were punched out sharply. Magnetic resonance (MR) images showed that the tumors contained mixed signal intensities: low intensity for T1-weighted images and high intensity for T2-weighted images. Histologically, the tumor was discerned as EHE with a spectrum of endothelial tumor, with the added feature of epithelioid hemangioma in a limited area. As for the initial treatment, curettage and bone cementing were performed for the epiphyseal tumor of the right femur, followed by surgical resections of the other tumors. Neither recurrence nor metastasis was observed 3 years after resection

Elimination of Specific miRNAs by Naked 14-nt sgRNAs

tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase ZL, which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5′-half-tRNA, RNA heptamer, hook RNA, and ∼14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase ZL cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ∼50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing

Dendritic retraction, but not atrophy, is consistent in amyotrophic lateral sclerosis-comparison between Onuf’s neurons and other sacral motor neurons-

BACKGROUND: Fundamental cytological changes of amyotrophic lateral sclerosis (ALS) were looked for by comparing relatively preserved Onuf’s nucleus (ON) and severely affected neighboring motor neuron groups (dorsolateral alpha motoneurons (DL) and other anterior horn neurons (OAH)). The second sacral segments from 11 ALS patients and 5 controls were initially quadruple-labeled for phosphorylated and non-phosphorylated TAR DNA-binding protein of 43 kDa (TDP43), and p62 with DAPI to identify TDP43-related changes. After digital recording of these fluorescence data encompassing the entire specimen at a high resolution, the same sections were stained with Klüver-Barrera method to obtain their exact bright-field counterparts. This novel approach facilitated exact identification of ON. Furthermore, this cell to cell comparison enabled to correlate quantitative indices of the neuronal cell bodies: perimeter, area and circularity index (CI) i.e. the ratio of (perimeter/2π) divided by the square root of (area/π), which decreases with dendritic retraction, overall number of neurons and inclusions. RESULTS: In addition to known preservation of ON neuron number relative to DL and OAH, size reduction of ON neurons was not significant even in the advanced stage. Significant size reduction in DL was counteracted in the presence of TDP43-positive inclusions. Early increase of neuronal size in OAH was further enhanced by the presence of TDP43-positive inclusions. Even with these heterogeneous cytopathological changes, a decrease in CI was consistent in all groups at an early phase and was correlated with neuronal loss. CONCLUSIONS: Among variable cytological changes of ALS, a decrease in CI is a consistent early feature shared between non-atrophic ON neurons and other anterior horn neurons with either decreased (DL) or even increased (OAH) size and profounder neuronal loss. This decrease in CI, representative of dendritic retraction, is fundamental to ALS pathogenesis, not necessarily linked to cell size and pathological inclusions

Apoptotic Cell Death and p53 Expression in Leiomyosarcoma of Soft-Tissue Origin

Leiomyosarcoma (LMS) of soft-tissue origin was studied on the expression and the biological significance of apoptosis in relation to p53 oncoprotein. Immunohistochemical analysis was performed initially on the paraffin-embbeded sections taken from 29 surgical tissues (20 cases) including 9 recurrent/metastatic tumors. The results are as follows: A positive correlation was observed between the p53 indices (PIs) and the proliferative markers designated by Ki-67, PCNA and MCM2, both of which increased significantly in the high-grade malignant LMS much more than in the low-grade one (P < 0.001). Apoptotic cells were detected by the TUNEL method and evaluated as the apoptotic index (AI). A high AI-level was shown in the high-grade malignant LMS, especially in cases of the recurrent/metastatic sites in comparison with the tumors of the primary site (P < 0.05). The AI was statistically higher in the p53-positive cases of high-grade malignant LMS than in the p53-negative cases of low-grade malignant LMS. In conclusion, apoptotic activity paralleled the overexpression of p53 protein along with an increasing grade of malignancy and may be related intimately to the increased malignant potential, especially to recurrence/metastasis

Traditional Japanese Formula Kigikenchuto Accelerates Healing of Pressure-Loading Skin Ulcer in Rats

We evaluated the effect of kigikenchuto (KKT), a traditional Japanese formula, in a modified rat pressure-loading skin ulcer model. Rats were divided into three groups, KKT extract orally administered (250 or 500 mg/kg/day for 35 days) and control. KKT shortened the duration until healing. Immunohistochemically, KKT increased CD-31-positive vessels in early phase and increased α-smooth muscle actin-(α-SMA-) positive fibroblastic cells in early phase and decreased them in late phase of wound healing. By Western blotting, KKT showed the potential to decrease inflammatory cytokines (MCP-1, IL-1β, and TNF-α) in early phase, decrease vascular endothelial growth factor in early phase and increase it in late phase, and modulate the expression of extracellular protein matrix (α-SMA, TGF-β1, bFGF, collagen III, and collagen I). These results suggested the possibility that KKT accelerates pressure ulcer healing through decreases of inflammatory cytokines, increase of angiogenesis, and induction of extracellular matrix remodeling