Melting temperature mapping method using imperfect-match linear long probes.

Identifying pathogenic microorganisms as early as possible is critical for selecting the appropriate antimicrobial therapy in infected patients. We previously reported the development of the Tm mapping method for identifying a broad range of pathogenic bacteria within 3 h of blood collection. However, the Tm mapping identification requires an analytical instrument with a tube-to-tube variation of no more than 0.1 °C, so we can only use a few instruments that have such high thermal accuracy. To address the problem, we developed the improved Tm mapping method using imperfect-match linear long quenching probes (IMLL Q-probes). Using IMLL Q-probes, almost all commercially available analytical instruments can be used for the Tm mapping method. Some bacterial species cannot be narrowed down to one species, but they can at least be narrowed down to the genus level. The Tm mapping method using IMLL Q-probes is useful for deciding on antimicrobial therapy in infected patients.

Novel rapid method for identifying and quantifying pathogenic bacteria within four hours of blood collection.

Sepsis is life-threatening organ dysfunction and is considered a major cause of health loss. However, since the current biomarkers of sepsis reflect the host's immune response to microorganisms, they would inevitably cause a time-lag. This means that there is still no truly reliable biomarker of sepsis. In the present study, we developed a novel method for identifying and quantifying unknown pathogenic bacteria within four hours of sample collection. The most important point of this study is that the novel method can be used to determine the number of bacteria in a sample as a novel biomarker of infectious diseases. Indeed, based on the number of bacteria, we were able to accurately estimate the severity of microbial infection. Furthermore, using the time-dependent changes in the number of bacteria, we were able to monitor the therapeutic effect accurately. The rapid identification and quantification of bacteria may change our approach to medical care.

Evolutionary Developments in Plant Specialized Metabolism, Exemplified by Two Transferase Families.

Plant specialized metabolism emerged from the land colonization by ancient plants, becoming diversified along with plant evolution. To date, more than 1 million metabolites have been predicted to exist in the plant kingdom, and their metabolic processes have been revealed on the molecular level. Previous studies have reported that rates of evolution are greater for genes involved in plant specialized metabolism than in primary metabolism. This perspective introduces topics on the enigmatic molecular evolution of some plant specialized metabolic processes. Two transferase families, BAHD acyltransferases and aromatic prenyltransferases, which are involved in the biosynthesis of paclitaxel and meroterpenes, respectively, have shown apparent expansion. The latter family has been shown to beinvolved in the biosynthesis of a variety of aromatic substances, including prenylated coumarins in citrus plants and shikonin in Lithospermum erythrorhizon. These genes have evolved in the development of each special subfamily within the plant lineage. The broadness of substrate specificity and the exon-intron structure of their genes may provide hints to explain the evolutionary process underlying chemodiversity in plants.

Melting Temperature Mapping Method: A Novel Method for Rapid Identification of Unknown Pathogenic Microorganisms within Three Hours of Sample Collection.

Acquiring the earliest possible identification of pathogenic microorganisms is critical for selecting the appropriate antimicrobial therapy in infected patients. We herein report the novel "melting temperature (Tm) mapping method" for rapidly identifying the dominant bacteria in a clinical sample from sterile sites. Employing only seven primer sets, more than 100 bacterial species can be identified. In particular, using the Difference Value, it is possible to identify samples suitable for Tm mapping identification. Moreover, this method can be used to rapidly diagnose the absence of bacteria in clinical samples. We tested the Tm mapping method using 200 whole blood samples obtained from patients with suspected sepsis, 85% (171/200) of which matched the culture results based on the detection level. A total of 130 samples were negative according to the Tm mapping method, 98% (128/130) of which were also negative based on the culture method. Meanwhile, 70 samples were positive according to the Tm mapping method, and of the 59 suitable for identification, 100% (59/59) exhibited a "match" or "broad match" with the culture or sequencing results. These findings were obtained within three hours of whole blood collection. The Tm mapping method is therefore useful for identifying infectious diseases requiring prompt treatment.

Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.

BACKGROUND: Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. METHODS: We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. RESULTS: Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. CONCLUSIONS: We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.

Extracts of Larix Leptolepis effectively augments the generation of tumor antigen-specific cytotoxic T lymphocytes via activation of dendritic cells in TLR-2 and TLR-4-dependent manner.

Type-1 immunity plays a crucial role in host defense against various tumors and infectious diseases. Here, we first demonstrated that extract of Larix Leptolepis (ELL), one of the most popular timbers at Hokkaido area in Japan, strongly activated Type-1 immunity. ELL induced production of Type-1 cytokines such as IL-12 and TNF-α from bone marrow-derived dendritic cells (BMDCs) in TLR2- and TLR4-dependent manner and remarkably up-regulated the expression of MHC and co-stimulatory molecules. In addition, antigen-specific CTLs were significantly augmented by the combined administration of ELL, antigen and BMDCs. Finally, we revealed that combination therapy using ELL, antigen and BMDCs significantly inhibited the growth of established tumor in mouse model. Thus, these findings suggested that ELL would be a novel adjuvant for inducing an activation of Type-1-dependent immunity including activation of BMDCs and induction of tumor-specific CTLs, which is applicable to the therapy of cancer and infectious diseases.

A novel eukaryote-made thermostable DNA polymerase which is free from bacterial DNA contamination.

To achieve the production of a thermostable DNA polymerase free from bacterial DNA contamination, we developed eukaryote-made thermostable DNA (Taq) polymerase. The novel eukaryote-made thermostable DNA polymerase resolves the problem of contaminating bacterial DNA in conventional bacterially made thermostable DNA polymerase as a result of its manufacture and incomplete purification. Using eukaryote-made thermostable DNA polymerase, the sensitive and reliable detection of bacteria becomes feasible for large fields, thereby making the development of a wide range of powerful applications possible.

Matrix metalloproteinase-2 inhibitors from Clinopodium chinense var. parviflorum.

From the aerial parts of Clinopodium chinense var. parviflorum, nine new phenylpropanoids, clinopodic acids A-I (2-10), were isolated together with a known phenylpropanoid, rosmarinic acid (1). The structures of these new compounds were elucidated on the basis of spectroscopic analysis. Clinopodic acid C (4) showed MMP-2 inhibitory activity (IC(50) 3.26 microM).

Production of paclitaxel and the related taxanes by cell suspension cultures of Taxus species.

Medicinal plants are the most promising source for the development of drugs, and many types of active ingredients from the plant resources have been studied in order to clarify the relationship between the chemical structure and the activity. However, it is not easy to develop drugs from those active compounds, and in many cases, the supply of active compounds can have some problems: 1) limited quantity of active compounds in plant; 2) low plant growth rate; 3) the limited localization of active ingredients in the specific organs; and 4) from the perspective of the conservation of natural resources. Therefore, the stable supply of the compounds commercially is very difficult and contains risk hedge. Plant cell culture is an attractive technology to solve these problems by securing the stable supply of the active compounds without damage to the natural plant resources. Recently, an efficient production process of anticancer drug paclitaxel by Taxus cell suspension cultures was constructed. The established Taxus cell lines produced paclitaxel and related taxanes by specific external stimuli, such as methyl jasmonate. The time-course analysis revealed that there are two regulatory steps existing in the paclitaxel biosynthesis: the taxane-ring formation step that is up-regulated by MeJA, and the acylation step at the C-13 position. By applying the data from the two-stage culture and the high-density culture, a large-scale culture process was developed with a stable paclitaxel production in the range of 140-295 mg L(-1), reaching 295 mg L(-1) at maximum.

Paclitaxel production by plant-cell-culture technology.

Plant cells catalyze multiple-step reactions of secondary metabolite biosynthesis, and selectively synthesize chiral compounds with polycyclic structures. Taking advantage of this characteristic, we studied the production of the anticancer drug paclitaxel, which is currently produced in limited supply. Callus culture investigations indicate that woody plant medium supplemented with 10(-5) mol L(-1) 1-naphthylacetic acid and without the NH4+ -type ion is the best condition for growth of the callus. The accumulation of paclitaxel and related taxanes in Taxus plants is thought to be a biological response to specific external stimuli. Several signal transducers were screened; taxane biosynthesis was strongly promoted by methyl jasmonate (MeJA) and silver thiosulfate (STS) as an anti-ethylene compound. Of ten taxane-type diterpenoids isolated from T. baccata suspension-cultured cells treated with MeJA, five have a phenylisoserine side-chain at the C-13 position of the taxane skeleton. Time-course analysis revealed two regulatory steps in taxane biosynthesis: the taxane-ring formation step and the acylation step of the C-13 position. Methyl jasmonate promoted the formation of the taxane-ring. The production of paclitaxel reached a maximum level of 295 mg L(-1) in a large-scale culture of T. x media cells using a two-stage process.