Application of principal component analysis and self-organizing map to the analysis of 2D fluorescence spectra and the monitoring of fermentation processes

2D fluorescence sensors produce a great deal of spectral data during fermentation processes, which can be analyzed using a variety of statistical techniques. Principal component analysis (PCA) and a self-organizing map (SOM) were used to analyze these 2D fluorescence spectra and to extract useful information from them. PCA resulted in scores and loadings that were visualized in the score-loading plots and used to monitor various fermentation processes with recombinantEscherichia coli andSaccharomyces cerevisiae. The SOM was found to be a useful and interpretative method of classifying the entire gamut of 2D fluorescence spectra and of selecting some significant combinations of excitation and emission wavelengths. The results, including the normalized weights and variances, indicated that the SOM network is capable of being used to interpret the fermentation processes monitored by a 2D fluorescence sensor.     

Proteome Analysis of Cellular Response of Pseudomonas putida KT2440 to Tetracycline Stress

Tetracycline-induced proteome of Pseudomonas putida KT2440 was analyzed by 2-D gel electrophoresis and matrix-assisted laser desorption ionization–time of flight/mass spectrum (NALDI-TOF/MS) in order to understand cellular response to tetracycline. Of the proteins upregulated in a culture medium containing subinhibitory concentration of tetracycline (50 μg/mL), we identified 38 proteins from cytosol and precipitated fractions by peptide mass fingerprinting and mass spectrum/mass spectrum analysis. Various amino acids ABC transporters, a ribose ABC transporter, and a sulfate ABC transporter were found to be upregulated. Protein synthesis-related proteins, stress proteins, energy metabolic enzymes, and unknown proteins were also strongly induced. Of the identified upregulated proteins, several proteins (isocitrate lyase, branched-chain amino acid ABC transporter, superoxide dismutase, etc.) were also upregulated under phenol-induced stress condition. These results demonstrate that tetracycline at a high concentration induced comprehensive stress in P. putida KT2440 and the global induction of proteins related to bacteria survival. Proteome analysis was found to be a useful tool for the elucidation of antibiotic-induced proteins in the present study.     

Cryopreservation of somatic embryos of the herbaceous peony (Paeonia lactiflora Pall.) by air drying

This study was carried out to establish a suitable method for the cryopreservation of somatic embryos of the herbaceous peony. The somatic embryos were obtained from cotyledon and anther cultures on a MS medium supplemented with abscisic acid (ABA) and phenylacetic acid (PAA), respectively. The frequency of somatic embryo formation was the greatest (61%) from the cotyledons cultured on a MS medium supplemented with 1.0 mg l(-1) of ABA. Embryos were also obtained directly from anthers cultured on a MS medium with or without 2.0 mg l(-1) of PAA. For the cryopreservation of peony somatic embryos, the embryos were dried under a stream of sterile air and frozen by immersion in liquid nitrogen. Thawed embryos were germinated into plantlets after placing on a medium containing 0.3 mg l(-1) of gibberellic acid (GA(3)). The frequency of the post-thaw regrowth of cryopreserved somatic embryos was related to their size and desiccation time, the latter ranging from 0 to 2 h. When the somatic embryos were desiccated for 1 h, the frequency of post-thaw regrowth was greater than 66%. The frequency of post-thaw regrowth of the cryopreserved somatic embryos from anthers and cotyledon tissues was generally high when they were 2-3 mm in size. Desiccation may be a suitable method for the cryopreservation of somatic embryos of the herbaceous peony.     

The serum IL-12:IL-6 ratio reliably distinguishes infectious from non-infectious causes of fever during autologous stem cell transplantation

BACKGROUND: Fever during neutropenia and after neutrophil engraftment (post-engraftment fever) occurs commonly during autologous transplantation (ASCT), but infections are infrequently identified. Tests that reliably exclude infection may reduce the cost and toxicity of unnecessary diagnostic testing and empiric treatment. We assessed whether serum levels of inflammatory cytokines could distinguish infectious from non-infectious causes of fever in patients undergoing ASCT. METHODS: Serum levels of IL-1beta, IL-2, IL-6, IL-8, IL-10, IL-12(p70), TNF-alpha and IFN-gamma were measured by sandwich ELISA at multiple pre-determined times and at the onset of the first fever during neutropenia and after neutrophil engraftment in patients with hematologic malignancies undergoing ASCT. Standard clinical criteria were used to assess for the presence of infection. RESULTS: Seventy-two febrile episodes occurred in 54 of 65 enrolled patients; 29 (40%) of the episodes occurred after neutrophil engraftment. Infections were identified as the cause of 28% and 24% of the neutropenic and post-engraftment febrile episodes, respectively. The level of IL-12 decreased and that of IL-6 increased significantly during fever because of infection, such that the IL-12:IL-6 ratio accurately excluded infection. The area under the ROC curve for the IL-12:IL-6 ratio was 0.88 (95% CI 0.79-0.97). The sensitivity, specificity, positive predictive and negative predictive values associated with a cut-off ratio of 4.1 were 95%, 75%, 60%, and 97%, respectively. DISCUSSION: The IL-12:IL-6 ratio effectively discriminates infectious from non-infectious causes of fever during ASCT. It may be useful in assessing the probability of infection in patients with post-engraftment fever.     

Thermogelling aqueous solutions of alternating multiblock copolymers of poly(L-lactic acid) and poly(ethylene glycol)

We are reporting alternating multiblock copolymers of poly(L-lactic acid)/poly(ethylene glycol) aqueous solution (> 15 wt %) undergoing sol-gel-sol transition as the temperature increases from 20 to 60 degrees C. Micelles of the multiblock copolymers (in water) are about 20 nm in radius at low temperature. They are aggregated to a larger size as the temperature increases, which should play a critical role in the sol-to-gel transition. The transition temperature and gel window were affected by the molecular weight and composition of the multiblock copolymer. In particular, the aqueous solution of an alternating multiblock copolymer (Mn approximately 6700 daltons) prepared from poly(ethylene glycol) (Mn approximately 600 daltons) and poly(L-lactic acid) (Mn approximately 1300 daltons) showed a maximum modulus at body temperature (37 degrees C). The in situ gel forming ability of the polymer aqueous solution in vivo as well as in vitro indicates that it can be a promising injectable biomaterial.     

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

BackgroundExposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood.PurposeIn this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells.MethodThe human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process.ResultsHyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown.ConclusionThis study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cells.     

S100A8 and S100A9 are messengers in the crosstalk between epidermis and dermis modulating a psoriatic milieu in human skin

S100A8 and S100A9 are members of the S100A8 protein family that exist as homodimers and heterodimers in neutrophils, monocytes, and macrophages. Recent studies have shown the pivotal roles of S100A8 and S100A9 in the propagation of inflammation and keratinocyte proliferation in psoriasis. We found significant up-regulation of S100A8 and S100A9 secretion from keratinocytes in psoriatic lesions. To mimic the in vivo secretory conditions of S100A8 and S100A9 from psoriatic epidermal keratinocytes, we used the culture medium (CM) of S100A8 and S100A8/A9 adenovirus-transduced keratinocytes to investigate the functions of S100A8 and S100A9. We detected increased levels of various pro-inflammatory cytokines in the CM, including IL-8 and TNF-α, which are involved in aggravating psoriatic skin lesions, and IL-6 and members of the CXCL family of pro-angiogenic cytokines. The CM increased immune cell migration and increased angiogenesis in human umbilical vein endothelial cells. In conclusion, we found that the upregulated production of S100A8 and S100A9 by psoriatic epidermal keratinocytes activated adjacent keratinocytes to produce several cytokines. Moreover, S100A8 and S100A9 themselves function as pro-angiogenic and chemotactic factors, generating a psoriatic milieu in skin.     

Discovery of 3,4-dihydropyrimidin-2(1H)-ones with inhibitory activity against HIV-1 replication

3,4-Dihydropyrimidin-2(1H)-ones (DHPMs) were selected and derivatized through a HIV-1 replication assay based on GFP reporter cells. Compounds 14, 25, 31, and 36 exhibited significant inhibition of HIV-1 replication with a good safety profile. Chiral separation of each enantiomer by fractional crystallization showed that only the S enantiomer retained anti-HIV activity. Compound (S)-40, a novel and potent DHPM analog, could serve as an advanced lead for further development and the determination of the mechanism of action.