Improving mass accuracy in matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of pathogenic proteins using 6xHIS-tagged internal calibration.

RATIONALE: Linear mode of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been routinely used for bacterial identification in the clinic, depending on the pattern analysis of spectral libraries rather than accurate mass measurement of ribosomal proteins (10-15 kDa). However, a demand for more accurate mass analysis of pathogens (e.g. KPC-2 carbapenemase) has been recently increasing for diagnostic purposes. METHODS: We introduced a 6xHIS-tagged KPC-2 (i.e. hKPC-2) and used it as an internal mass calibrator for the mass calibration of target proteins. After internal mass calibration (In-Cal), we evaluated the observed mass of KPC-2 against the theoretical mass of hKPC-2, which has 823 Da mass difference from the target protein. We further assessed the accuracy and precision of our calibration method regarding the identification of KPC-2 and other pathogens in clinical isolates (n = 42). RESULTS: Among several candidates for internal mass calibrators, the In-Cal using a 6xHIS-tagged protein on the target showed the highest mass accuracy and precision in the detection of target proteins (e.g. KPC-2). The application of hKPC-2 as an internal calibrator showed substantial improvement of mass accuracy, mass precision and also quantification of KPC in linearity and repeatability for KPC detection in the clinical isolates. CONCLUSIONS: Our In-Cal method using 6xHIS-tagged protein in MALDI-TOFMS allows successful mass calibration (<3.5 Da) of pathogenic proteins (>20 kDa) and provides high mass accuracy as much as that of medium- and high-resolution mass spectrometry.

Adolescents' Attitudes and Intentions toward Help-Seeking and Computer-Based Treatment for Depression.

OBJECTIVE: Many depressed adolescents do not seek professional help despite there being evidence-based treatments for depression, such as cognitive behavioral therapy or computer-based therapy. To increase professional help-seeking behavior in depressed adolescents, it is necessary to positively change help-seeking attitudes. This study aimed to explore the effect of sub-groups of help-seeking attitudes, gender, and depression level on adolescents' help-seeking intentions and their perceptions of computer-based psychotherapy. METHODS: Participants were 246 adolescents aged 13-18 years recruited from six middle and high schools in South Korea. Measures were self-administered questionnaires, and included the Patient Health Questionnaire-9, the Attitudes Toward Seeking Professional Psychological Help Scale, the Intention to Seek Counseling Inventory, Preferences for Depression Treatment, and the Perceptions of Computerized Therapy Questionnaire. RESULTS: Help-seeking intentions were positively related with female gender and the recognition of the need for help. A higher level of confidence in therapists was related to high preference for computer-based therapy and face-to-face therapy. Adolescents with more severe depression were more likely to prefer pharmacotherapy. The perceptions of computer-based therapy were more positive in male adolescents, and in adolescents with a higher level of confidence in therapists yet a lower level of interpersonal openness. CONCLUSION: To promote adolescents' help-seeking behavior, improvement of the recognition of the need for help is required, especially among male adolescents. Computer-based therapy provides an alternative for male adolescents with high confidence in therapists yet low interpersonal openness. Consideration of the help-seeking attitudes and gender is needed when providing therapeutic intervention to depressed adolescents.

Fabrication of dihydroxyflavone-conjugated hyaluronic acid nanogels for targeted antitumoral effect.

We prepared hyaluronic acid (HA)-based nanogels conjugated with dihydroxyflavone (DHF) and evaluated their cellular uptake and antitumoral efficiency. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was used as a conjugation agent for esterification between DHF and HA as well as crosslinking among HA. The conjugations were confirmed by nuclear magnetic resonance spectroscopy, UV/vis spectroscopy, and high-performance liquid chromatography. The size and Zeta-potential of the DHF/HA nanogels were reduced with an increase in the concentration of DMTMM due to the involvement of more HA molecules for the conjugation reactions. The DHF/HA nanogel with a smaller size was greatly taken up by two kinds of tumor cells (HeLa and HepG2), compared to NIH3T3. The cell viabilities were reduced to approximately 60% for HeLa and HepG2 cells after 48 h post treatment with DHF/HA nanogels.

Fabrication and optimization of Nanodiamonds-composited poly(ε-caprolactone) fibrous matrices for potential regeneration of hard tissues.

BACKGROUND: Electrospun fibrous matrices are of great importance for tissue engineering and drug delivery device. However, relatively low mechanical strength of the fibrous matrix is one of the major disadvantages. NDs with a positive charge were selected to enhance the mechanical property of a composited fibrous matrix by inducing the intermolecular interaction between NDs and polymer chain. We prepared ND-composited poly (ε-caprolactone) (PCL) fibrous matrices by electrospinning and evaluated their performance in terms of mechanical strength and cell behaviors. METHODS: A predetermined amounts of NDs (0.5, 1, 2 and 3 wt%) were added into PCL solution in a mixture of chloroform and 2,2,2-trifluoroethanol (8:2). ND-composited PCL (ND/PCL) fibrous matrices were prepared by electrospinning method. The tensile properties of the ND/PCL fibrous matrices were analyzed by using a universal testing machine. Mouse calvaria-derived preosteoblast (MC3T3-E1) was used for cell proliferation, alkaline phosphatase (ALP) assay, and Alizarin Red S staining. RESULTS: The diameters of the fibrous matrices were adjusted to approximately 1.8 µm by changing process variables. The intermolecular interaction between NDs and PCL polymers resulted in the increased tensile strength and the favorable interfacial adhesion in the ND/PCL fibrous matrices. The ND/PCL fibrous matrix with 1 wt% of ND had the highest tensile strength among the samples and also improved proliferation and differentiation of MC3T3-E1 cells. CONCLUSIONS: Compared to the other samples, the ND/PCL fibrous matrix with 1 wt% of ND concentration exhibited superior performances for MC3T3 cells. The ND/PCL fibrous matrix can be potentially used for bone and dental tissue engineering.