Using peptide barcodes for simultaneous profiling of protein expression from mRNA.

RATIONALE: mRNA technology has begun to play a significant role in the areas of therapeutic intervention and vaccine development. However, optimizing the mRNA sequence that influences protein expression levels is a resource-intensive and time-consuming process. This study introduces a new method to accelerate the selection of sequences of mRNA for optimal protein expression. METHODS: We designed the mRNA sequences in such a way that a unique peptide barcode, corresponding to each mRNA sequence, is attached to the expressed protein. These barcodes, cleaved off by a protease and simultaneously quantified by mass spectrometry, reflect the protein expression, enabling a parallel analysis. We validated this method using two mRNAs, each with different untranslated regions (UTRs) but encoding enhanced green fluorescence protein (eGFP), and investigated whether the peptide barcodes could analyze the differential eGFP expression levels. RESULTS: The fluorescence intensity of eGFP, a marker of its expression level, has shown noticeable changes between the two UTR sequences in mRNA-transfected cells when measured using flow cytometry. This suggests alterations in the expression level of eGFP due to the influence of different UTR sequences. Furthermore, the quantified amount of peptide barcodes that were released from eGFP showed consistent patterns with these changes. CONCLUSIONS: The experimental findings suggest that peptide barcodes serve as a valuable tool for assessing protein expression levels. The process of mRNA sequence selection, aimed at maximizing protein expression, can be enhanced by the parallel analysis of peptide barcodes using mass spectrometry.

Transfer learning and wavelength selection method in NIR spectroscopy to predict glucose and lactate concentrations in culture media using VIP-Boruta.

Regression models are constructed to predict glucose and lactate concentrations from near-infrared spectra in culture media. The partial least-squares (PLS) regression technique is employed, and we investigate the improvement in the predictive ability of PLS models that can be achieved using wavelength selection and transfer learning. We combine Boruta, a nonlinear variable selection method based on random forests, with variable importance in projection (VIP) in PLS to produce the proposed variable selection method, VIP-Boruta. Furthermore, focusing on the situation where both culture medium samples and pseudo-culture medium samples can be used, we transfer pseudo media to culture media. Data analysis with an actual dataset of culture media and pseudo media confirms that VIP-Boruta can effectively select appropriate wavelengths and improves the prediction ability of PLS models, and that transfer learning with pseudo media enhances the predictive ability. The proposed method could reduce the prediction errors by about 61% for glucose and about 16% for lactate, compared to the traditional PLS model.

Glucose Monitoring in Cell Culture with Online Ultrasound-Assisted Near-Infrared Spectroscopy.

Near-infrared (NIR) spectroscopic analysis for suspensions often requires a pretreatment by dilution or filtering of suspended solids to maintain the sensitivity of the measurements. An online ultrasound-assisted spectroscopy (UAS) unit enabling pretreatment-free and noncontact analysis for bioprocessing is proposed and evaluated with a model suspension containing 3-µm-diameter polystyrene latex particles (PSLs) with the density of 5.1 × 108 counts/ml and Chinese hamster ovary (CHO) cell culture whose cell density was 3.2 × 107 cells/ml. The online UAS uses acoustic radiation force generated by ultrasonic standing waves. Suspended matter such as the PSLs and CHO cells can be localized at nodal planes in the suspensions by the acoustic radiation force. Hence, in the case of the online UAS, incoming light can pass through the suspensions more easily than that in the case of a conventional spectroscopy. Its effectiveness was evaluated by the predictive capability of a calibration model for glucose concentrations in the model suspensions and the CHO cell cultures. The calibration models were constructed by use of a partial least-squares regression (PLS-R) in the range of 4900-4200 cm-1 region after the pretreatment of second-order Savitzky-Golay filter. The calibration model built from the NIR spectra acquired with the online UAS could predict the glucose concentration in the CHO cell cultures with a measurement error of 0.6%. It was validated that the glucose concentrations in the flowing model suspension were able to be monitored by the online UAS with a measurement error of 8%. The newly developed online UAS for cell culture monitoring allows us to promote a wider use of NIR spectroscopy. For example, in the applications to the biopharmaceutical and cell-therapy industries, the online UAS enables simpler and easier monitoring of cell cultures because cleaning and sterilization of monitoring tools after cell culturing can be eliminated.

Distribution of chloroplast DNA haplotypes in the contact zone of Fagus crenata in the southwest of Kanto District, Japan.

We have investigated cpDNA haplotype distribution in 24 populations of Fagus crenata in the southwest of Kanto District, Japan, and clarified the extent of intermixing of haplotypes in the contact zone by additional fine-scale analysis of two areas. Two cpDNA haplotypes belonging to different lineages were detected, and their distribution had geographical structure. Intermixed populations with the two haplotypes were limited to a narrow area. The geographical boundary between the haplotypes extended from Hakone to the west of the Kanto Mountains through the northern foot of Mt Fuji. No relationship was observed between the boundary location and the current topography of the southwest of Kanto District.

Structural study on highly oxidized states of a water oxidation complex [RuIII(bpy)2(H2O)]2(mu-O)4+ by ruthenium K-edge X-ray absorption fine structure spectroscopy.

The oxidation-induced structural change of a water-oxidizing diruthenium complex, [(bpy)(2)(H(2)O)Ru(III)(micro-O)Ru(III)(OH(2))(bpy)(2)](4+) (bpy = 2,2'-bipyridine), was investigated by means of X-ray absorption spectroscopy. Ru K-edge XANES (X-ray absorption near-edge structure) spectra from the acidic solution and solid precipitates obtained by oxidation showed that the absorption edge shifts toward higher energy with a preedge feature slightly more enhanced than those of the lower oxidation states. This indicates that the higher oxidation state has a lower symmetry due to shortening of the Ru-O bonds that originated from the water ligands. The EXAFS (extended X-ray absorption fine structure) spectra were similar to those of the lower oxidation states, whose analysis revealed the existence of short Ru-O double bonds and an almost linear Ru-O-Ru angle (169 +/- 2 degrees ). Ab initio EXAFS simulations for several possible structural models suggest that the dimeric structure is maintained during the water oxidation reaction.

Development of light-modulated XAFS spectroscopy.

The light-modulation method was applied to XAFS spectroscopy as a useful tool for electronic and structural studies of light-excited states. The visible light for electronic excitation was modulated by an optical chopper and the concomitant change in the X-ray absorbance was extracted by a lock-in amplifier. It is essential to reduce any noise arising from several sources, especially those generated by the X-ray monochromator. Fe and Co K-edge XANES and EXAFS spectra were measured using the present approach for spin transition compounds. The light-modulated XAFS method is found to be applicable to systems with an excited-state ratio of less than several percent. Detailed discussion is also given concerning quantitative measurements, including the signal amplitude and phase shift information.