Automatic Determination of the Sequential Order of Dynamic Data and Its Application to Vibrational Spectroscopy.

For over the past 30 years, generalized two-dimensional correlation spectroscopy has formed an active and widespread research area. One of the most attractive properties of this method is that one can determine the sequential order of signal changes. But the determination of the sequential order has only been done manually for several arbitrarily chosen bands. In this paper, we develop a method to automatically determine the sequential order of all of the band intensity changes, and we applied this method to band changes of vibration spectra. This method will open a door to analyze more complicated signals often seen in life phenomena.

Raman spectral dynamics of single cells in the early stages of growth factor stimulation.

Cell fates change dynamically in response to various extracellular signals, including growth factors that stimulate differentiation and proliferation. The processes underlying cell-fate decisions are complex and often include large cell-to-cell variations, even within a clonal population in the same environment. To understand the origins of these cell-to-cell variations, we must detect the internal dynamics of single cells that reflect their changing chemical milieu. In this study, we used the Raman spectra of single cells to trace their internal dynamics during the early stages of growth factor stimulation. This method allows nondestructive and inclusive time-series analyses of chemical compositions of the same single cells. Applying a Gaussian mixture model to the major principal components of the single-cell Raman spectra, we detected the dynamics of the chemical states in MCF-7 cancer-derived cells in the absence and presence of differentiation and proliferation factors. The dynamics displayed characteristic variations according to the functions of the growth factors. In the differentiation pathway, the chemical composition changed directionally between multiple states, including both reversible and irreversible state transitions. In contrast, in the proliferation pathway, the chemical composition was homogenized into a single state. The differentiation factor also stimulated fluctuations in the chemical composition, whereas the proliferation factor did not.

Raman and autofluorescence spectrum dynamics along the HRG-induced differentiation pathway of MCF-7 cells.

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space.

[A case with chronic hepatitis C who developed liver cirrhosis due to liver dysfunction caused by pegylated interferon plus ribavirin treatment despite negativity of serum HCV RNA, during therapy].

We report a case of chronic hepatitis C in whom liver cirrhosis was later diagnosed following abnormality of ALT levels during pegylated interferon α2a and ribavirin treatment. A 62-year-old woman with chronic hepatitis C was treated with pegylated interferon α2a plus ribavirin for 72 weeks. Her HCV RNA became negative 16 weeks after the start of treatment and continued to be negative for most of the treatment duration. Her AST/ALT, ALP/γ-GTP levels became elevated soon after the initiation of treatment and thereafter remained unchanged. However, most of these levels normalized after the end of treatment. Post-treatment liver biopsy showed liver cirrhosis, probably due to the interferon treatment itself. This unusual therapeutic outcome should be considered if the levels of hepatic dysfunction during interferon treatment are severe.

Severe intraocular inflammation after intravitreal injection of bevacizumab.

PURPOSE: To report 5 cases of severe intraocular inflammation that developed after an intravitreal injection of the same lot of bevacizumab. DESIGN: Retrospective case series. PARTICIPANTS: Patients treated with an intravitreal injection of bevacizumab (lot B3003B01). METHODS: The clinical charts of 35 eyes of 35 consecutive patients who were treated with intravitreal injection of lot B3003B01 bevacizumab from December 18, 2008, through January 20, 2009, were reviewed. MAIN OUTCOME MEASURES: Incidence of intraocular inflammation, results of bacterial cultures, best-corrected visual acuity (BCVA), and endothelial cell density. RESULTS: Five (14.3%) of the 35 cases had severe intraocular inflammation, and the inflammation had some characteristics of toxic anterior segment syndrome (TASS). Five of the 5 cases had a predominantly anterior chamber reaction, and 4 of the 5 cases were accompanied by hypopyon. Undiluted samples collected from both the aqueous and vitreous of the 5 cases were culture negative. The BCVA was 0.66+/-0.29 (mean+/-standard deviations) logarithm of the minimum angle resolution (logMAR) units, and the endothelial cell density was 2683.6+/-97.3/mm(2) before the intravitreal bevacizumab. At the final visit, the BCVA was 0.44+/-0.36 logMAR units, and the cell density was 2679.0+/-217.5/mm(2). These differences were not significant (P = 0.171 and 0.964). CONCLUSIONS: These observations indicate that an intravitreal injection of bevacizumab can induce sterile endophthalmitis that has characteristics of TASS.

[Efficacy of intravitreal bevacizumab for age-related macular degeneration].

PURPOSE: To investigate the efficacy of intravitreal bevacizumab (IVB) for neovascular age-related macular degeneration (AMD). METHODS: We conducted a retrospective study of 29 eyes of 29 patients with AMD (19 eyes) and polypoidal choroidal vasculopathy (PCV; 10 eyes), who were followed up at least 1 year after the initial IVB (1.0 mg/0.04 ml). The eyes were classified according to the lesion type and size. Best-corrected visual acuity (BCVA) and central retinal thickness were examined before and 3 months, 6 months and 12 months after the IVB. RESULTS: The mean application times of IVB were 2.1 in 1 year. When classifying the eyes according to the lesion type, BCVA improved in 5 (26.3%) eyes with AMD and 1 (10.0%) eye with PCV by over 0.2 logarithmic minimum angle of resolution (logMAR) units. The BCVA decreased significantly 1 year after the IVB in eyes with PCV (p = 0.032). When classifying the eyes according to the lesion size, BCVA improved by over 0.2 logMAR units in the 4 (50.0%) eyes with a size of less than 1 disc diameter, 1 (10.0%) eye with the size of 1 to 3 disc diameters, and 1 (9.1%) eye with the size of over 4 disc diameters. The BCVA decreased significantly 1 year after the IVB in the eyes with the size of 1 to 3 disc diameters and with the size of over 4 disc diameters (p = 0.028, 0.013, respectively). The central retinal thickness did not change significantly at any time point compared to that before the IVB. CONCLUSIONS: These results suggest that IVB may be efficacious in preserving visual acuity in AMD eyes and in eyes with the size of less than 1 disc diameter.

Automatic Background Removal and Correction of Systematic Error Caused by Noise Expecting Bio-Raman Big Data Analysis.

Spectral pretreatments, such as background removal from Raman big data, are crucial to have a smooth link to advanced spectral analysis. Recently, we developed an automated background removal method, where we considered the shortest length of a spectrum by changing the scaling factor of the background spectrum. Here, we propose a practical way to correct the systematic error caused by noise from measurements. This correction has been realized to be more effective and accurate for automatic background removal.

Practical Computation of FFT-based Generalized Two-dimensional Correlation Spectroscopy.

A practical computation of fast Fourier transformation (FFT) based generalized two-dimensional (2D) correlation spectroscopy is described. Using simple sinusoids, we tested and confirmed that the method served effectively and properly, invariant to the changes of the number of data points of the time profiles. This computation is applicable to any type of waveforms in a versatile manner.

[Efficacy of treatments on vision-related quality of life in patients with diabetic retinopathy].

PURPOSE: To evaluate changes in the vision-related quality of life in patients with diabetic retinopathy with the 25-item National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25). Patients were categorized into three groups : without treatment, receiving pan-retinal photocoagulation, and those who had vitrectomy. SUBJECTS AND METHODS: Altogether 327 patients (131 without treatment, [observation group]; 60 receiving pan-retinal photocoagulation, [photocoagulation group]; and 136 patients who had vitrectomy, [vitrectomy group]). The VFQ-25 was recorded at the time of entry and 1 year later. The VFQ-25 score was compared between the time of entry and 1 year later for each group and among the three groups at both the time of entry and 1 year later. RESULTS: VFQ-25 scores (mean +/- standard deviations) at the time of entry and 1 year later were 91.3 +/- 7.8 and 92.2 +/- 7.8 in the observation group, 80.7 +/- 15.7 and 77.6 +/-19.1 in the photocoagulation group, and 67.4 +/-17.3 and 75.4 +/- 17.5 in the vitrectomy group. VFQ-25 scores in the observation group and in the photocoagulation group did not change statistically between the time of entry and 1 year later (p = 0.113, 0.169, respectively), while the score of the vitrectomy group increased statistically (p < 0.001). In addition, although VFQ-25 scores were statistically significant among the three groups at the time of entry (p < 0.05), the scores 1 year later were not statistically different between the photocoagulation group and vitrectomy group. CONCLUSIONS: These results suggest that vitrectomy for diabetic retinopathy may be effective in increasing the quality of life of patients with diabetic retinopathy.

Attomole detection of hemagglutinin molecule of influenza virus by combining an electrochemiluminescence sensor with an immunoliposome that encapsulates a Ru complex.

An immunoliposome (80 nm in diameter) encapsulating a Ru complex with two aminobutyl moieties was prepared to detect the presence of hemagglutinin molecules, which play an important role in influenza virus infection. The highly sensitive detection was accomplished by electrochemiluminescence (ECL) from the Ru complex adsorbed onto Au electrodes after competitive immunoreactions. This method clarified that the adsorption of the Ru complex onto the electrode was an important factor in obtaining high sensitivity. Optimization of the analytical conditions enabled determination of the hemagglutinin molecules of the influenza virus in the concentration range of 3 x 10(-14) (6 x 10(-19) mol/50 microL sample) to 2 x 10(-12) g/mL. The sensitivity was far superior to that obtained by conventional ELISA as well as to that obtained by biosensors and reported thus far.

Generalized Two-dimensional Correlation Analysis for Unimodal Waveforms Modeled by Quadratic Polynomials.

We describe further potential of generalized 2D correlation analysis, aiming to realize the automation of the sequential order determination of signal variations. By modeling unimodal waveforms using quadratic functions, we can analytically express 2D correlation functions to yield an index to determine the sequential order. Based on the obtained results, we find an exception for determining the sequential order of signal variations. To resolve the exception, we suggest an extended way of interpreting the sequential order of signal intensity changes.

Human corneal epithelial cell proliferation by epiregulin and its cross-induction by other EGF family members.

PURPOSE: To investigate the effects of epiregulin, a newly identified member of the epidermal growth factor (EGF) family, on the proliferation of human corneal epithelial cells (HCECs). METHODS: The proliferation of HCECs was determined by cell counting and BrdU incorporation assays at specific times after exposure to different concentrations of human recombinant epiregulin (0 to 20 ng/ml). Immunohistochemical staining was used to localize epiregulin in cadaveric corneas. RT-PCR and real-time PCR were used to determine the expression levels of epiregulin in cultured and cadaveric HCECs. To examine the interaction between epiregulin and epidermal growth factor receptors (EGFRs), the phosphorylation of ErbB1 and ERK1/ERK2 (ERK1/2) was estimated by western blot analysis in the presence or absence of AG1478, a specific inhibitor of EGFR kinase activity. To search for cross-induction of epiregulin by other EGF family members, the expressions of EGF, heparin-binding epidermal growth factor-like growth factor (HB-EGF), amphiregulin (AR), and transforming growth factor-alpha (TGF-alpha) mRNA were determined by real-time PCR in the presence of 10 ng/ml of epiregulin. Conversely, the expression of epiregulin was also determined following the incubation of HCECs with 10 nM of either of EGF, HB-EGF, TGF-alpha, or AR. RESULTS: The mRNA of epiregulin was expressed in cultured HCECs and HCECs obtained from cadaveric eyes. Epiregulin was strongly detected in the limbal epithelium and basal epithelium of the peripheral cornea, but it was weakly detected in the central corneal epithelium. HCECs proliferated in the presence of epiregulin in a dose-dependent manner as detected by an increase in cell numbers or in BrdU incorporation. When HCECs were incubated with exogenous epiregulin, the expression of the mRNA of epiregulin was upregulated as detected by real-time PCR, and the phosphorylation of ErbB1 and ERK1/2 was upregulated in a dose-dependent manner as shown by western blot analysis. These upregulations were inhibited by AG1478, a specific inhibitor of EGFR kinase activity. Epiregulin increased the expression of HB-EGF and AR, while TGF-alpha, HB-EGF, AR, and EGF increased the expression of epiregulin in HCECs. CONCLUSIONS: These findings indicate that epiregulin played an autocrine role in the proliferation of HCECs presumably through cross-induction with other EGF family members.

A Proposal for Automated Background Removal of Bio-Raman Data.

The subtraction of background components from observed spectra is essential in performing multivariable analysis, frequently applied to Raman spectra of cells. The subtraction procedure, however, is manual and time consuming, especially for big data. Here, we propose an automated method for removing background information from measured spectra of cells, exhibiting the theoretical framework and an experimental application.

A general model-based approach to two-dimensional infrared correlation spectroscopy incorporating the global phase angle.

We describe a theoretical framework for a model-based approach to two-dimensional correlation spectroscopy that is generally applicable to any arbitrary model function. The method is based on the correlation between spectral data and a set of model waveforms with a varying correlation index, the global phase angle Theta. When experimental spectral intensity variations are expressed as sinusoidal, exponential, Lorentzian, or quadratic functions, the proposed approach allows us to estimate the quantitative values of the target parameters in those expressions. In addition, this method enables us to assess the sequential order in a series of bands undergoing non-identical intensity changes in a dynamic data set. We present both simulated and experimentally obtained data that illustrate that the deviations from linearity of the absorption band intensity waveforms are clearly detected and can be quantitatively estimated using quadratic functions.

Early vitreous hemorrhage after vitrectomy with preoperative intravitreal bevacizumab for proliferative diabetic retinopathy.

PURPOSE: To evaluate the effect of preoperative intravitreal bevacizumab (IVB) on surgical procedures, visual prognosis, and postoperative complications, especially postoperative vitreous hemorrhage, in cases with proliferative diabetic retinopathy (PDR). MATERIALS AND METHODS: Seventy-one eyes of 54 consecutive patients (23 eyes of 18 women, 48 eyes of 36 men) were investigated in this study. Twenty-five eyes received IVB one to 30 days before the vitrectomy (Bevacizumab Group) and the other 46 eyes had the vitrectomy alone (Control Group). The surgical procedures, best-corrected visual acuities at baseline, 1, 3, and 6 months after the vitrectomy, and postoperative complications in the Bevacizumab Group were compared to the Control Group. RESULTS: The patients were significantly younger in the Bevacizumab Group compared to the Control Group (P = 0.008). The incidence of preoperative vitreous hemorrhage, tractional retinal detachment, and iris neovascularization was significantly higher in the Bevacizumab Group than in the Control Group (P = 0.017, 0.041, and 0.018, respectively). The surgical procedures performed and the visual acuity at all time points was not significantly different between groups (P > 0.05, all comparisons). The incidence of early (≤ 4 weeks) postoperative vitreous hemorrhage was significantly higher in the Bevacizumab Group (27%) than in the Control Group (7%; P = 0.027) although the rate of late (>4 weeks) postoperative vitreous hemorrhage was not significantly different between groups (P > 0.05). CONCLUSION: Vitrectomy with preoperative IVB may have no detrimental effect on surgical procedures and achieves the surgical outcomes for repair of PDR equal to vitrectomy alone despite the obvious selection bias of the patients in this study. However, special monitoring is highly recommended for early postoperative vitreous hemorrhage because bevacizumab in the vitreous may be washed out during vitrectomy.

Corneal epithelial wound healing impaired in keratinocyte-specific HB-EGF-deficient mice in vivo and in vitro.

PURPOSE: To study the role played by HB-EGF in corneal epithelial wound healing. METHODS: A 2-mm corneal epithelial wound was created in keratinocyte-specific, HB-EGF-deficient mice--HB(lox/lox):K-5Cre (HB(-/-))--and the speed of wound healing was compared with that in wild-type (WT) mice. Cultured confluent mouse corneal epithelial cells (MCECs) from WT and HB(-/-) mice were scraped, and the bare area was measured. The proliferation of MCECs was determined by BrdU incorporation. The degree of attachment of WT and HB(-/-) MCECs was also determined. The mRNA expression of EGF family and cell adhesion molecules was determined by real-time PCR. RESULTS: Corneal epithelial wound healing was significantly delayed in HB(-/-) mice, and the expression of HB-EGF was detected at the leading edge of the wound in HB(lox/+):K5-Cre (HB(+/-)) mice by the presence of lacZ staining. The wound closure was significantly impaired in HB(-/-) MCECs and was improved by adding HB-EGF. The number of BrdU-positive MCECs of WT and HB(-/-) mice was not significantly different, and both increased to different degrees when HB-EGF was added. The adhesion of isolated HB(-/-) MCECs was lower than that of WT MCECs, but the degree of adhesion was restored by adding HB-EGF. The expression of epiregulin was upregulated in HB(-/-) MCECs, and α6- and ß1-integrin were upregulated by adding HB-EGF. CONCLUSIONS: HB-EGF plays an important role in corneal epithelial cell healing by enhancing cellular attachment and part of cell proliferation.

Clonally expanding thymocytes having lineage capability in gamma-ray-induced mouse atrophic thymus.

PURPOSE: To characterize, in the setting of gamma-ray-induced atrophic thymus, probable prelymphoma cells showing clonal growth and changes in signaling, including DNA damage checkpoint. METHODS AND MATERIALS: A total of 111 and 45 mouse atrophic thymuses at 40 and 80 days, respectively, after gamma-irradiation were analyzed with polymerase chain reaction for D-J rearrangements at the TCRbeta locus, flow cytometry for cell cycle, and Western blotting for the activation of DNA damage checkpoints. RESULTS: Limited D-J rearrangement patterns distinct from normal thymus were detected at high frequencies (43 of 111 for 40-day thymus and 21 of 45 for 80-day thymus). Those clonally expanded thymocytes mostly consisted of CD4(+)CD8(+) double-positive cells, indicating the retention of lineage capability. They exhibited pausing at a late G1 phase of cell cycle progression but did not show the activation of DNA damage checkpoints such as gammaH2AX, Chk1/2, or p53. Of interest is that 17 of the 52 thymuses showing normal D-J rearrangement patterns at 40 days after irradiation showed allelic loss at the Bcl11b tumor suppressor locus, also indicating clonal expansion. CONCLUSION: The thymocytes of clonal growth detected resemble human chronic myeloid leukemia in possessing self-renewal and lineage capability, and therefore they can be a candidate of the lymphoma-initiating cells.