On-the-fly Raman microscopy guaranteeing the accuracy of discrimination.

Accelerating the measurement for discrimination of samples, such as classification of cell phenotype, is crucial when faced with significant time and cost constraints. Spontaneous Raman microscopy offers label-free, rich chemical information but suffers from long acquisition time due to extremely small scattering cross-sections. One possible approach to accelerate the measurement is by measuring necessary parts with a suitable number of illumination points. However, how to design these points during measurement remains a challenge. To address this, we developed an imaging technique based on a reinforcement learning in machine learning (ML). This ML approach adaptively feeds back "optimal" illumination pattern during the measurement to detect the existence of specific characteristics of interest, allowing faster measurements while guaranteeing discrimination accuracy. Using a set of Raman images of human follicular thyroid and follicular thyroid carcinoma cells, we showed that our technique requires 3,333 to 31,683 times smaller number of illuminations for discriminating the phenotypes than raster scanning. To quantitatively evaluate the number of illuminations depending on the requisite discrimination accuracy, we prepared a set of polymer bead mixture samples to model anomalous and normal tissues. We then applied a home-built programmable-illumination microscope equipped with our algorithm, and confirmed that the system can discriminate the sample conditions with 104 to 4,350 times smaller number of illuminations compared to standard point illumination Raman microscopy. The proposed algorithm can be applied to other types of microscopy that can control measurement condition on the fly, offering an approach for the acceleration of accurate measurements in various applications including medical diagnosis.

Near-infrared spectroscopy of low-transmittance samples by a high-power time-stretch spectrometer using an arrayed waveguide grating (AWG).

Although time-stretch spectroscopy is an emerging ultrafast spectroscopic technique, the applications in industrial fields have been limited due to the low output power caused by undesirable nonlinear effects occurred in a long optical fiber used for pulse chirping. Here, we developed a high-power time-stretch near infrared (NIR) spectrometer utilizing arrayed waveguide gratings (AWGs). The combination of AWGs and short optical fibers allowed large amounts of chromatic dispersion to be applied to broadband supercontinuum pulses without the power limitation imposed by employing the long optical fiber. With the proposed configuration, we achieved chirped pulses with the output power of 60 mW in the 900-1300 nm wavelength region, which is about 10 times higher than conventional time-stretch spectrometers using long optical fibers. With the developed spectrometer, the NIR absorption spectra of a standard material and liquid samples were observed with high accuracy and precision within sub-millisecond measurement time even with four orders of magnitude optical attenuation by a neutral density filter. We also confirmed the quantitative spectral analysis capability of the developed spectrometer for highly scattering samples of an oil emulsion. The qualitative comparison of the measurement precision between the developed spectrometer and the previous time-stretch spectrometer was also conducted.

Multiwell Raman plate reader for high-throughput biochemical screening.

Although Raman spectroscopy has been used for the quantitative analysis of samples in many fields, including material science, biomedical, and pharmaceutical research, its low sensitivity hindered the application of the analytical capability for high-throughput screening. Here, we developed a high-throughput Raman screening system that can analyze hundreds of specimens in a multiwell plate simultaneously. Multiple high numerical aperture (NA) lenses are assembled under each well in the multiwell plate to detect Raman scattering simultaneously with high sensitivity. The Raman spectrum of 192 samples loaded on a standard 384-well plate can be analyzed simultaneously. With the developed system, the throughput of Raman measurement was significantly improved (about 100 times) compared to conventional Raman instruments based on a single-point measurement. By using the developed system, we demonstrated high-throughput Raman screening to investigate drug polymorphism and identify a small-molecule binding site in a protein. Furthermore, the same system was used to demonstrate high-speed chemical mapping of a centimeter-sized pork slice.

Label-free monitoring of crystalline chitin hydrolysis by chitinase based on Raman spectroscopy.

We demonstrate a method for label-free monitoring of hydrolytic activity of crystalline-chitin-degrading enzyme, chitinase, by means of Raman spectroscopy. We found that crystalline chitin exhibited a characteristic Raman peak at 2995 cm-1, which did not appear in the reaction product, N,N'-diacetylchitobiose. We used this Raman peak as a marker of crystalline chitin degradation to monitor the hydrolytic activity of chitinase. When the crystalline chitin suspension and chitinase were mixed together, the peak intensity of crystalline chitin at 2995 cm-1 was linearly decreased depending on incubation time. The decrease in peak intensity was inversely correlated with the increase in the amount of released N,N'-diacetylchitobiose, which was measured by conventional colorimetric assay with alkaline ferricyanide. Our result, presented here, provides a new method for simple, in situ, and label-free monitoring of enzymatic activity of chitinase.

Detecting nitrile-containing small molecules by infrared photothermal microscopy.

The use of infrared (IR) photothermal microscopy (IR-PTM) is emerging for imaging chemical substances in various samples. In this research, we demonstrated the use of a nitrile group as a vibrational tag to image target molecules in the low water-background region. We performed IR photothermal imaging of trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) in cells and confirmed the high spatial resolution by photothermal detection using visible light as a probe beam. We imaged FCCP-treated HeLa cells and confirmed that the photothermal signal was indeed produced from the vibrational tag in lipid droplets. We also compared the results with nitrile imaging by stimulated Raman scattering (SRS) microscopy. From both the calculated and experimental results, IR-PTM demonstrated a signal-to-noise ratio (SNR) several tens of times better than that of SRS microscopy on the basis of the same power input.

Using redox-sensitive mitochondrial cytochrome Raman bands for label-free detection of mitochondrial dysfunction.

Mitochondrial activity is a widely used criterion to judge the metabolic condition of a living specimen. Numerous methods have been developed for related analyses, including the detection of O2 consumption, trans-membrane potential, and ATP production. In this study, we demonstrate that the redox state of cytochromes can serve as a sensitive mitochondrial activity indicator in glutamate-stressed neuronal cells. Mitochondrial dysfunction was detected by Raman imaging as early as 30 min after glutamate-stress induction. By comparing this result with other commonly used mitochondrial function assays, we found Raman imaging has a similar sensitivity to ATP production and trans-membrane potential assays. Other viability tests, such as MTT assay and ROS production tests, showed a slower response than our method. A thorough understanding of cytochrome dynamics with our new method will help establish Raman spectroscopy as a competitive clinical diagnosis tool for neurodegenerative diseases involving mitochondrial dysfunction.

Axial resolution and signal-to-noise ratio in deep-tissue imaging with 1.7-µm high-resolution optical coherence tomography with an ultrabroadband laser source.

We investigated the axial resolution and signal-to-noise ratio (SNR) characteristics in deep-tissue imaging by 1.7-µm optical coherence tomography (OCT) with the axial resolution of 4.3 µm in tissue. Because 1.7-µm OCT requires a light source with a spectral width of more than 300 nm full-width at half maximum to achieve such high resolution, the axial resolution in the tissue might be degraded by spectral distortion and chromatic dispersion mismatching between the sample and reference arms. In addition, degradation of the axial resolution would also lead to reduced SNR. Here, we quantitatively evaluated the degradation of the axial resolution and the resulting decrease in SNR by measuring interference signals through a lipid mixture serving as a turbid tissue phantom with large scattering and absorption coefficients. Although the axial resolution was reduced by a factor of ∼6 after passing through a 2-mm-thick tissue phantom, our result clearly showed that compensation of the dispersion mismatching allowed us to achieve an axial resolution of 4.3 µm in tissue and improve the SNR by ∼5 dB compared with the case where dispersion mismatching was not compensated. This improvement was also confirmed in the observation of a hamster's cheek pouch in a buffer solution.

Optical coherence microscopy in 1700 nm spectral band for high-resolution label-free deep-tissue imaging.

Optical coherence microscopy (OCM) is a label-free, high-resolution, three-dimensional (3D) imaging technique based on optical coherence tomography (OCT) and confocal microscopy. Here, we report that the 1700-nm spectral band has the great potential to improve the imaging depth in high-resolution OCM imaging of animal tissues. Recent studies to improve the imaging depth in OCT revealed that the 1700-nm spectral band is a promising choice for imaging turbid scattering tissues due to the low attenuation of light in the wavelength region. In this study, we developed high-resolution OCM by using a high-power supercontinuum source in the 1700-nm spectral band, and compared the attenuation of signal-to-noise ratio between the 1700-nm and 1300-nm OCM imaging of a mouse brain under the condition of the same sensitivity. The comparison clearly showed that the 1700-nm OCM provides larger imaging depth than the 1300-nm OCM. In this 1700-nm OCM, the lateral resolution of 1.3 µm and the axial resolution of 2.8 µm, when a refractive index was assumed to be 1.38, was achieved.

Overexpression of N-Myc rapidly causes acute myeloid leukemia in mice.

N-MYC encodes a basic helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor that is frequently overexpressed in human neuroblastoma. N-MYC overexpression has also been reported in human acute myeloid leukemias (AML), which we show here is a frequent event. Myeloid cells in N-Myc-overexpressing mouse bone marrow hyperproliferate but those in c-MYC-overexpressing bone marrow do not. The NH(2)-terminal transactivation domain, nuclear localization signal, and bHLH/LZ domain of N-Myc are essential for this effect. Microarray analysis revealed 969 differentially expressed genes between N-Myc- and c-MYC-overexpressing myeloid cells. N-Myc-overexpressing cells showed decreased transforming growth factor beta signaling and increased c-Jun-NH(2)-kinase signaling, both of which are associated with proliferation and leukemic transformation of myeloid cells. Mice transplanted with bone marrow expressing wild-type N-Myc developed clonal and transplantable AML after approximately 1 month; those transplanted with bone marrow expressing mutant N-Myc did not. Twist, a known suppressor of the p19Arf/p53 pathway, was up-regulated in all tumors. These results show that N-Myc overexpression is highly oncogenic in mouse myeloid cells and suggest that N-MYC up-regulation contributes to human myeloid leukemogenesis.

Conditional MN1-TEL knock-in mice develop acute myeloid leukemia in conjunction with overexpression of HOXA9.

The chromosomal translocation t(12; 22)(p13;q11) in human myeloid leukemia generates an MN1-TEL (meningioma 1-translocation-ETS-leukemia) fusion oncoprotein. This protein consists of N-terminal MN1 sequences, a transcriptional coactivator fused to C-terminal TEL sequences, an ETS (E26 transformation-specific) transcription factor. Enforced expression of MN1-TEL in multipotent hematopoietic progenitors in knock-in mice perturbed growth and differentiation of myeloid as well as lymphoid cells. Depending on obligatory secondary mutations, these mice developed T-cell lympholeukemia. Here we addressed the role of MN1-TEL in myeloid leukemogenesis using the same mouse model. Expression of MN1-TEL enhanced the growth of myeloid progenitors in an interleukin 3/stem cell factor (IL-3/SCF)-dependent manner in vitro whereas 10% of MN1-TEL-expressing mice developed altered myelopoiesis with severe anemia after long latency. Coexpression of MN1-TEL and IL-3, but not SCF, rapidly caused a fatal myeloproliferative disease rather than acute myeloid leukemia (AML). Because MN1-TEL+ AML patient cells overexpress HOXA9 (homeobox A9), we tested the effect of coexpression of MN1-TEL and HOXA9 in mice and found that 90% of MN1-TEL+/HOXA9+ mice developed AML much more rapidly than control HOXA9+ mice. Thus, the leukemogenic effect of MN1-TEL in our knock-in mice is pleiotropic, and the type of secondary mutation determines disease outcome.

MN1-TEL myeloid oncoprotein expressed in multipotent progenitors perturbs both myeloid and lymphoid growth and causes T-lymphoid tumors in mice.

The MN1-TEL (meningioma 1-translocation-ETS-leukemia) fusion oncoprotein is the product of the t(12;22)(p13;q11) in human myeloid leukemia consisting of N-terminal MN1 sequences, a transcriptional coactivator, fused to C-terminal TEL sequences, an E26-transformation-specific (ETS) transcription factor. To analyze the role of MN1-TEL in leukemogenesis, we created a site-directed transgenic (knock-in) mouse model carrying a conditional MN1-TEL transgene under the control of the Aml1 regulatory sequences. After induction, MN1-TEL expression was detected in both myeloid and lymphoid cells. Activation of MN1-TEL expression enhanced the repopulation ability of myeloid progenitors in vitro as well as partially inhibited their differentiation in vivo. MN1-TEL also promoted the proliferation of thymocytes while it blocked their differentiation from CD4-/CD8- to CD4+/CD8+ in vivo. After long latency, 30% of the MN1-TEL-positive mice developed T-lymphoid tumors. This process was accelerated by N-ethyl-N-nitrosourea-induced mutations. MN1-TEL-positive T-lymphoid tumors showed elevated expression of the Notch-1, Hes-1, c-Myc, and Lmo-2 genes while their Ink4a/pRB and Arf/p53 pathways were impaired, suggesting that these alterations cooperatively transform T progenitors. We conclude that MN1-TEL exerts its nonlineage-specific leukemogenic effects by promoting the growth of primitive progenitors and blocking their differentiation, but cooperative mutations are necessary to fully induce leukemic transformation.

TEL2, an ETS factor expressed in human leukemia, regulates monocytic differentiation of U937 Cells and blocks the inhibitory effect of TEL1 on ras-induced cellular transformation.

TEL2 is a member of the ETS family of transcription factors, which is highly similar to TEL1/ETV6. It binds to DNA via the ETS domain and interacts with itself or TEL1 via the pointed domain. The expression of TEL2 in normal and leukemic hematopoietic cells suggests a role in hematopoietic development. In this article, we describe the role of TEL2 in hematopoietic differentiation and cellular transformation. Quantitative reverse transcription-PCR showed that the expression of TEL2 mRNA was down-regulated during monocytic differentiation of U937 and HL60 induced by 1,25-(OH)2 vitamin D3 and 12-O-tetradecanoylphorbol 13-acetate, respectively. Overexpression of TEL2 in U937 cells inhibited differentiation induced by vitamin D3. In contrast, overexpression of a TEL2 mutant lacking either the pointed domain or a functional ETS domain induced both differentiation of U937 cells and inhibited their growth in vitro and in vivo. In addition, these mutants blocked TEL2-mediated transcriptional repression of a synthetic promoter containing TEL2 binding sites. These data suggest that dominant-negative inhibition of TEL2 might cause differentiation. Quantitative reverse transcription-PCR demonstrated that TEL2 is expressed at higher level in some primary human leukemia samples than in normal bone marrow. Furthermore, overexpression of TEL2 in NIH3T3-UCLA cells blocked the inhibitory effect of TEL1 on Ras-induced cellular transformation. These results suggest that TEL2 may play an important role in hematopoiesis and oncogenesis.

Pathophysiology and treatment for cervical flexion myelopathy.

Previous studies have suggested that spinal cord compression by the vertebral bodies and intervertebral discs during neck flexion cause cervical flexion myelopathy (CFM). However, the exact pathophysiology of CFM is still unknown, and surgical treatment for CFM remains controversial. We examined retrospectively patients with CFM based on studies of the clinical features, neuroradiological findings, and neurophysiological assessments. The objectives of this paper are to investigate the pathophysiology of CFM, and to examine an optimal surgical treatment. Twenty-three patients (20 male, three female) with age of onset ranging from 11 to 23 years (mean 15.7 years) were examined for the study. All patients were inspected by magnetic resonance imaging (MRI), myelogram, or computed tomographic myelogram (CTM) of the cervical spine. In eight patients, dynamic motor evoked potentials (MEP) studies were performed. Five patients underwent surgical treatment; two patients had cervical duraplasty with laminoplasty, two patients had musculotendinous transfer, one patient had both of these procedures, and the remaining 18 patients were treated conservatively. Amyotrophy of the hand intrinsic and flexor muscle group of the forearm except the brachioradial muscle was observed hemilaterally in 20 patients and bilaterally in three patients. In three patients, T1-weighted MRI with neck flexion showed linear high intensity regions in the epidural space. In all patients, axial MRI/CTM demonstrated flattening of the spinal cord with the posterior surface of the dura mater shifting anteriorly. The amplitude of MEPs decreased after cervical flexion in two patients with progressive muscular atrophy. In three patients, dysesthesia of the upper extremities disappeared following cervical duraplasty. Musculotendinous transfer for three patients significantly improved the performance of their upper extremity. The findings of this study suggest that degenerative changes of the dura mater may be a characteristic pathology of CFM. Cervical duraplasty with laminoplasty is effective for cases at an early stage, and musculotendinous transfer should be selected in patients at a late stage.

Prophylactic effect of inactivated influenza vaccine on young children.

BACKGROUND: The effectiveness of inactivated influenza vaccine in healthy infants and young children has been controversial. The aim of this study was to determine the prophylactic effect of inactivated influenza vaccine in young children. METHODS: Eighty-six healthy infants and children younger than 7-years-old were immunized by a subcutaneous injection of inactivated influenza vaccine before the 1999/2000 influenza season. Ninety-four age-matched children were randomly assigned as the control. These children were followed-up from January to April, 2000. A diagnosis of influenza A virus infection was made rapidly by a positive result of the the enzyme immunoassay membrane test using enzyme-conjugated monoclonal antibodies specific for a conserved epitope of influenza A nucleoprotein. The incidence of influenza A infection was compared and statistically assessed. RESULTS: The prevalence of influenza A virus infection, diagnosed by the influenza A rapid detection test, was 5.8% in the vaccine group and 17.0% in the control group, that is significantly lower in the vaccine receiving group than the non-receiving group (P = 0.016). However, four out of five infected children in the vaccine group were younger than 2-years-old. CONCLUSION: We conclude that inactivated influenza vaccine reduces the incidence of influenza A virus infection in 2-6-year-old children.

Molecular basis for exaggerated sensitivity to mexiletine in the cardiac isoform of the fast Na channel.

Cardiac sodium channels have been shown to have a higher sensitivity to local anesthetic agents, such as lidocaine, than the sodium channels of other tissues. To examine if this is also true for mexiletine, we have systematically measured mexiletine sensitivity of the Na channel isoforms, rH1, (mu)1, and rBII, which were transiently expressed in human embryonic kidney (HEK) 293 cells. We confirmed that the cardiac isoform rH1 exhibited the highest sensitivity among the three tested channel isoforms. In rH1, (mu)1, and rBII, the respective IC(50) values were 62, 294, and 308 microM mexiletine, in regard to tonic block, and 18, 54, and 268 microM mexiletine, in relation to use (8 Hz)-dependent block. The relatively high drug sensitivity of rH1 was an invariant finding, irrespective of channel state or whether channels were subjected to infrequent or frequent depolarizing stimuli. Mutating specific amino acids in the skeletal muscle isoform (mu)1 (namely, (mu)1-I433V and (mu)1-S251A) to those of the cardiac isoform at putative binding sites for local anesthetic agents revealed that only one of the point mutations ((mu)1-S251A) has relevance to the high cardiac drug sensitivity, because mexiletine produced significantly more use-dependent and tonic block in (mu)1-S251A than wild-type (mu)1.

Microsurgical transdural discectomy with laminoplasty: New treatment for paracentral and paracentroforaminal cervical disc herniation associated with spinal canal stenosis.

STUDY DESIGN: A clinical study of the surgical procedure for cervical disc herniation was conducted. OBJECTIVES: To describe microsurgical transdural discectomy with laminoplasty, and to assess the clinical outcome of this surgical technique. SUMMARY OF BACKGROUND DATA: A posterior approach for cervical disc herniation has been considered risky, and few reports on a transdural approach to this disorder have appeared in the past decade. However, a transdural approach with recent innovations (a microsurgical technique, intraoperative spinal cord monitoring, and laminoplasty) has not been reported. METHODS: For this study, 30 patients with myelopathy or radiculomyelopathy accompanied by cervical disc herniation, aged 30 to 77 years (mean, 55 years), underwent microsurgical transdural discectomy with laminoplasty. Preoperative images showed multisegmental disc degeneration, developmental canal stenosis, or both for all the patients. The intraoperative evoked spinal cord potentials were recorded for neurophysiologic assessment. The follow-up period averaged 52 months (range, 24-118 months). RESULTS: The operative time averaged 239 minutes (range, 160-340 minutes), and the mean blood loss was 169 mL (range, 30-701 mL). The Japanese Orthopedic Association score improved from 3.5 to 15 (mean, 11.4) before surgery to 9 to 17 (mean, 15.2) after surgery. The intraoperative evoked spinal cord potentials indicated the affected spinal cord level and reflected the severity of myelopathy. Postoperative cerebrospinal fluid leakage, pseudomeningocele, and progression to cervical deformity were not observed. Transient palsy of the C5 nerve root was observed in two patients with C4-C5 central cervical disc herniation. CONCLUSIONS: Microsurgical transdural discectomy with laminoplasty can be performed safely as a selected surgical option for paracentral and paracentroforaminal cervical disc herniation with multisegmental canal stenosis.

Valproic acid induces apoptosis in human leukemia cells by stimulating both caspase-dependent and -independent apoptotic signaling pathways.

We investigated the effects of valproic acid (VPA) on the growth and survival of human leukemia cell lines. VPA induced cell death in all of the nine cell lines tested in a dose dependent manner. VPA-treatment induced apoptotic changes in MV411 cells including DNA fragmentation, phosphatidylserine externalization, cytochrome c release from mitochondria, and activation of caspases-3, -8, and -9. A caspase inhibitor, zVAD-FMK, inhibited the DNA fragmentation induced by VPA but not cell death. These findings suggest that VPA exerts an anti-leukemic effect by both caspase-dependent and -independent apoptotic signaling pathways.