Quantitative evaluation of endometrium-expressed mRNAs for the purpose of discriminating between menstruation and traumatic vaginal injury in sexual assault cases.

In sexual assault cases, it is crucial to discriminate between peripheral blood and menstrual blood to provide evidence for vaginal intercourse with traumatic injury. In this study, the menstrual blood mRNA markers progestagen-associated endometrial protein (PAEP), matrix metallopeptidase 7 (MMP7), and left-right determination factor 2 (LEFTY2) were evaluated by quantitative RT-PCR (RT-qPCR) for the discrimination of menstrual blood from peripheral blood and vaginal fluid. As a result, all markers with cutoff delta cycle quantification (ΔCq) values were specifically determined in menstrual blood among forensically relevant body fluids. Even though the changes in the expression levels of each marker differed during the menstrual cycle, all markers were determined to be positive in most of the randomly collected menstrual blood samples that were analyzed. Additionally, the markers with proposed cutoff ΔCq values could discriminate between menstrual blood and peripheral blood-mixed vaginal fluid samples. The determination of positive markers was less affected by storage temperature under dry conditions than under wet conditions, while PAEP was detectable in samples stored below room temperature under wet conditions. The detectability of PAEP was considered to be the result of its higher expression level compared with MMP7 and LEFTY2. In conclusion, menstrual blood markers for the RT-qPCR procedure evaluated in this study were highly specific for menstrual blood. The proposed procedure could be useful for discriminating between menstruation and traumatic bleeding in the female genital tract. In particular, PAEP is expected to be applicable to forensic casework samples because of its high specificity and robustness.

Molecular basis of host plant recognition by silkworm larvae.

Herbivorous insects can identify their host plants by sensing plant secondary metabolites as chemical cues. We previously reported the two-factor host acceptance system of the silkworm Bombyx mori larvae. The chemosensory neurons in the maxillary palp (MP) of the larvae detect mulberry secondary metabolites, chlorogenic acid (CGA), and isoquercetin (ISQ), with ultrahigh sensitivity, for host plant recognition and feeding initiation. Nevertheless, the molecular basis for the ultrasensitive sensing of these compounds remains unknown. In this study, we demonstrated that two gustatory receptors (Grs), BmGr6 and BmGr9, are responsible for sensing the mulberry compounds with attomolar sensitivity for host plant recognition by silkworm larvae. Calcium imaging assay using cultured cells expressing the silkworm putative sugar receptors (BmGr4-10) revealed that BmGr6 and BmGr9 serve as receptors for CGA and ISQ with attomolar sensitivity in human embryonic kidney 293T cells. CRISPR/Cas9-mediated knockout (KO) of BmGr6 and BmGr9 resulted in a low probability of making a test bite of the mulberry leaves, suggesting that they lost the ability to recognize host leaves. Electrophysiological recordings showed that the loss of host recognition ability in the Gr-KO strains was due to a drastic decrease in MP sensitivity toward ISQ in BmGr6-KO larvae and toward CGA and ISQ in BmGr9-KO larvae. Our findings have revealed that the two Grs, previously considered to be sugar receptors, are molecules responsible for detecting plant phenolics in host plant recognition.

Development and comparison of forensic interval age prediction models by statistical and machine learning methods based on the methylation rates of ELOVL2 in blood DNA.

Age estimation can be useful information for narrowing down candidates of unidentified donors in criminal investigations. Various age estimation models based on DNA methylation biomarkers have been developed for forensic usage in the past decade. However, many of these models using ordinary least squares regression cannot generate an appropriate estimation due to the deterioration in prediction accuracy caused by an increased prediction error in older age groups. In the present study, to address this problem, we developed age estimation models that set an appropriate prediction interval for all age groups by two approaches: a statistical method using quantile regression (QR) and a machine learning method using an artificial neural network (ANN). Methylation datasets (n = 1280, age 0-91 years) of the promoter for the gene encoding ELOVL fatty acid elongase 2 were used to develop the QR and ANN models. By validation using several test datasets, both models were shown to enlarge prediction intervals in accordance with aging and have a high level of correct prediction (>90 %) for older age groups. The QR and ANN models also generated a point age prediction with high accuracy. The ANN model enabled a prediction with a mean absolute error (MAE) of 5.3 years and root mean square error (RMSE) of 7.3 years for the test dataset (n = 549), which were comparable to those of the QR model (MAE = 5.6 years, RMSE = 7.8 years). Their applicability to casework was also confirmed using bloodstain samples stored for various periods of time (1-14 years), indicating the stability of the models for aged bloodstain samples. From these results, it was considered that the proposed models can provide more useful and effective age estimation in forensic settings.

Potential application of Staphylococcus species detection in the specific identification of saliva.

When found at crime scenes, saliva constitutes forensically relevant evidence. Although several tests have been developed to effectively identify saliva in such circumstances, most cannot discriminate between saliva and nasal secretion. Recently, studies have developed saliva tests involving oral bacteria as salivary markers. Although the specificity of such tests has been evaluated on most biological specimens, their specificity for nasal secretion samples remains to be tested. Herein, to improve the specificity of the saliva detection tests for nasal secretion samples, we reanalyzed a public microbiome dataset and conducted inhouse 16S rRNA sequencing to identify a new marker to distinguish between saliva and nasal secretions. The sequencing data indicated the existence of oral bacteria such as Streptococcus in nasal secretion samples, which may be responsible for the false positives in the saliva tests. Furthermore, we found that including the 16S rRNA gene of the genus Staphylococcus as a nasal secretion marker may improve the specificity of PCR-based saliva tests for nasal secretion samples. In addition, we assessed the specificity of previously developed salivary bacteria detection tests for nasal secretion samples and oral bacterial markers were detected in two of eight nasal secretion samples, which led to the false positive results for saliva detection. Thus, the specificity of such tests can be improved by adding Staphylococcus as a nasal marker, as revealed by our sequencing analysis.

The roles of enteroendocrine cell distribution and gustatory receptor expression in regulating peptide hormone secretion in the midgut of Bombyx mori larvae.

To regulate physiological homeostasis and behavior in Bombyx mori, more than 20 peptide hormones in the midgut of larvae are secreted upon detection of food substances at the lumen. Although it is logical to assume that the timings of peptide hormone secretions are regulated, little is known about the mechanisms. In this study, the distributions of enteroendocrine cells (EECs) producing five peptide hormones and EECs expressing gustatory receptors (Grs), as candidate receptors for luminal food substances and nutrients, were examined via immunostaining in B. mori larvae. Three patterns of peptide hormone distribution were observed. Tachykinin (Tk)- and K5-producing EECs were located throughout the midgut; myosuppressin-producing EECs were located in the middle-to-posterior midgut; and allatostatin C- and CCHamide-2-producing EECs were located in the anterior-to-middle midgut. BmGr4 was expressed in some Tk-producing EECs in the anterior midgut, where food and its digestive products arrived 5 min after feeding began. Enzyme-linked immunosorbent assay (ELISA) revealed secretion of Tk starting approximately 5 min after feeding began, suggesting that food sensing by BmGr4 may regulate Tk secretion. BmGr6 was expressed in a few Tk-producing EECs in the middle-to-posterior midgut, although its significance was unclear. BmGr6 was also expressed in many myosuppressin-producing EECs in the middle midgut, where food and its digestive products arrived 60 min after feeding began. ELISA revealed secretion of myosuppressin starting approximately 60 min after feeding began, suggesting that food sensing by BmGr6 may regulate myosuppressin secretion. Finally, BmGr9 was expressed in many BmK5-producing EECs throughout the midgut, suggesting that BmGr9 may function as a sensor for the secretion of BmK5.

Validation of a novel fluorescent probe-based real-time PCR assay to detect saliva-specific unmethylated CpG sites for saliva identification.

The identification of saliva from forensic samples is often important to establish what happened at a crime scene, especially in sexual assault cases. Recently, CpG sites that are specifically methylated or unmethylated in saliva have been reported as markers for saliva identification. In this study, we designed a fluorescent probe-based real-time polymerase chain reaction (PCR) assay for analyzing the methylation status of two neighboring CpG sites, which we previously found were saliva-specifically unmethylated. Specificity analysis using various types of body fluid/tissue samples demonstrated a probe detecting the unmethylation of the two CpG sites reacted only to saliva DNA, indicating this probe as an all-or-nothing marker for the presence of saliva DNA. Sensitivity analysis demonstrated that the detection limit was 0.5 ng saliva DNA as input for bisulfite conversion, while we confirmed a negative effect of larger amounts of non-saliva DNA on sensitivity in the analysis of saliva-vaginal DNA mixtures. We finally validated the applicability of this test to swabs from licked skin and bottles after drinking as mock forensic samples in comparison with other saliva-specific markers. We confirmed the potential usefulness of this test for skin samples, from which a saliva-specific mRNA was not detected reliably, while the ingredients in several beverages might affect methylation analysis. Given the simplicity of real-time PCR as well as the high specificity and sensitivity of the test, we believe the developed method is suitable for routine forensic analysis and can play an important role in saliva identification.

Dietary compounds activate an insect gustatory receptor on enteroendocrine cells to elicit myosuppressin secretion.

Sensing of midgut internal contents is important for ensuring appropriate hormonal response and digestion following the ingestion of dietary components. Studies in mammals have demonstrated that taste receptors (TRs), a subgroup of G protein-coupled receptors (GPCRs), are expressed in gut enteroendocrine cells (EECs) to sense dietary compounds and regulate the production and/or secretion of peptide hormones. Although progress has been made in identifying expression patterns of gustatory receptors (GRs) in gut EECs, it is currently unknown whether these receptors, which act as ligand-gated ion channels, serve similar functions as mammalian GPCR TRs to elicit hormone production and/or secretion. A Bombyx mori Gr, BmGr6, has been demonstrated to express in cells by oral sensory organs, midgut and nervous system; and to sense isoquercitrin and chlorogenic acid, which are non-nutritional secondary metabolites of host mulberry. Here, we show that BmGr6 co-expresses with Bommo-myosuppressin (BMS) in midgut EECs, responds to dietary compounds and is involved in regulation of BMS secretion. The presence of dietary compounds in midgut lumen after food intake resulted in an increase of BMS secretions in hemolymph of both wild-type and BmGr9 knockout larvae, but BMS secretions in BmGr6 knockout larvae decreased relative to wild-type. In addition, loss of BmGr6 led to a significant decrease in weight gain, excrement, hemolymph carbohydrates levels and hemolymph lipid levels. Interestingly, although BMS is produced in both midgut EECs and brain neurosecretory cells (NSCs), BMS levels in tissue extracts suggested that the increase in hemolymph BMS during feeding conditions is primarily due to secretion from midgut EECs. Our studies indicate that BmGr6 expressed in midgut EECs responds to the presence of dietary compounds in the lumen by eliciting BMS secretion in B. mori larvae.

BmGr4 responds to sucrose and glucose and expresses in tachykinin-related peptide-secreting enteroendocrine cells.

The regulatory hormones known as tachykinin-related peptides (TRPs) are identified as brain-gut peptides in insects. Dietary components from mulberry leaves, including glucose, induce secretion of TRPs from Bombyx mori midgut. However, the sensory molecules that recognize these compounds are still unknown. Here, we identified the gustatory receptor, BmGr4, as a sucrose and glucose receptor using Ca2+ imaging. Immunostaining revealed BmGr4 expression not only in the midgut, but also in the brain. In addition, BmGr4 expression was found to co-localize with TRP-expressing cells in both midgut enteroendocrine cells (EECs) and brain neurosecretory cells (NSCs). Furthermore, dietary nutrients after food intake result in an increase of TRP-level in hemolymph of silkworm larvae. These results provide significant circumstantial evidence for the involvement of the sucrose and glucose receptor, BmGr4, in the elicitation of TRP secretion in midgut EECs and brain NSCs.

Precise and comprehensive determination of multiple body fluids by applying statistical cutoff values to a multiplex reverse transcription-PCR and capillary electrophoresis procedure for forensic purposes.

Body fluid identification from crime scene evidence is an essential procedure in forensic investigations. Among various procedures, multiplex reverse transcription PCR assays have a clear advantage over conventional methods because different types of body fluids can be analyzed simultaneously. For more precise, comprehensive, and objective identification of forensically relevant body fluids, 15 target genes for blood, saliva, semen, vaginal fluid, and nasal secretion were selected; their primers were re-designed and multiplex PCR conditions were optimized to prioritize specificity for those body fluids. Multiple amplicons were separated and determined by the SeqStudio Genetic Analyzer with an all-in-one and easy-to-use cartridge. Then, the cutoff value was set for each marker to eliminate the detection of slight amplification in non-targeted body fluids. As a result, the targeted body fluid specificities of the developed procedure were drastically improved. Although successful determination of the target gene depends on sample condition and marker sensitivity, our procedure was applicable for the precise determination of body fluids in mixed body fluid stains, aged samples, and various mock casework samples. Therefore, it could be a powerful and convenient tool for the precise identification of multiple body fluids in forensic laboratories.

Evaluation and simultaneous determination of rectal mucosa markers by multiplex reverse transcription-PCR for biological evidence of sexual assault with anal penetration.

Sexual assault with anal penetration is closely related to child sexual abuse or male victims. However, it is difficult to prove such an act by using biological samples collected from the surface of a suspected object because procedures for identifying rectal mucosa have not been developed sufficiently. Therefore, for the specific identification of rectal mucosa, mRNA markers reported to be characteristically expressed in the rectum were screened and a multiplex RT-PCR procedure was developed for the simultaneous determination of those candidate markers. The detectability and specificity of rectal mucosa candidate markers were evaluated using rectal mucosa samples and forensically relevant body fluids. Diluted or mixed samples were also tested to evaluate the applicability of this procedure for forensic casework. As a result, simultaneous amplification and determination of the selected candidates (PHGR1, MUC13, CLCA1, MEP1A, CDX1, and ZG16) and reference gene were successfully performed using a multiplex RT-PCR assay combined with capillary electrophoresis and fragment analysis. Applying the cutoff values, none of the other body fluids cross-reacted with rectal mucosa candidate markers. Because the low sensitivity and detectability of some candidate markers could be compensated for by their simultaneous detection, all six candidate markers were considered to be applicable as rectal mucosa markers. Besides, the developed assay should not be performed on suspicious fecal samples directly because these markers could be positive in the fecal samples themselves. The developed multiplex RT-PCR procedure might not be suitable for minute or diluted samples; however, it might be resistant to contamination with sexual assault-related body fluids. In conclusion, the simultaneous determination of selected rectal mucosa markers with a biological sample collected from the surface of a suspected object could be beneficial for criminal investigation of sexual assault with anal penetration.

A humoral factor, hemolymph proteinase 8, elicits a cellular defense response of nodule formation in Bombyx mori larvae in association with recognition by C-type lectins.

Previously, we found that nodule formation, a cellular defense response in insects, is regulated by humoral factors called C-type lectins in the hemolymph. To elucidate the factors that elicit nodule formation following the recognition of microorganisms by C-type lectins, a reproducible quantitative in vitro assay system was constructed. Then, using this system, the inhibitory activities of antisera raised against hemolymph proteases (HPs), serine protease homologues (SPHs), and pathogen-associated molecular pattern (PAMP)-recognition proteins were assessed. Among the antisera raised against HP and SPH, only that against HP8, a terminal proteinase that activates Spätzle, consistently inhibited in-vitro nodule-like aggregate formation in all three tested microorganisms, Micrococcus luteus, Escherichia coli, and Saccharomyces cerevisiae. Antisera raised against C-type lectins, BmLBP, and BmMBP also inhibited nodule-like aggregate formation, while those against ß-glucan recognition proteins and peptidoglycan recognition protein-S1 did not. Microorganisms pretreated with hemolymph, which contains HP8 and C-type lectins, also induced nodule-like aggregate formation, indicating that nodulation factors are present on microbial cells. Furthermore, antisera raised against HP8, BmLBP, and BmMBP showed inhibitory activities in the in vivo nodule formation system using Bombyx mori larvae. Thus, two humoral factors in the hemolymph of B. mori larvae, BmHP8 and C-type lectins, were found to play significant roles in eliciting the cellular defense response of nodule formation.

Investigating matrix effects of different combinations of lipids and peptides on TOF-SIMS data.

Matrix effects, which cause a change in ion intensity, occur in mass spectrometry methods including time-of-flight secondary ion mass spectrometry (TOF-SIMS). Matrix effects often cause large issues in quantitative analysis because secondary ions related to a particular molecule could be dramatically enhanced or suppressed regardless of the concentration. To investigate matrix effects in biological samples, the authors evaluated mixed lipid {POPC [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine, molecular weight (MW) 759.6]}, peptide [leu-enkephalin, neo-leu-enkephalin (amino acid sequence: YAGFL, MW 569.3), and neo-angiotensin II (amino acid sequence: DRVYIHAF, MW 1019.5)] samples. Matrix effect features were investigated by analyzing the concentration dependence of secondary ions in lipid-peptide mixed samples to develop a method that enables quantitative analysis using TOF-SIMS. Matrix effects depended on the lipid-peptide combination. Interestingly, some secondary ions possessed an intensity that was highly dependent on concentration.

Evaluation of secondary ions related to plant tissue using least absolute shrinkage and selection operator.

With regard to life sciences, it is important to understand biological functions such as metabolic reactions at the cellular level. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) that can provide chemical mappings at 100 nm lateral resolutions is useful for obtaining three-dimensional maps of biological molecules in cells and tissues. TOF-SIMS spectra generally contain several hundred to several thousand secondary ion peaks that provide detailed chemical information. In order to manage such a large number of peaks, data analysis methods such as multivariate analysis techniques have been applied to TOF-SIMS data of complex samples. However, the interpretation of the data analysis results is sometimes still difficult, especially for biological samples. In this study, TOF-SIMS data of resin-embedded plant samples were analyzed using one of the sparse modeling methods, least absolute shrinkage and selection operator (LASSO), to directly select secondary ions related to biological structures such as cell walls and nuclei. The same sample was measured by optical microscopy and the same measurement area as TOF-SIMS was extracted in order to prepare a target image for LASSO. The same area of the TOF-SIMS and microscope data were fused to evaluate the influence of the image fusion on the TOF-SIMS spectrum information using principal component analysis. Specifically, the authors examined onion mycorrhizal root colonized with Gigaspora margarita (an arbuscular mycorrhizal fungus). The results showed that by employing this approach using LASSO, important secondary ions from biological samples were effectively selected and could be clearly distinguished from the embedding resin.

Factors determining the reaction temperature of the solvent-free enzymatic synthesis of trehalose esters.

Solvent-free synthesis encourages the design of processes and products that reduce the use and generation of hazardous chemicals. Given the importance of developing greener methodologies, we sought to determine the factors influencing the reaction temperature required for solvent-free, enzymatic synthesis of sugar esters such as trehalose (TRE) esters, using Novozyme 435 as the enzyme catalyst. The use of lauric acid (La) and ethyl laurate (LaEt) as acyl donors did not affect the activation temperature for the generation of trehalose diesters (TDEs), despite the differences in corresponding by-products (water and ethanol). However, when glucose (GLU) and La were employed as reaction substrates as a comparison, glucose monoester (GME) generation readily occurred at much lower temperatures than with the TRE esters, even without a water collection device. Moreover, when the glass transition temperature (Tg) of the sugar substrates increased, a higher reaction temperature was required. These results suggest that while the activation temperature of the reaction did not correlate with the boiling point of the by-product, it did correlate with the glass transition temperature (Tg) of the trehalose substrates. Thus, our work demonstrates the importance of the physical state of amorphous matrices in determining the optimal reaction temperature of a solvent-free sugar synthesis.

Glucose, some amino acids and a plant secondary metabolite, chlorogenic acid induce the secretion of a regulatory hormone, tachykinin-related peptide, from the silkworm midgut.

Enteroendocrine cells in the insect midgut are thought to secrete peptide hormones in response to the nutritional state. However, the role of dietary compounds in inducing peptide hormone secretion from enteroendocrine cells in insects remains unknown. In the present study, we demonstrated that several dietary compounds from mulberry leaves, including glucose, amino acids, and the secondary metabolite chlorogenic acid, induced significant secretion of tachykinin-related peptides from isolated silkworm midguts at the luminal concentrations measured in fed larvae. This study provides evidence that the insect midgut senses a non-nutritious secondary metabolite in addition to nutrient metabolites to monitor luminal food status and secretes a feeding regulatory hormone, suggesting that a unique dietary sensory system modulates insect feeding via enteroendocrine control.

Insect taste receptors relevant to host identification by recognition of secondary metabolite patterns of non-host plants.

The taste sensing system is crucial for food recognition in insects and other animals. It is commonly believed that insect gustatory receptors (Grs) expressed in gustatory organs are indispensable for host plant selection. Many behavioral studies have shown that mono- or oligo-phagous lepidopteran insects use the balance between feeding attractants and feeding deterrents in host plants and that these are sensed by taste organs for host plant recognition. However, the molecular mechanism underlying taste recognition, especially of feeding deterrents, remains to be elucidated. To better understand this mechanism, we studied orphan Grs, including Bombyx mori Gr (BmGr) 16, BmGr18, and BmGr53, from the mono-phagous insect, Bombyx mori. Using Calcium imaging in mammalian cells, we first confirmed in lepidoptera insects that three of the putative bitter Grs widely responded to structurally different feeding deterrents. Although the phylogenetic distance of these Grs was considerable, they responded to partially overlapping deterrents of plant secondary metabolites. These findings suggest that not only these three Grs but also most of the Grs that have been assigned to putative bitter Grs are feeding-deterrent receptors that play a role in host plant recognition.

Evaluation of matrix effects on TOF-SIMS data of leu-enkephalin and 1,2-dioleoyl-sn-glycero-3-phosphocholine mixed samples.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is one of the most powerful methods to analyze biomolecules in biological tissues and cells because it provides detailed chemical structure information and chemical images with a high spatial resolution. However, in terms of quantitative analysis, there are issues such as matrix effects that often cause secondary ion intensity changes regardless of the actual concentration in a sample. For instance, the intensity of secondary ions related to peptides is generally suppressed when lipids coexist. Since the evaluation of biomolecules is crucial to understand biological phenomena, it is required to analyze peptides or lipids without matrix effects. Therefore, the mechanism of matrix effects regarding peptides and lipids in TOF-SIMS was investigated in this study. Leu-enkephalin (YGGFL, molecular weight of 555.3 Da) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, C44H84NO8P, molecular weight 785.6 Da) were employed to prepare model samples. Model samples contain different weight ratios of these two molecules. The intensity of secondary ions related to the peptide or the lipid was compared with control samples containing pure leu-enkephalin or DOPC. As a result, it is indicated that the intensity of DOPC related secondary ions is strongly enhanced by coexisting leu-enkephalin, while the intensity of leu-enkephalin related secondary ions is suppressed by coexisting DOPC especially in a low concentration range of the peptide.

Evaluation of blood adsorption onto dialysis membranes by time-of-flight secondary ion mass spectrometry and near-field infrared microscopy.

Blood adsorption onto the inside surface of hollow fiber dialysis membranes was investigated by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and near-field infrared microscopy (NFIR) in order to evaluate the biocompatibility and permeability of dialysis membranes. TOF-SIMS is useful for the imaging of particular molecules with a high spatial resolution of approximately 100 nm. In contrast, infrared spectra provide quantitative information and NFIR enables analysis with a high spatial resolution of less than 1 µm, which is close to the resolution of TOF-SIMS. A comparison was made of one of the most widely used dialysis membranes made of polysulfone (PSf), that has an asymmetric and inhomogeneous pore structure, and a newly developed asymmetric cellulose triacetate (ATA) membrane that also has an asymmetric pore structure, even though the conventional cellulose triacetate membrane has a symmetric and homogeneous pore structure. As a result, it was demonstrated that blood adsorption on the inside surface of the ATA membrane is more reduced than that on the PSf membrane. Graphical abstract Analysis of blood adsorption on inside surface of hollow fiber membrane.

Development on smart suit for dairy work assistance.

Our purpose in this study is to achieve an independent life and a social involvement for the elderly using KEIROKA Technology(fatigue-reduction) which makes it possible to improve the quality of chores and occupations by removing excessive strain and tiredness. The authors have developed power assist suits named "smart suit". The authors have evaluated the effect that the purpose of dairy work assistance, to measure EMG of the worker, compared to the potential of the surface of the non-wearing and wearing "smart suit".

IR-SNOM analysis of occluding substances in lumina of xylem elements in sapwood of Quercus serrata attacked by Platypus quercivorus.

A new microspectroscopic technique was applied to the analysis of occluding deposits in xylem elements of Quercus serrata. The production of this substance is believed to be a defense response of the sapwood against fungal infection. An occluding substance about 10 µm across was analyzed by Infrared-Scanning Near-field Optical Microscopy (IR-SNOM), which allows for the measurement of IR spectrum with high spatial resolution. The near-field IR spectrum of an occluding substance was different from those of xylem elements and featured a lack of the clear C-H absorption band that should appear at 3000-2850 cm(-1). On the other hand, the absorption band of ester bond exhibited a very strong peak. Among the near-field IR spectra of related compounds, a similar ester absorption peak was observed in the spectrum of pectin and tannic acid. The presence of a C-H absorption band as a very week peak was similar to (+)-catechin and tannic acid.