Machine learning-based evaluation of spontaneous pain and analgesics from cellular calcium signals in the mouse primary somatosensory cortex using explainable features.

Introduction: Pain that arises spontaneously is considered more clinically relevant than pain evoked by external stimuli. However, measuring spontaneous pain in animal models in preclinical studies is challenging due to methodological limitations. To address this issue, recently we developed a deep learning (DL) model to assess spontaneous pain using cellular calcium signals of the primary somatosensory cortex (S1) in awake head-fixed mice. However, DL operate like a "black box", where their decision-making process is not transparent and is difficult to understand, which is especially evident when our DL model classifies different states of pain based on cellular calcium signals. In this study, we introduce a novel machine learning (ML) model that utilizes features that were manually extracted from S1 calcium signals, including the dynamic changes in calcium levels and the cell-to-cell activity correlations. Method: We focused on observing neural activity patterns in the primary somatosensory cortex (S1) of mice using two-photon calcium imaging after injecting a calcium indicator (GCaMP6s) into the S1 cortex neurons. We extracted features related to the ratio of up and down-regulated cells in calcium activity and the correlation level of activity between cells as input data for the ML model. The ML model was validated using a Leave-One-Subject-Out Cross-Validation approach to distinguish between non-pain, pain, and drug-induced analgesic states. Results and discussion: The ML model was designed to classify data into three distinct categories: non-pain, pain, and drug-induced analgesic states. Its versatility was demonstrated by successfully classifying different states across various pain models, including inflammatory and neuropathic pain, as well as confirming its utility in identifying the analgesic effects of drugs like ketoprofen, morphine, and the efficacy of magnolin, a candidate analgesic compound. In conclusion, our ML model surpasses the limitations of previous DL approaches by leveraging manually extracted features. This not only clarifies the decision-making process of the ML model but also yields insights into neuronal activity patterns associated with pain, facilitating preclinical studies of analgesics with higher potential for clinical translation.

Approval status and characteristics of work-related musculoskeletal disorders among Korean workers in 2020.

Background: This study aimed to investigate the characteristics of work-related musculoskeletal disorders (WRMSDs) in occupational disease claims and identify patterns of WRMSDs for each body part by industry and occupation. Methods: This study analyzed the raw data of occupational disease claims for musculoskeletal disorders deliberated by the Occupational Disease Decision Committee of the Korea Workers' Compensation & Welfare Service in 2020. The data was classified into 6 body parts with the highest numbers of occupational disease cases by using the complete enumeration data on principal diagnoses and 4 types of subdiagnoses in the raw data. The characteristics and approval rates of WRMSDs by body part, industry and occupation were examined and summarized. Results: A total of 13,015 occupational disease cases for WRMSDs were classified, and lumbar spinal (back) diseases accounted for the largest proportion of claimed diseases, followed by shoulder, elbow, wrist, knee, and neck diseases in a descending order. The occupations with the highest and second highest numbers of occupational disease cases by body part were found to be automobile assemblers and production-related elementary workers for the neck, school meal service workers and cooks for the back, construction frame mold carpenters and school meal service workers for the shoulder, elementary workers in mining and food service workers for the elbow, food service workers and automobile parts assemblers for the wrist, and ship welders and school meal service workers for the knee. Conclusions: This study examined the characteristics and approval status of WRMSDs by body part and occupation. Based on the study results, management strategies for the prevention of WRMSDs should be established regarding occupations with a high risk of WRMSDs for each body part.

5-HT1A receptors mediate the analgesic effect of rosavin in a mouse model of oxaliplatin-induced peripheral neuropathic pain.

Oxaliplatin, a third-generation platinum derivative, is the mainstay of current antineoplastic medications for advanced colorectal cancer therapy. However, peripheral neuropathic complications, especially cold allodynia, undermine the lifeprolonging outcome of this anti-cancer agent. Rosavin, a phenylpropanoid derived originally from Rhodiola rosea, exhibits a wide range of therapeutic properties. The present study explored whether and how rosavin alleviates oxaliplatin-induced cold hypersensitivity in mice. In the acetone drop test, cold allodynia behavior was observed from days 3 to 5 after a single injection of oxaliplatin (6 mg/kg, i.p.). Cold allodynia was significantly attenuated following rosavin treatment (10 mg/kg, i.p.). Specific endogenous 5-HT depletion by three consecutive pretreatments with parachlorophenylalanine (150 mg/kg/day, i.p.) abolished the analgesic action of rosavin; this effect was not observed following pretreatment with naloxone (opioid receptor antagonist, 10 mg/kg, i.p.). Furthermore, 5-HT1A receptor antagonist WAY-100635 (0.16 mg/kg, i.p.), but not 5-HT3 receptor antagonist MDL-72222 (1 mg/kg, i.p.), blocked rosavin-induced analgesia. These results suggest that rosavin may provide a novel approach to alleviate oxaliplatin-induced cold allodynia by recruiting the activity of 5-HT1A receptors.

Analysis of the Change Patterns of Sagittal Alignment Values After Selective Thoracic Fusion in Lenke 1 Adolescent Idiopathic Scoliosis According to Preoperative Thoracic Kyphosis Status.

STUDY DESIGN: This was a retrospective study. OBJECTIVE: The objective of this study was to demonstrate the different change patterns in reciprocal sagittal alignment values after selective thoracic fusion (STF) in Lenke type 1 adolescent idiopathic scoliosis (AIS) according to preoperative thoracic kyphosis (TK). SUMMARY OF BACKGROUND DATA: Several studies have found significant increase in TK after STF, while other studies have reported decrease in TK postoperatively. Similar inconclusive results on changes in lumbar lordosis (LL) have been reported, showing LL increase, decrease, or no change. MATERIALS AND METHODS: Ninety-three patients presenting with Lenke type 1 AIS treated by posterior STF with a minimum follow-up of 2 years were included in this study. Using whole spine radiographs, sagittal parameters including TK, LL, and upper lumbar lordosis (ULL) were compared preoperatively and at the last follow-up between a hypokyphosis group (preoperative TK<20 degrees) and a normokyphosis group (preoperative TK≥20 degrees). Health-related quality of life (HRQOL) was assessed using scoliosis research society health-related quality of life-30 (SRS-30) and short from health survey-36 questionnaire at the last visit. RESULTS: The mean follow-up duration was 74.9 months. In the hypokyphosis group (35 patients), TK, LL, and ULL statistically significantly increased after surgery by mean 7.7, 5.1, and 3.7 degrees (P<0.001, <0.001, and 0.001). In the normokyphosis group (58 patients), these parameters did not show significant changes after STF. Final TK was significantly lower in hypokyphosis group than that in the normokyphosis group (21.2 vs. 30.9 degrees, P<0.001) while final LL did not differ between 2 groups (52.4 vs. 54.6 degrees, P=0.194). HRQOL did not differ significantly between the 2 groups. CONCLUSIONS: After STF in Lenke 1 AIS, TK, and LL statistically significantly increased through an increase in the mean ULL in the hypokyphosis group while those mean values did not change in the normokyphosis group. Despite the final mean value of the TK in the hypokyphosis group increasing by 7.7 degrees, it was statistically significantly lower than the final mean TK value in the normokyphosis group which did not increase after STF surgery by posterior approach. However, HRQOL showed no significant difference between the 2 groups.

Minimum Three-year Follow-up of Specific Functional Disabilities After Multilevel Lumbar Fusion: Comparison of Long-level and Short-level Fusion.

STUDY DESIGN: Retrospective case-control study. OBJECTIVE: To evaluate specific functional disabilities after short- and long-level lumbar fusion. SUMMARY OF BACKGROUND DATA: The Oswestry Disability Index (ODI) cannot represent all types of functional disabilities observed after lumbar fusion and a region-specific Functional Disability Index (SFDI) is necessary. METHODS: We examined the differences in postoperative functional disability between 81 patients who underwent ≥3-level lumbar fusion (group I) and 70 age- and sex-matched patients who underwent one- or two-level lumbar fusion (group II). The ODI and Visual Analogue Scale (VAS) were assessed pre- and postoperatively. The SFDI was assessed after lumbar fusion. We evaluated intergroup differences in postoperative VAS, ODI, and SFDI scores during 3-year follow-up. Each mean score was evaluated separately for the 10 ODI and the 12 SFDI items, and we evaluated the changes observed in these scores over the 3-year follow-up. RESULTS: The mean intergroup preoperative ODI and VAS scores were similar. The mean postoperative intergroup VAS scores were similar; however, the mean postoperative ODI and SFDI scores were significantly higher in group I than in group II at 1-year (P<0.001, P<0.001, respectively) and 3-year follow-up (P = 0.037, P<0.001, respectively). Among 10 ODI items, group I showed significant disability with regard to six items at the 1-year follow-up compared with group II, but only showed significant disability with regard to one item at the 3-year follow-up. Among the 12 SFDI items, group I showed significant disability with regard to all 12 items at 1-year follow-up compared with group II, as well as significant disability with regard to nine items at 3-year follow-up. CONCLUSION: The SFDI is more sensitive than the ODI in assessing functional disabilities based on the levels of fusion. Most SFDI items indicated continued significant disability in patients with long-level lumbar fusion even 3 years postoperatively. LEVEL OF EVIDENCE: 3.

Occupational cancer claims in Korea from 2010 to 2016.

BACKGROUND: Research on carcinogens causing occupational cancer has been updated. Further, social interest in occupational cancer has increased. In addition, the standard for recognizing cancer as a work-related disease has also been revised. The present study aims to describe the distribution of occupational cancer claims or its approval rate and their association with work-related variables. METHODS: We analyzed 1299 claim cases for occupational cancer from 2010 to 2016 provided by the Korea Workers' Compensation and Welfare Service (KCOMWEL). The status of approval rate was shown by year, sex, industry, occupation, age of diagnosis, duration from employment to diagnosis, and cancer site. RESULTS: The approval rate was 39.0% from 2010 to 2016 and tended to increase annually since 2011. Both the number of claims and the approval rate were higher in men. Mining and quarrying showed the highest approval rate (78.4%). The approval rates by age of diagnosis and duration from employment to diagnosis increased as the time periods increased. Respiratory organ had the highest number of claims and the highest approval rate by cancer site. CONCLUSIONS: The approval rate of occupational cancer has shown an increasing trend since 2011. The increase of occupational carcinogens and cancer sites and the improvement of social awareness about occupational cancer could have resulted in this trend. The present study provides unique, and the latest and most accurate findings on occupational cancer data of recent 7 years that could be helpful to researchers or policy makers on occupational cancer.

Job Stress-attributable Burden of Disease in Korea.

BACKGROUND: As job stress is associated with various diseases and psychiatric conditions, we aimed to estimate the job stress-attributable burden of disease in Korea based on the concept of disability-adjusted life years (DALY). METHODS: We selected ischemic heart disease (IHD), stroke, major depressive disorder (MDD), and suicide as health outcomes from job stress, because of the ease of access to data estimating burdens and of important meaning of them in Korean occupational background. RESULTS: Our findings demonstrated that approximately 21% of Korean workers were exposed to high job strain, which was attributable for approximately 6.7% of IHDs, 6.9% of strokes, 13.6% of MDDs, and 4% of suicides. In terms of job stress-attributable DALY, the burdens of disease per 100,000 people were 38 for IHD, 72 for stroke, 168 for MDDs, and 44 for suicides. CONCLUSION: The present findings suggested that one-fifth of Korean workers were suffering from high job strain. Although the figures may not be accurate due to several assumptions, job stress is an important risk factor for health in working environment in Korea.

Radiological and clinical long-term results of heterotopic ossification following lumbar total disc replacement.

BACKGROUND: The long-term results of heterotopic ossification (HO) following lumbar total disc replacement (TDR) and the corresponding clinical and radiological outcomes are unclear. PURPOSE: This study aimed to report the long-term results of HO following lumbar TDR and to analyze the clinical and radiological outcomes. STUDY DESIGN/SETTING: A retrospective case review was performed for the consecutive patients who underwent lumbar TDR. PATIENT SAMPLE: The study included 48 patients (60 segments) who underwent lumbar TDR. OUTCOME MEASURES: The time and location of HO development, segmental range of motion (ROM) of index level, the visual analog scale (VAS), and the Oswestry Disability Index (ODI) were analyzed. METHODS: Forty-eight patients (60 segments) were divided into HO and non-HO groups, and radiographs were used to measure the time and location of HO development. We compared segmental ROM between two groups using flexion-extension radiographs. Clinical outcomes were assessed using the VAS and the ODI. Furthermore, the segmental ROM, VAS, and ODI scores of each HO class were compared with those of the non-HO group. RESULTS: The mean follow-up duration was 104.4 months. Heterotopic ossification was detected in 30 of 60 segments following lumbar TDR, and HO progression was noted in six segments. The mean segmental ROM was significantly lower in the HO group than in the non-HO group. The mean VAS and ODI scores were not significantly different between the two groups. Segmental ROM was significantly lower in the class III and IV of the HO group than in the non-HO group. The VAS and ODI scores were not significantly different among the different classes. CONCLUSIONS: We found that the incidence of HO is the highest within 12 months after lumbar TDR, and the incidence might increase 5 years after surgery. Furthermore, HO progressed over time. Segmental ROM was decreased in the HO groups; however, the limitation in motion might have little clinical influence.

Characteristics and Efficacy of a New 3-Dimensional Printed Mesh Structure Titanium Alloy Spacer for Posterior Lumbar Interbody Fusion.

This study evaluated the characteristics of a newly developed 3-dimensional printed mesh structure titanium spacer and its efficacy for posterior lumbar interbody fusion. Posterior lumbar interbody fusion with this spacer was performed at 53 segments (40 patients; mean age, 64 years; range, 51-73 years). Data were collected prospectively. Radiographic characteristics were analyzed with changes in interbody height, instability of the segments, formation of bone bridges around the implants, and pseudarthrosis, as determined by dynamic radiographs and postoperative computed tomography scans. Clinical outcomes were evaluated with the visual analog scale for the low back and extremities, the Oswestry Disability Index, and the 36-Item Short Form Survey. Radiographically, preoperative anterior and posterior interbody height was significantly increased immediately postoperatively (P<.05), and this increase was maintained until the last follow-up. No segmental motion of 3° or greater was noted at the last follow-up. Sagittal computed tomography images showed complete anterior bone bridges for 94.3% of cases and complete posterior bone bridges for 86.7% of cases. Coronal computed tomography images showed bilateral complete bone bridges for 94.3% of cases and unilateral bone bridges for 5.7% of cases without incomplete bilateral bone bridges. No pseudarthrosis or revision, particularly including posterior lumbar interbody fusion at L5-S1, was noted. Compared with preoperative values, the visual analog scale score for the low back and extremities, the Oswestry Disability Index, and the 36-Item Short Form Survey score showed significant improvement at the last follow-up (P<.05). Posterior lumbar interbody fusion with a newly developed 3-dimensional printed mesh structure titanium spacer showed satisfactory radiographic and clinical results, with no cases of pseudarthrosis or revision, including posterior lumbar interbody fusion at L5-S1. [Orthopedics. 2017; 40(5):e880-e885.].

Reducing false-positive incidental findings with ensemble genotyping and logistic regression based variant filtering methods.

As whole genome sequencing (WGS) uncovers variants associated with rare and common diseases, an immediate challenge is to minimize false-positive findings due to sequencing and variant calling errors. False positives can be reduced by combining results from orthogonal sequencing methods, but costly. Here, we present variant filtering approaches using logistic regression (LR) and ensemble genotyping to minimize false positives without sacrificing sensitivity. We evaluated the methods using paired WGS datasets of an extended family prepared using two sequencing platforms and a validated set of variants in NA12878. Using LR or ensemble genotyping based filtering, false-negative rates were significantly reduced by 1.1- to 17.8-fold at the same levels of false discovery rates (5.4% for heterozygous and 4.5% for homozygous single nucleotide variants (SNVs); 30.0% for heterozygous and 18.7% for homozygous insertions; 25.2% for heterozygous and 16.6% for homozygous deletions) compared to the filtering based on genotype quality scores. Moreover, ensemble genotyping excluded > 98% (105,080 of 107,167) of false positives while retaining > 95% (897 of 937) of true positives in de novo mutation (DNM) discovery in NA12878, and performed better than a consensus method using two sequencing platforms. Our proposed methods were effective in prioritizing phenotype-associated variants, and an ensemble genotyping would be essential to minimize false-positive DNM candidates.

Prioritizing disease-linked variants, genes, and pathways with an interactive whole-genome analysis pipeline.

Whole-genome sequencing (WGS) studies are uncovering disease-associated variants in both rare and nonrare diseases. Utilizing the next-generation sequencing for WGS requires a series of computational methods for alignment, variant detection, and annotation, and the accuracy and reproducibility of annotation results are essential for clinical implementation. However, annotating WGS with up to date genomic information is still challenging for biomedical researchers. Here, we present one of the fastest and highly scalable annotation, filtering, and analysis pipeline-gNOME-to prioritize phenotype-associated variants while minimizing false-positive findings. Intuitive graphical user interface of gNOME facilitates the selection of phenotype-associated variants, and the result summaries are provided at variant, gene, and genome levels. Moreover, the enrichment results of specific variants, genes, and gene sets between two groups or compared with population scale WGS datasets that is already integrated in the pipeline can help the interpretation. We found a small number of discordant results between annotation software tools in part due to different reporting strategies for the variants with complex impacts. Using two published whole-exome datasets of uveal melanoma and bladder cancer, we demonstrated gNOME's accuracy of variant annotation and the enrichment of loss-of-function variants in known cancer pathways. gNOME Web server and source codes are freely available to the academic community (http://gnome.tchlab.org).

50 years of publication in the field of medical education.

BACKGROUND: The advent of new medical education (ME) journals makes evident the growth of the field of ME. However, the nature and context of growth is undefined. AIM: To analyze the evolution of publication in ME. METHODS: MEDLINE retrieval using medical subject headings was used to analyze patterns of ME publications from 1960-2010: changes in number of ME publications; number of journals publishing ME articles; co-topics occurring frequently in ME articles; differences among journals' publication of co-topics. RESULTS: Annual publication of ME articles increased from 279 in 1960 to 3760 in 2010. 81,531 articles were published in 4208 different journals. 104 journals published ME articles in 1960, 855 in 2010. Despite an increase in journals in all fields, ME journals now account for a larger proportion of all journals indexed in MEDLINE than in 1960. One-quarter of all ME articles were indexed as internship/residency; 16% as graduate ME; 15% as undergraduate ME; and 14% as continuing ME. The five journals that published the most ME articles distinguished themselves by publishing some topics with greater or less frequency. CONCLUSIONS: The increase in the number of ME publications and in the number of journals publishing ME articles suggests a supportive environment for a growing field; but variation in journals' foci has implications for readers, editors and authors.

gSearch: a fast and flexible general search tool for whole-genome sequencing.

BACKGROUND: Various processes such as annotation and filtering of variants or comparison of variants in different genomes are required in whole-genome or exome analysis pipelines. However, processing different databases and searching among millions of genomic loci is not trivial. RESULTS: gSearch compares sequence variants in the Genome Variation Format (GVF) or Variant Call Format (VCF) with a pre-compiled annotation or with variants in other genomes. Its search algorithms are subsequently optimized and implemented in a multi-threaded manner. The proposed method is not a stand-alone annotation tool with its own reference databases. Rather, it is a search utility that readily accepts public or user-prepared reference files in various formats including GVF, Generic Feature Format version 3 (GFF3), Gene Transfer Format (GTF), VCF and Browser Extensible Data (BED) format. Compared to existing tools such as ANNOVAR, gSearch runs more than 10 times faster. For example, it is capable of annotating 52.8 million variants with allele frequencies in 6 min. AVAILABILITY: gSearch is available at http://ml.ssu.ac.kr/gSearch. It can be used as an independent search tool or can easily be integrated to existing pipelines through various programming environments such as Perl, Ruby and Python.

Coordination of trunk and pelvis in young and elderly individuals during axial trunk rotation.

It has been well known that complex tasks such as walking and arm reaching can be achieved by the coordination of the trunk and pelvis. However, understanding of the effect of aging on the coordination of the trunk and pelvis during axial trunk rotation is still lacking. The present study examined relative phase of the trunk and pelvis during axial trunk rotation, and compared it between young and older groups. 22 healthy young (age: 23.9±4.6, gender: 11 male and 11 female) and 22 healthy elderly (age: 68.4±4.9, gender: 11 male and 11 female) individuals participated in the experiment and performed axial trunk rotation. Relative phase between the trunk and pelvis was calculated based on the angular displacements of the two segments. The results demonstrated age-related changes in coordination pattern of the trunk and pelvis during axial trunk rotation.

Tendon impingement of the extensor digiti minimi: clinical cases series and cadaveric study.

The authors describe two unique clinical cases of closed extensor digiti minimi (EDM) tendon injuries after hyperflexion of the wrist with full finger flexion and one case of chronic tenosynovitis around the EDM tendon. All three cases were thought to be related to the bifurcation of the EDM tendon and synovial septum. Subsequently, variations in EDM tendons were investigated in 49 cadaveric hands with a focus on patterns of tendon bifurcation and their relationships with the surrounding synovial sheath. The EDM tendon was found to be bifurcated in 74% (n = 36) of hands and all of these hands contained a synovial septum. In 9 (25%) hands, the EDM tendon bifurcated proximal to the retinaculum, in 15 (42%), it bifurcated distal to the retinaculum, and in the other 12 hands (33%), the tendon bifurcated at the retinacular level. In 6 of the 15 hands with an infraretinacular bifurcation, the tendon was found to impinge on the synovial septum during passive flexion of the wrist with full finger flexion, and the mean distance between the synovial septum and the bifurcation point in these specimens was 0.6 cm (range, 0.4-0.7 cm), which was differed significantly from hands not showing impingement (P = 0.01). This study shows that distal bifurcation of the EDM tendon may lead to tendon impingement on the septum and suggests that this is a potential etiology of chronic tenosynovitis of the fifth compartment and of acute closed tendon injuries.

An assessment of histone-modification antibody quality.

We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/).

CompleteMOTIFs: DNA motif discovery platform for transcription factor binding experiments.

UNLABELLED: CompleteMOTIFs (cMOTIFs) is an integrated web tool developed to facilitate systematic discovery of overrepresented transcription factor binding motifs from high-throughput chromatin immunoprecipitation experiments. Comprehensive annotations and Boolean logic operations on multiple peak locations enable users to focus on genomic regions of interest for de novo motif discovery using tools such as MEME, Weeder and ChIPMunk. The pipeline incorporates a scanning tool for known motifs from TRANSFAC and JASPAR databases, and performs an enrichment test using local or precalculated background models that significantly improve the motif scanning result. Furthermore, using the cMOTIFs pipeline, we demonstrated that multiple transcription factors could cooperatively bind to the upstream of important stem cell differentiation regulators. AVAILABILITY: http://cmotifs.tchlab.org.

Does collocation inform the impact of collaboration?

BACKGROUND: It has been shown that large interdisciplinary teams working across geography are more likely to be impactful. We asked whether the physical proximity of collaborators remained a strong predictor of the scientific impact of their research as measured by citations of the resulting publications. METHODOLOGY/PRINCIPAL FINDINGS: Articles published by Harvard investigators from 1993 to 2003 with at least two authors were identified in the domain of biomedical science. Each collaboration was geocoded to the precise three-dimensional location of its authors. Physical distances between any two coauthors were calculated and associated with corresponding citations. Relationship between distance of coauthors and citations for four author relationships (first-last, first-middle, last-middle, and middle-middle) were investigated at different spatial scales. At all sizes of collaborations (from two authors to dozens of authors), geographical proximity between first and last author is highly informative of impact at the microscale (i.e. within building) and beyond. The mean citation for first-last author relationship decreased as the distance between them increased in less than one km range as well as in the three categorized ranges (in the same building, same city, or different city). Such a trend was not seen in other three author relationships. CONCLUSIONS/SIGNIFICANCE: Despite the positive impact of emerging communication technologies on scientific research, our results provide striking evidence for the role of physical proximity as a predictor of the impact of collaborations.

A case of sparganosis in the leg.

The life-span of the sparganum in humans is not exactly known, but it may survive longer than 5 years in some patients. We experienced a case infected with a sparganum that is presumed to have lived for 20 years in a patient's leg. The patient was a 60-year-old woman, and she was admitted to a hospital due to ankle pain that was aggravated on dorsiflexion. She had noticed a mass on her knee some 20 years ago, but she received no medical management for it. The mass moved into the ankle joint 3 months before the current admission, and then the aforementioned symptoms appeared. A living sparganum was recovered by surgery, and the calcified tract near the knee was proved to be the pathway along which the larva had passed.

Identification of genes upregulated by pinewood nematode inoculation in Japanese red pine.

Pine wilt disease caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, has destroyed huge areas of pine forest in East Asia, including Japan, China and Korea. No protection against PWN has been developed, and the responses of pine trees at the molecular level are unrecorded. We isolated and analyzed upregulated or newly induced genes from PWN-inoculated Japanese red pine (Pinus densiflora Sieb. et Zucc.) by using an annealing control primer system and suppression subtractive hybridization. Significant changes occurred in the transcript abundance of genes with functions related to defense, secondary metabolism and transcription, as the disease progressed. Other gene transcripts encoding pathogenesis-related proteins, pinosylvin synthases and metallothioneins were also more abundant in PWN-inoculated trees than in non-inoculated trees. Our report provides fundamental information on the molecular mechanisms controlling the biochemical and physiological responses of Japanese red pine trees to PWN invasion.