Uncovering Endolysins against Methicillin-Resistant Staphylococcus aureus Using a Microbial Single-Cell Genome Database.

Endolysins, peptidoglycan hydrolases derived from bacteriophages (phages), are being developed as a promising alternative to conventional antibiotics. To obtain highly active endolysins, a diverse library of these endolysins is vital. We propose here microbial single-cell genome sequencing as an efficient tool to discover dozens of previously unknown endolysins, owing to its culture-independent sequencing method. As a proof of concept, we analyzed and recovered endolysin genes within prophage regions of Staphylococcus single-amplified genomes in human skin microbiome samples. We constructed a library of chimeric endolysins by shuffling domains of the natural endolysins and performed high-throughput screening against Staphylococcus aureus. One of the lead endolysins, bbst1027, exhibited desirable antimicrobial properties, such as rapid bactericidal activity, no detectable resistance development, and in vivo efficacy. We foresee that this endolysin discovery pipeline is in principle applicable to any bacterial target and boost the development of novel antimicrobial agents.

Impact of spinal sagittal malalignment on locomotive syndrome and physical function in community-dwelling middle aged and older women.

BACKGROUND: Adult spinal deformity has a substantially debilitating effect on older people's physical and mental health. However, the impact of sagittal malalignment on locomotive syndrome (LS), sarcopenia, and physical function in community-dwelling older women has not yet been clarified. This study aimed to investigate the association between these factors in community-dwelling middle aged and older women. METHODS: A total of 361 women were recruited from participants performing aquatic exercises in a rural area of Japan. The body mass index, skeletal muscle mass index, trunk muscle mass, spinal inclination angle (SIA), grip strength, timed up-and-go test (TUG), maximum stride of the participants, and one-leg standing time were measured. Low back pain (LBP)- and health-related quality of life (HRQOL) were evaluated using the Oswestry Disability Index (ODI) and the Short-Form 8 questionnaire. Associations between the global sagittal alignment using SIA and investigating parameters were analyzed. RESULTS: The prevalence of sarcopenia was 3.6%. The prevalence of LS (stages 1, 2, and 3) was 43.8% (158 of 361), and the number of participants in each LS stage was 203 (stage 0), 95 (stage 1), 28 (stage 2), and 35 (stage 3). The SIA was significantly correlated with the 25-question geriatric locomotive function scale (r' = 0.292, p < 0.001), ODI (r' = 0.267, p < 0.001), and TUG (r' = 0.453, p < 0.001) after adjusting for age. In the receiver-operating characteristic curve analysis, the cutoff values of SIA for LS ≥ stage 2 and ODI ≥ 20% were 5°. CONCLUSIONS: LBP-related QOL and physical performance were significantly associated with global sagittal alignment. Global sagittal alignment was correlated with the three-stage category of LS. The spinal inclination of 5° was a cutoff value to predict exacerbation of mobility function and HRQOL status.

Target enrichment of uncultured human oral bacteria with phage-derived molecules found by single-cell genomics.

Advances in culture-independent microbial analysis, such as metagenomics and single-cell genomics, have significantly increased our understanding of microbial lineages. While these methods have uncovered a large number of novel microbial taxa, many remain uncultured, and their function and mode of existence in the environment are still unknown. This study aims to explore the use of bacteriophage-derived molecules as probes for detecting and isolating uncultured bacteria. Here, we proposed multiplex single-cell sequencing to obtain massive uncultured oral bacterial genomes and searched prophage sequences from over 450 obtained human oral bacterial single-amplified genomes (SAGs). The focus was on the cell wall binding domain (CBD) in phage endolysin, and fluorescent protein-fused CBDs were generated based on several CBD gene sequences predicted from Streptococcus SAGs. The ability of the Streptococcus prophage-derived CBDs to detect and enrich specific Streptococcus species from human saliva while maintaining cell viability was confirmed by magnetic separation and flow cytometry. The approach to phage-derived molecule generation based on uncultured bacterial SAG is expected to improve the process of designing molecules that selectively capture or detect specific bacteria, notably from uncultured gram-positive bacteria, and will have applications in isolation and in situ detection of beneficial or pathogenic bacteria.

Determining factors that maintain physical function or increase frailty using the Kihon checklist among community-dwelling older adults: a six-year longitudinal study in Agano, Japan.

BACKGROUND: A significant increase in the older adult population in Japan will significantly increase healthcare costs. This study aimed to examine the risk factors contributing to robustness transitioning to frailty in older residents. METHODS: Participants were aged 70 in 2016 and 76 in 2022. Participants were evaluated using the Kihon Checklist (KCL). RESULTS: Participants for this longitudinal study included 444 older persons who completed the KCL surveys in 2016 and 2022. The follow-up rate was 80.6%; therefore, 358 participants were included in the analysis. The median KCL score increased significantly from 2 to 2016 to 3 in 2022 (p < 0.001). The prevalence of robustness significantly decreased from 60.9 to 48.6% (p = 0.042). In a stepwise logistic regression analysis, robustness was independently associated with regular continuous walks for 15 min and a body mass index of above 18.5%. The following variables were associated with the transition to prefrailty: experiencing a fall in the past year and not going out at least once a week. For the transition to frailty, the variables were turned to family or friends for advice, experienced a fall in the past year, and felt helpless in the last two weeks. The independent factor for the transition from prefrailty to frailty was having a BMI of less than 18.5. In contrast, the independent factor for improving from frailty to robustness or prefrailty was going out at least once a week. CONCLUSIONS: We recommend maintaining continuous walking for more than 15 min, maintaining a BMI of at least 18.5, and going out more than once a week to improve being house-bounded and depressive mood, not only to prevent the transition to prefrailty or frailty but also to improve frailty.

Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read sequencing.

Obtaining complete and accurate bacterial genomes is vital for studying the characteristics of uncultured bacteria. Single-cell genomics is a promising approach for the culture-independent recovery of bacterial genomes from individual cells. However, single-amplified genomes (SAGs) often have fragmented and incomplete sequences due to chimeric and biased sequences introduced during the genome amplification process. To address this, we developed a single-cell amplified genome long-read assembly (scALA) workflow to construct complete circular SAGs (cSAGs) from long-read single-cell sequencing data of uncultured bacteria. We used the SAG-gel platform, which is both cost-effective and high-throughput, to obtain hundreds of short-read and long-read sequencing data for specific bacterial strains. The scALA workflow generated cSAGs by repeated in silico processing for sequence bias reduction and contig assembly. From 12 human fecal samples, including two cohabitant groups, scALA generated 16 cSAGs of three specifically targeted bacterial species: Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. We discovered strain-specific structural variations shared among cohabiting hosts, while all cSAGs of the same species showed high homology in aligned genomic regions. A. hadrus cSAGs exhibited 10 kbp-long phage insertions, various saccharide metabolic capabilities, and different CRISPR-Cas systems in each strain. The sequence similarity of A. hadrus genomes did not necessarily correspond with orthologous functional genes, while host geographical regionality seemed to be highly related to gene possession. scALA allowed us to obtain closed circular genomes of specifically targeted bacteria from human microbiota samples, leading to an understanding of within-species diversities, including structural variations and linking mobile genetic elements, such as phages, to hosts. These analyses provide insight into microbial evolution, the adaptation of the community to environmental changes, and interactions with hosts. cSAGs constructed using this method can expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.

Low back pain significantly influences locomotive syndrome in older people: Evaluation using the 3-stage categories.

BACKGROUND: The Japanese Orthopaedic Association (JOA) introduced the concept of locomotive syndrome (LS), which indicates a decline in mobility function by musculoskeletal disorders with new 3-staged category. Additionally, sarcopenia indicates a decline in the quantity and/or quality of skeletal muscle. However, the relationship between low back pain (LBP) and LS or sarcopenia in older people has not been sufficiently understood. This study aimed to investigate the association between them through a cross-sectional locomotorium survey. METHODS: A total of 302 participants were drawn from the aquatic exercise participants in a rural area of Japan. The body mass index, body fat percentage, skeletal muscle mass index (SMI), spinal inclination angle (SIA), grip strength, timed up-and-go test (TUG), and maximum stride of the participants were measured. LBP and LBP-related quality of life (QOL) were evaluated using the Oswestry Disability Index (ODI), visual analogue scale (VAS) of LBP, and the Short-Form 8 (SF-8). Associations between the investigating parameters and sarcopenia or LS were analyzed. RESULTS: There were no significant differences in the findings except grip strength between the non-sarcopenia and sarcopenia groups. However, the LS group showed significantly larger SIA, higher ODI, higher VAS of LBP, lower physical component score (PCS) of the SF-8, longer time in TUG, and lower value in maximum stride than the non-LS group. In addition, the ODI and PCS of the SF-8 significantly deteriorated as the LS stage progressed, and the GLFS-25 score was significantly correlated with ODI (r = 0.706, p < 0.001) and PCS (r = -0.643, p < 0.001) scores. CONCLUSIONS: LBP, LBP-related QOL, and physical performance were found to be significantly associated with LS, not sarcopenia, with LBP-related QOL and physical function being closely correlated with 3-stage categories of LS. Thus, these results suggested that LBP is a key factor for LS prevalence.

Exploring strain diversity of dominant human skin bacterial species using single-cell genome sequencing.

To understand the role of the skin commensal bacterial community in skin health and the spread of pathogens, it is crucial to identify genetic differences in the bacterial strains corresponding to human individuals. A culture-independent genomics approach is an effective tool for obtaining massive high-quality bacterial genomes. Here we present a single-cell genome sequencing to obtain comprehensive whole-genome sequences of uncultured skin bacteria from skin swabs. We recovered 281 high-quality (HQ) and 244 medium-quality single-amplified genomes (SAGs) of multiple skin bacterial species from eight individuals, including cohabiting group. Single-cell sequencing outperformed in the genome recovery from the same skin swabs, showing 10-fold non-redundant strain genomes compared to the shotgun metagenomic sequencing and binning approach. We then focused on the abundant skin bacteria and identified intra-species diversity, especially in 47 Moraxella osloensis derived HQ SAGs, characterizing the strain-level heterogeneity at mobile genetic element profiles, including plasmids and prophages. Even between the cohabiting individual hosts, they have unique skin bacterial strains in the same species, which shows microdiversity in each host. Genetic and functional differences between skin bacterial strains are predictive of in vivo competition to adapt bacterial genome to utilize the sparse nutrients available on the skin or produce molecules that inhibit the colonization of other microbes or alter their behavior. Thus, single-cell sequencing provides a large number of genomes of higher resolution and quality than conventional metagenomic analysis and helps explore the skin commensal bacteria at the strain level, linking taxonomic and functional information.

Variations in Strain Distribution at Distal Radius under Different Loading Conditions.

Distal radial fractures exhibit various fracture patterns. By assuming that the strain distribution at the distal radius affects the diversification of the fracture pattern, a parameter study using the finite element model of a wrist developed from computed tomography (CT) images was performed under different loading conditions. The finite element model of the wrist consisted of the radius, ulna, scaphoid, lunate, triquetrum, and major carpal ligaments. The material properties of the bone models were assigned on the basis of the Hounsfield Unit (HU) values of the CT images. An impact load was applied to the scaphoid, lunate, and triquetrum to simulate boundary conditions during fall accidents. This study considered nine different loading conditions that combine three different loading directions and three different load distribution ratios. According to the analysis results, the strain distribution at the distal radius changed with respect to the change in the loading condition. High strain concentration occurred in regions where distal radius fractures are commonly developed. The direction and distribution of the load acting on the radius were considered to be factors that may cause variations in the fracture pattern of distal radius fractures.

Identification of two cancer stem cell-like populations in triple-negative breast cancer xenografts.

Gene expression analysis at the single-cell level by next-generation sequencing has revealed the existence of clonal dissemination and microheterogeneity in cancer metastasis. The current spatial analysis technologies can elucidate the heterogeneity of cell-cell interactions in situ. To reveal the regional and expressional heterogeneity in primary tumors and metastases, we performed transcriptomic analysis of microtissues dissected from a triple-negative breast cancer (TNBC) cell line MDA-MB-231 xenograft model with our automated tissue microdissection punching technology. This multiple-microtissue transcriptome analysis revealed three cancer cell-type clusters in the primary tumor and axillary lymph node metastasis, two of which were cancer stem cell (CSC)-like clusters (CD44/MYC-high, HMGA1-high). Reanalysis of public single-cell RNA-sequencing datasets confirmed that the two CSC-like populations existed in TNBC xenograft models and in TNBC patients. The diversity of these multiple CSC-like populations could cause differential anticancer drug resistance, increasing the difficulty of curing this cancer.

Strain-level profiling of viable microbial community by selective single-cell genome sequencing.

Culture-independent analysis with high-throughput sequencing has been widely used to characterize bacterial communities. However, signals derived from non-viable bacteria and non-cell DNA may inhibit its characterization. Here, we present a method for viable bacteria-targeted single-cell genome sequencing, called PMA-SAG-gel, to obtain comprehensive whole-genome sequences of surviving uncultured bacteria from microbial communities. PMA-SAG-gel uses gel matrixes that enable sequential enzymatic reactions for cell lysis and genome amplification of viable single cells from the microbial communities. PMA-SAG-gel removed the single-amplified genomes (SAGs) derived from dead bacteria and enabled selective sequencing of viable bacteria in the model samples of Escherichia coli and Bacillus subtilis. Next, we demonstrated the recovery of near-complete SAGs of eight oxygen-tolerant bacteria, including Bacteroides spp. and Phocaeicola spp., from 1331 human feces SAGs. We found the presence of two different strains in each species and identified their specific genes to investigate the metabolic functions. The survival profile of an entire population at the strain level will provide the information for understanding the characteristics of the surviving bacteria under the specific environments or sample processing and insights for quality assessment of live bacterial products or fecal microbiota transplantation and for understanding the effect of antimicrobial treatments.

Recovery of strain-resolved genomes from human microbiome through an integration framework of single-cell genomics and metagenomics.

BACKGROUND: Obtaining high-quality (HQ) reference genomes from microbial communities is crucial for understanding the phylogeny and function of uncultured microbes in complex microbial ecosystems. Despite improvements in bioinformatic approaches to generate curated metagenome-assembled genomes (MAGs), existing metagenome binners obtain population consensus genomes but they are nowhere comparable to genomes sequenced from isolates in terms of strain level resolution. Here, we present a framework for the integration of single-cell genomics and metagenomics, referred to as single-cell (sc) metagenomics, to reconstruct strain-resolved genomes from microbial communities at once. RESULTS: Our sc-metagenomics integration framework, termed SMAGLinker, uses single-cell amplified genomes (SAGs) generated using microfluidic technology as binning guides and integrates them with metagenome-assembled genomes (MAGs) to recover improved draft genomes. We compared sc-metagenomics with the metagenomics-alone approach using conventional metagenome binners. The sc-metagenomics approach showed precise contig binning and higher recovery rates (>97%) of rRNA and plasmids than conventional metagenomics in genome reconstruction from the cell mock community. In human microbiota samples, sc-metagenomics recovered the largest number of genomes with a total of 103 gut microbial genomes (21 HQ, with 65 showing >90% completeness) and 45 skin microbial genomes (10 HQ, with 40 showing >90% completeness), respectively. Conventional metagenomics recovered one Staphylococcus hominis genome, whereas sc-metagenomics recovered two S. hominis genomes from identical skin microbiota sample. Single-cell sequencing revealed that these S. hominis genomes were derived from two distinct strains harboring specifically different plasmids. We found that all conventional S. hominis MAGs had a substantial lack or excess of genome sequences and contamination from other Staphylococcus species (S. epidermidis). CONCLUSIONS: SMAGLinker enabled us to obtain strain-resolved genomes in the mock community and human microbiota samples by assigning metagenomic sequences correctly and covering both highly conserved genes such as rRNA genes and unique extrachromosomal elements, including plasmids. SMAGLinker will provide HQ genomes that are difficult to obtain using metagenomics alone and will facilitate the understanding of microbial ecosystems by elucidating detailed metabolic pathways and horizontal gene transfer networks. SMAGLinker is available at https://github.com/kojiari/smaglinker . Video abstract.

Five-year longitudinal study of frailty prevalence and course assessed using the Kihon Checklist among community-dwelling older adults in Japan.

The aim of this study was to analyze the 5-year natural course of frailty status assessed with the Kihon Checklist (KCL) and the risk factors of transition towards frailty in community-dwelling older adults. We used the data from the postal KCL survey conducted by the municipal government between 2011 and 2016. The sample of the current study consisted of 551 older adults (265 men and 286 women) aged 65-70 years in 2011. The median KCL score increased from 2 (interquartile range 1-3) in 2011 to 3 (1-5) in 2016 (p < 0.001). Hence, the prevalence of frailty increased from 8.0 to 12.3% (p < 0.001). Regarding the 5-year transitions in frailty status, 68.3% of participants remained unchanged, while 21.4% transitioned towards a worse frailty status, and 10.3% towards an improved status. Of the 507 respondents who were robust or prefrail at the baseline, 44 experienced a transition towards frailty, indicating that the 5-year incidence of frailty was 8.7%. These 44 individuals had higher body mass indexes (BMI) and lower physical activity scores on the KCL than others (p < 0.05), the latter of which was an independent predictor of transition toward frailty in the multivariate analysis. This study was the first to evaluate the 5-year natural course of frailty status assessed using the KCL in community-dwelling elderly adults, in which the prevalence of frailty increased by 4.3%. To prevent transition towards frailty, maintaining optimal physical activity is recommended.

Seronegative Oligoarthritis Preceding Psoriasis by 9.5 Years.

We report a case of psoriatic arthritis where oligoarthritis preceded the skin lesions. A 57-year-old man complained of left third-finger pain. Laboratory examinations were negative for anti-cyclic citrullinated peptide antibodies and rheumatoid factor; he was treated for suspected rheumatoid arthritis. Six years later, X-ray revealed enthesitis of his fingers and wrist joint. At 9.5 years after the initial visit, skin lesions appeared in the left auricular region and buttock and dermatopathology findings indicated psoriasis vulgaris. The final diagnosis was psoriatic arthritis. In cases of seronegative oligoarthritis, psoriatic arthritis must be considered because some patients demonstrate osteoarticular lesions preceding skin lesions.

High-Quality Draft Single-Cell Genome Sequences of Two Gammaproteobacteria Strains Sampled from Soil in a Strawberry Farm.

Here, we present high-quality draft single-cell genome sequences of Gammaproteobacteria strains BBSC-SA01 and BBSC-SA02, obtained from uncultivated cells of soil in a strawberry farm using the single-cell sequencing platform bit-MAP. These draft genomes putatively represent novel species within Gammaproteobacteria and allow further investigation into the soil microbiome.

Effective microtissue RNA extraction coupled with Smart-seq2 for reproducible and robust spatial transcriptome analysis.

Spatial transcriptomics is useful for understanding the molecular organization of a tissue and providing insights into cellular function in a morphological context. In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 µm in a diameter and 10 µm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. This resulted in reproducible sequence read mapping in exon regions and the detection of more than 2000 genes compared to non-fixed samples in the RNA-seq analysis. Our method would be applied to various transcriptome analyses, providing the information for region specific gene expression in tissue specimens.

Association between bone mineral density and ulnar styloid fracture in older Japanese adults with low-energy distal radius fracture.

We investigated the association of the ulnar styloid fracture (USF) with the bone mineral status and fractured radial displacement in elderly patients. The presence of USF correlates with decreased BMD and severe displacement of the radius. These findings are helpful in treating osteoporosis to prevent subsequent fragility fracture. PURPOSE: The pathogenesis of ulnar styloid fracture (USF), which often occurs with distal radius fracture (DRF), is unclear. This study aimed to investigate whether USF concomitant with low-energy DRF was associated with the bone mineral status and the degree of radiographically observed pretreatment radius displacement in Japanese adults above 50 years of age. METHODS: The study subjects were 45 (44 female, 1 male) consecutive patients aged > 50 years with DRF caused by falls from June 2015 to May 2016. Fractures due to high-energy injuries were excluded. Patients were divided into two groups according to the presence or absence of USF. Radius displacement was assessed on anteroposterior and lateral radiographs by measuring ulnar variance, radial inclination, and volar tilt at initial examination before manual reduction of the bone. Bone mineral density (BMD) of the lumbar spine, femoral neck, and distal radius was also measured by dual-energy X-ray absorptiometry within 1 week of injury. RESULTS: Significant differences in the BMD values of femoral neck, ulnar variance, radial inclination, and volar tilt were found between patients with USF and those without USF (all comparisons, p < 0.05). Logistic regression analysis of all subject data identified that volar tilt was significantly associated with the presence of USF (p = 0.048). CONCLUSIONS: The presence of USF in low-energy DRF correlates with the decreased BMD of femoral neck and severe displacement of radius in elderly patients. These findings are helpful for the treatment of osteoporosis to prevent subsequent fragility fracture.

Time-lapse single-cell transcriptomics reveals modulation of histone H3 for dormancy breaking in fission yeast.

How quiescent cells break dormancy is a key issue in eukaryotic cells including cancer. Fungal spores, for example, remain quiescent for long periods until nourished, although the mechanisms by which dormancy is broken remain enigmatic. Transcriptome analysis could provide a clue, but methods to synchronously germinate large numbers of spores are lacking, and thus it remains a challenge to analyse gene expression upon germination. Hence, we develop methods to assemble transcriptomes from individual, asynchronous spore cells of fission yeast undergoing germination to assess transcriptomic changes over time. The virtual time-lapse analyses highlights one of three copies of histone H3 genes whose transcription fluctuates during the initial stage of germination. Disruption of this temporal fluctuation causes defects in spore germination despite no visible defects in other stages of the life cycle. We conclude that modulation of histone H3 expression is a crucial 'wake-up' trigger at dormancy breaking.

The Dynamic Photometric Stereo Method Using a Multi-Tap CMOS Image Sensor.

The photometric stereo method enables estimation of surface normals from images that have been captured using different but known lighting directions. The classical photometric stereo method requires at least three images to determine the normals in a given scene. However, this method cannot be applied to dynamic scenes because it is assumed that the scene remains static while the required images are captured. In this work, we present a dynamic photometric stereo method for estimation of the surface normals in a dynamic scene. We use a multi-tap complementary metal-oxide-semiconductor (CMOS) image sensor to capture the input images required for the proposed photometric stereo method. This image sensor can divide the electrons from the photodiode from a single pixel into the different taps of the exposures and can thus capture multiple images under different lighting conditions with almost identical timing. We implemented a camera lighting system and created a software application to enable estimation of the normal map in real time. We also evaluated the accuracy of the estimated surface normals and demonstrated that our proposed method can estimate the surface normals of dynamic scenes.

Bone Formation Parameters of the Biopsied Ilium Differ between Subtrochanteric and Diaphyseal Atypical Femoral Fractures in Bisphosphonate-Treated Patients.

Atypical femoral fractures (AFFs) are defined as atraumatic or low-trauma fractures located in the subtrochanteric or diaphyseal sites. Long-term bisphosphonates (BPs) are administered to prevent fragility fractures in patients with primary osteoporosis or collagen diseases who are already taking glucocorticoids (GCs). Long-term BP use is one of the most important risk factors for AFFs. Its pathogenesis is characterized by severely suppressed bone turnover (SSBT), but whether the characteristics of patients are different regarding to location of fracture site remains unknown. In this study, we compared the characteristics and bone histomorphometric findings between subtrochanteric and diaphyseal sites in patients with BP-associated AFFs. Nine women with BP-associated AFFs were recruited, including 3 with systemic lupus erythematosus, 2 with rheumatoid arthritis, 2 with primary osteoporosis, 1 with polymyalgia rheumatica, and 1 with sarcoidosis. Patients were divided into the subtrochanteric group (n = 5; average age, 52 years; BP treatment, 5.9 years) and the diaphyseal group (n = 4; average age, 77 years; BP treatment, 2.6 years). Compared with the diaphyseal group, the subtrochanteric group had significantly higher daily GC doses (average, 10.9 vs. 2.3 mg/day) and significantly lower serum 25-hydroxyvitamin-D levels (17.8 vs. 25.6 ng/mL). Bone histomorphometry of the biopsied iliac bone showed SSBT in 3 cases (subtrochanteric, n = 1; diaphyseal, n = 2). Osteoid volume and trabecular thickness were significantly lower in the subtrochanteric group than in the diaphyseal group. Bone formation was inhibited more severely in subtrochanteric than in the diaphyseal group due to the higher GC doses used.

Site-specific gene expression analysis using an automated tissue micro-dissection punching system.

Site-specific gene expression analyses are important for understanding tissue functions. Despite rapid developments in DNA-related technologies, the site-specific analysis of whole genome expression for a tissue remains challenging. Thus, a new tool is required for capturing multiple tissue micro-dissections or single cells while retaining the positional information. Here, we describe the development of such a system, which can pick up micro-dissections by punching a tissue repeatedly in a very short period, e.g., 5 s/sampling cycle. A photo of the punched tissue provides information on the dissected positions, allowing site-specific gene expression analysis. We demonstrate the site-specific analysis of a frozen tissue slice of mouse brain by analyzing many micro-dissections produced from the tissue at a 300-µm pitch. The site-specific analysis provided new insights into the gene expression profiles in a tissue and on tissue functions. The analysis of site-specific whole genome expression may therefore, open new avenues in life science.