Foot morphology and correlation with lower extremity pain in Japanese children: A cross-sectional study of the foot posture Index-6.

BACKGROUND: Abnormal foot morphology in children and adolescents is a possible risk factor for lower extremity pain. Foot posture index-6 (FPI-6) is a valid and reliable tool to assess foot morphology. However, the normative data on the age distribution for FPI-6 in Asian children and adolescents are still minimal. Further, the correlation of FPI-6 with lower extremity pain is poorly understood. We aimed to investigate the normative distribution for FPI-6 and the relationship between FPI-6 scores and knee and heel pain in Japanese children. METHODS: We included 2569 Japanese children, aged 9-15 years, at a single school from 2016 to 2018. We summarized the age distribution of children and their mean bilateral FPI-6 scores. Additionally, we assessed the tenderness at the apophysis or tendon insertions at the knee and heel. We performed a cross-sectional analysis to investigate the correlations between FPI-6 scores and sex, age, and knee and heel pain for the data obtained each year. RESULTS: The mean FPI-6 score was 3.1 ± 2.4, 3.4 ± 2.0, and 3.2 ± 1.9 for the left foot and 3.0 ± 2.4, 3.2 ± 1.9, and 3.1 ± 1.9 for the right foot in 2016, 2017, and 2018, respectively. Boys tended to have higher scores than girls, and the FPI-6 score of the left foot was significantly higher than that of the right foot (p < 0.05). There was no correlation between FPI-6 scores and knee and heel pain. CONCLUSION: Children and adolescents between 9 and 15 years of age have neutral to slightly pronated foot morphology and an average FPI-6 score of 3.0-3.4. In addition, there was no relationship between foot morphology and knee and heel pain. This normative distribution for FPI-6 in Japanese children could serve as a reference value for future research and clinical evaluation.

SOX9 and SRY binding sites on mouse mXYSRa/Enh13 enhancer redundantly regulate Sox9 expression to varying degrees.

Sox9 plays an essential role in mammalian testis formation. It has been reported that gene expression in the testes is regulated by enhancers. Among them, mXYSRa/Enh13-which is located at far upstream of the transcription start site-plays a critical role, wherein its deletion causes complete male-to-female sex reversal in mice. It has been proposed that the binding sites (BSs) of SOX9 and SRY, the latter of which is the sex determining gene on the Y chromosome, are associated with mXYSRa/Enh13. They function as an enhancer, whereby the sequences are evolutionarily conserved and in vivo binding of SOX9 and SRY to mXYSRa/Enh13 has been demonstrated previously. However, their precise in vivo functions have not been examined to date. To this end, this study generated mice with substitutions on the SOX9 and SRY BSs to reveal their in vivo functions. Homozygous mutants of SOX9 and SRY BS were indistinguishable from XY males, whereas double mutants had small testes, suggesting that these functions are redundant and that there is another functional sequence on mXYSRa/Enh13, since mXYSRa/Enh13 deletion mice are XY females. In addition, the majority of hemizygous mice with substitutions in SOX9 BS and SRY BS were female and male, respectively, suggesting that SOX9 BS contributes more to SRY BS for mXYSRa/Enh13 to function. The additive effect of SOX9 and SRY via these BSs was verified using an in vitro assay. In conclusion, SOX9 BS and SRY BS function redundantly in vivo, and at least one more functional sequence should exist in mXYSRa/Enh13.

Turnover of mammal sex chromosomes in the Sry-deficient Amami spiny rat is due to male-specific upregulation of Sox9.

Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry, leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis, in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9. The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.

Risk factors for the occurrence and protraction of patellar and patellar tendon pain in children and adolescents: a prospective cohort study of 3 years.

BACKGROUND: Patellar and patellar tendon pain is a common limitation to children's participation in social and physical activities. Some factors have been implicated in the occurrence and protraction of knee pain, but the causal relationship is unknown. The purpose of this study was to investigate whether participants' physical characteristics and activity level are risk factors for the occurrence and protraction of patellar and patellar tendon pain in children and adolescents. METHODS: A three-year prospective cohort study was conducted with healthy students who were aged 8-14 years old, in Japan. Height, weight, heel-buttock distance, straight leg raising angle, and dorsiflexion angle of the ankle joint were collected as individual physical factors at the beginning of each year. The presence of self-reported patellar and patellar tendon pain and the Hospital for Special Surgery Pediatric Functional Activity Brief Scale (HSS Pedi-FABS) was collected every month. Protraction was defined as either (1) pain lasting for more than three continuous months or (2) recurrent pain after more than three months of complete recovery. Participants who did not have any pain at the beginning of the observation period were included in the analysis. We analyzed the odds ratio (OR) of pain occurrence within a year of registration and protraction throughout the study period for all physical factors and HSS Pedi-FABS. RESULTS: We included 1133 participants in the analysis and 252 participants developed knee pain within a year. 34.8% of participants with pain experienced protraction during the follow-up period. A high HSS Pedi-FABS significantly predicted knee pain occurrence (OR 1.03, 95% confidence interval [CI] 1.01-1.05) and protraction (OR 1.03, 95% CI 1.00-1.05). In addition, younger children and girls were at a significantly higher risk of patellar and patellar tendon pain protraction (age, OR 0.81, 95% CI, 0.73-0.90; sex, OR 1.69, 95% CI, 1.09-2.64). Other physical factors did not significantly predict the occurrence or protraction of knee pain. CONCLUSIONS: This study showed that a greater physical activity level was a risk factor for the occurrence and protraction of patellar and patellar tendon pain in childhood. In addition, younger age and female sex predicted higher risk of protraction of pain.

Human SRY Expression at the Sex-determining Period is Insufficient to Drive Testis Development in Mice.

The sex-determining region of the Y chromosome, Sry/SRY, is an initiation factor for testis development in both humans and mice. Although the functional compatibility between murine SRY and human SRY was previously examined in transgenic mice, their equivalency remains inconclusive. Because molecular interaction and timeline of mammalian sex determination were mostly described in murine experiments, we generated a mouse model in which Sry was substituted with human SRY to verify the compatibility. The mouse model had the human SRY open reading frame at the locus of murine Sry exon 1-Sry(SRY) mice-and was generated using the CRISPR/Cas9 system. The reproductive system of the mice was analyzed. The expression of human SRY in the fetal gonadal ridge of Sry(SRY) mice was detected. The external and internal genitalia of adult Sry(SRY) mice were similar to those of wild-type females, without any significant difference in anogenital distance. Sry(SRY) mice obtained gonads, which were morphologically considered as ovaries. Histological analysis revealed that the cortical regions of gonads from adult Sry(SRY) mice contained few follicles. We successfully replaced genes on the Y chromosome with targeted genome editing using the CRISPR/Cas9 system. Because the Sry(SRY) XY mice did not develop testis, we concluded that human SRY was insufficient to drive testis development in mouse embryos. The difference in response elements and lack of glutamine-rich domains may have invalidated human SRY function in mice. Signal transduction between Sry/SRY expression and Sox9/SOX9 activation is possibly organized in a species-specific manner.

Effect of Systemic Administration of Granulocyte Colony-Stimulating Factor on a Chronic Partial-Thickness Cartilage Defect in a Rabbit Knee Joint.

OBJECTIVE: Cartilage lesions in the knee joint can lead to joint mechanics changes and cause knee pain. Bone marrow stimulation (BMS) promotes cartilage regeneration by perforating the subchondral bone just below the injury and inducing bone marrow cells. This study aimed to investigate whether systemic administration of granulocyte colony-stimulating factor (G-CSF) with BMS improves repair of chronic partial-thickness cartilage defects (PTCDs). DESIGN: Eighteen 6-month-old New Zealand white rabbits were divided into 3 groups: control (C, n = 6), BMS alone (n = 6), and BMS + G-CSF (n = 6). Partial cartilage defects with 5 mm diameter were created in the trochlear region of both knees; after 4 weeks, the BMS alone and BMS + G-CSF groups underwent BMS; G-CSF (50 µg/kg) or saline was administered subcutaneously for 5 days starting from 3 days before BMS. At 8 and 16 weeks after cartilage defect creation, the area of cartilage defects was macroscopically and histologically evaluated. RESULTS: International Cartilage Repair Society (ICRS) grades for macroscopic assessment were 0, 0.7, and 0.7 at 8 weeks and 0, 1.2, and 1.3 at 16 weeks in the C, BMS, and BMS + G-CSF groups, respectively. Wakitani scores for histological assessment were 9.8, 8.7, and 8.2 at 8 weeks and 9.5, 9, and 8.2 at 16 weeks in the C, BMS, and BMS + G-CSF groups, respectively. The BMS + G-CSF group showed significantly more repair than the C group, but there was no difference from the BMS group. CONCLUSIONS: The effect of BMS and G-CSF on chronic PTCDs in mature rabbit knees was limited.

Humanization of a tandem repeat in IG-DMR causes stochastic restoration of paternal imprinting at mouse Dlk1-Dio3 domain.

The Dlk1-Dio3 imprinted domain, regulated by an intergenic differentially methylated region (IG-DMR), is important for mammalian embryonic development. Although previous studies have reported that DNA methylation of a tandem repeated array sequence in paternal IG-DMR (IG-DMR-Rep) plays an essential role in the maintenance of DNA methylation in mice, the function of a tandem repeated array sequence in human IG-DMR (hRep) is unknown. Here, we generated mice with a human tandem repeated sequence, which replaced the mouse IG-DMR-Rep. Mice that transmitted the humanized allele paternally exhibited variable methylation status at the IG-DMR and were stochastically rescued from the lethality of IG-DMR-Rep deficiency, suggesting that hRep plays a role in human IG-DMR for the regulation of imprinted expression. Moreover, chromatin immunoprecipitation analysis showed that TRIM28 was enriched in hypermethylated paternal hRep without ZFP57. Our results suggest that hRep contributes to the maintenance of human IG-DMR methylation imprints via the recruitment of TRIM28.

Nitrogen Availability and Microbial Communities of Canopy Soils in a Large Cercidiphyllum japonicum Tree of a Cool-Temperate Old Growth Forest.

Canopy soils on large trees are important for supporting the lives of many canopy plants, and thereby increasing regional biodiversity. However, because of the less accessibility to canopy soils, there is insufficient knowledge on how canopy soils produce available nitrogen (N) for canopy plants through the activity of canopy soil microbes. Canopy soils usually have different soil properties from ground soils, so we hypothesized that canopy soils would have unique microbial communities compared to ground soils, but still provide available N for canopy plants. Here, we compared soil N availability, including net N mineralization and nitrification rate, and microbial communities between canopy soils (organic soils) collected at various heights of a large Cercidiphyllum japonicum tree and ground soils (organic and mineral soils) in a cool-temperate old-growth forest of Japan. The canopy soils had significantly different N availability (mass-based higher but volume-based lower) and microbial communities from the ground mineral soils. Among organic soils, the height of the soil had an impact on the microbial communities but not on the N availability, which agreed with our hypothesis. Despite the decrease in fungal abundance in the higher soils, the increase in certain components of the cellulose-decomposing fungi and oligotrophic bacteria may contribute to the available N production. Also, the abundance of ammonia-oxidizers did not change with the height, which would be important for the nitrification rate. Our study implied canopy soils could provide N to canopy plants partly through the functional redundancy within different microbial communities and constant population of ammonia-oxidizers.

Identification of the first promoter-specific gain-of-function SOX9 missense variant (p.E50K) in a patient with 46,XX ovotesticular disorder of sex development.

SOX9, a transcription factor, is expressed in the undifferentiated XX and XY gonads. SRY induces significant upregulation of SOX9 expression in XY gonads. Loss-of-function SOX9 variants cause testicular dysgenesis in 46,XY patients, while duplication of the total gene or the upstream regulatory region results in testicular development in 46,XX patients. However, gain-of-function (GoF) SOX9 variants have not been reported previously. We report the case of a 16-year-old female patient with a 46,XX karyotype who had masculinized external genitalia and unilateral ovotestis. Next-generation sequencing-based genetic screening for disorders of sex development led to the identification of a novel SOX9 variant (p.Glu50Lys), transmitted from the phenotypically normal father. Expression analysis showed that E50K-SOX9 enhanced transactivation of the luciferase reporter containing the testis enhancer sequence core element compared with that containing the wildtype-SOX9. This GoF activity was not observed in the luciferase reporter containing Amh, the gene for anti-Müllerian hormone. We genetically engineered female mice (Sox9E50K/E50K ), and they showed no abnormalities in the external genitalia or ovaries. In conclusion, a novel SOX9 variant with a promoter-specific GoF activity was identified in vitro; however, the disease phenotype was not recapitulated by the mouse model. At present, the association between the GoF SOX9 variant and the ovotestis phenotype remains unclear. Future studies are needed to verify the possible association.

A Quick Immuno-FISH Protocol for Detecting RNAs, Proteins, and Chromatin Modifications.

Immunofluorescence and fluorescence in situ hybridization (FISH) are widely used cytogenetic techniques for visualization of protein and RNA/DNA molecules. Here, we describe an experimental procedure for quick sequential immunofluorescence and RNA FISH (immuno-FISH), which enables the simultaneous detection of proteins, chromatin modifications, and RNAs on the inactive X-chromosome (Xi) using female mouse embryonic fibroblast (MEF) and tail-tip 3T3 cell lines. Using a pooled array of oligonucleotides labeled with a single fluorophore as an RNA FISH probe, we can reduce the time for RNA FISH from an overnight process to 1-2 h without losing its sensitivity. This protocol could be applied to visualization of various protein and RNA molecules, and chromatin modifications.

Possible role of MRI-detected osteophytes as a predictive biomarker for development of osteoarthritis of the knee: A study using data from the Osteoarthritis Initiative.

Objective: To elucidate the possible role of MRI-detected osteophytes as a predictive imaging biomarker for knee osteoarthritis (KOA). Design: Subjects (n â€‹= â€‹303) were selected according to the following inclusion criteria from the Osteoarthritis Initiative (OAI) data set: (1) â€‹< â€‹55 years old; (2) Western Ontario and McMaster Universities Arthritis Index pain score of 0; (3) Kellgren-Lawrence (KL) system grade 0 or 1; and (4) Complete MRI data set of the right knee. A pre-OA group (POA) consisted of subjects who developed KL grade 2 or more within 96 months, and a non-OA group (NOA) that remained KL 0 or 1 during that period. Baseline MRIs were assessed for osteophyte formation. Twenty-five locations were examined according to the MOAKS osteophyte score. Osteophytes at each location were assessed in terms of their predictive value for OA development. Results: Thirty-two subjects were POA and 271 were NOA. Age, BMI, and sex did not differ between the two groups. In the POA group, the number of subjects with osteophytes tended to be higher at all 25 sites. Forward stepwise regression analysis revealed five locations - medial patella, lateral intra-condylar notch of the femur, lateral femoral condyle, tibial spine, and lateral posterior condyle - were important for the prediction of KOA development. Having more than two osteophytes at these five locations predicted KOA development with a sensitivity of 0.75 and specificity of 0.79. Conclusions: MRI-detected osteophytes could serve as a predictive biomarker of KOA development within 96 months after detection.

The indirect γ-aminobutyric acid (GABA) receptor agonist gabaculine-induced loss of the righting reflex may inhibit the descending analgesic pathway.

In the spinal cord, γ-aminobutyric acid (GABA) interneurons play an essential role in antinociception. However, not all actions of GABA favor antinociception at the supraspinal level. We previously reported that gabaculine, which increases endogenous GABA in the synaptic clefts, induces loss of the righting reflex (LORR) that is one indicator of hypnosis, but not immobility in response to noxious stimulus. A slow pain is transmitted to the spinal cord via C fibers and evokes substance P (SP) release from their terminals. However, the antinociceptive effects of gabaculine are still unknown. Our study examined whether the analgesic effects of the opioid morphine or the α2-adrenoceptor agonist dexmedetomidine, whose actions are mediated through facilitation of the descending analgesic pathway, are affected by gabaculine-induced LORR. We also explored the effects of GABA receptor agonists on SP release from cultured dorsal root ganglion (DRG) neurons. All drugs were administered systemically to mice. To assess antinociception, loss of nociceptive response (analgesia) and immobility were evaluated. DRG cells were dissected from rats. Gabaculine produced no analgesia. Either morphine or dexmedetomidine in combination with gabaculine induced immobility; however, the doses of each drug required to induce immobility were much higher than those required to induce analgesia. Capsaicin significantly increased SP release from DRG cells, but a high concentration (1 mM) of the GABA receptor agonist muscimol, propofol, gaboxadol, or baclofen did not inhibit the capsaicin-induced SP release, suggesting that their antinociceptive effects were not through this mechanism. Thus, the gabaculine-induced LORR may inhibit the descending analgesic pathway.

Timing of Intra-Articular Injection of Synovial Mesenchymal Stem Cells Affects Cartilage Restoration in a Partial Thickness Cartilage Defect Model in Rats.

OBJECTIVE: We investigated the effect of administration of intra-articular mesenchymal stem cells (MSCs) on cartilage repair at different timings, and the distribution of MSCs in the knee. DESIGN: A partial thickness cartilage defect (PTCD) was created on the medial femoral condyle in 14-week-old Sprague-Dawley rats. Intra-articular injection of 1 × 106 MSCs was performed at 3 time points, namely at the time of surgery (0w group), at 1 week after surgery (1w group), and at 2 weeks after surgery (2w group). For the control, 50 µL phosphate-buffered saline was injected at the time of surgery. The femoral condyles were collected at 6 weeks after creation of PTCD and assessed histologically. To investigate the distribution of MSCs, fluorescent-labeled MSCs were injected into the knee joint. RESULTS: In the control group, the cartilage lesion was distinguishable from surrounding cartilage. In the 0w group, hypocellularity and a slight decrease in safranin O stainability were observed around the injured area, but cartilage was restored to a nearly normal condition. In contrast, in the 1w and 2w groups, the cartilage surface was irregular and safranin O stainability in the injured and surrounding areas was poor. Histological score in the 0w group was significantly better than in the control, 1w, and 2w groups. At 1 day postinjection, fluorescent-labeled MSCs were mostly distributed in synovium. However, no migration into the PTCD was observed. CONCLUSIONS: Early intra-articular injection of MSCs was effective in enhancing cartilage healing in a rat PTCD model. Injected MSCs were distributed in synovium, not in cartilage surrounding the PTCD.

The effect of induced mood on word imagery processing: An ERP study.

The effect of induced mood on language processing has been examined in behavioral and event-related potential (ERP) studies. A previous study examined the effects of induced mood on word imagery processing by the N400 and N700 components of the ERP and behavioral performance in an imageability judgment task in which participants decided whether a word easily evoked visual imagery or not (Ogawa and Nittono, 2019). The N400 amplitude was larger (more negative-going) under positive mood than under negative mood, while reaction time and the N700 amplitude were not affected by induced moods. These results were interpreted as evidence that, compared to negative mood, positive mood facilitates semantic memory activation during word imagery processing. However, it remains unclear whether positive mood facilitates the phenomenological experience of imagery. To replicate and extend the previous findings, this study examined the effects of mood on subjective ratings of word imageability and ERP components. Single words with moderate imageability were used to avoid floor or ceiling effects. If a positive mood facilitates word imageability processing, subjective imageability ratings would be higher under positive mood than under negative mood. The N400 amplitude, but not the N700 amplitude, would be larger under positive mood than under negative mood. Contrary to predictions, an experiment with a sufficient sample size (N = 41) did not replicate the previous findings regarding N400 amplitude. Induced moods also did not affect the subjective imageability ratings and the N700 amplitude. These results suggest that the effects of induced mood on language processing may not occur at the level of single-word processing. Rather, moods may change the strength of association between word concepts in semantic memory.

Structural Insights into the Substrate Specificity Switch Mechanism of the Type III Protein Export Apparatus.

Bacteria use a type III protein export apparatus for construction of the flagellum, which consists of the basal body, the hook, and the filament. FlhA forms a homo-nonamer through its C-terminal cytoplasmic domains (FlhAC) and ensures the strict order of flagellar assembly. FlhAC goes through dynamic domain motions during protein export, but it remains unknown how it occurs. Here, we report that the FlhA(G368C) mutation biases FlhAC toward a closed form, thereby reducing the binding affinity of FlhAC for flagellar export chaperones in complex with their cognate filament-type substrates. The G368C mutations also restrict the conformational flexibility of a linker region of FlhA (FlhAL), suppressing FlhAC ring formation. We propose that interactions of FlhAL with its neighboring subunit converts FlhAC in the ring from a closed conformation to an open one, allowing the chaperon/substrate complexes to bind to the FlhAC ring to form the filament at the hook tip.

Comparison of meniscal extrusion and osteophyte formation at the intercondylar notch as a predictive biomarker for incidence of knee osteoarthritis-Data from the Osteoarthritis Initiative.

BACKGROUND: Medial meniscal extrusion (ME) is a biomarker to predict later development of knee osteoarthritis (KOA). On the other hand, we have reported osteophyte formation at the posterior condylar notch of the femur served as a biomarker for the same purpose. The purpose of this study is to compare capacity of the two biomarkers in predicting KOA development. METHODS: Two cohort of knees were established utilizing publicly available data from the Osteoarthritis Initiative (OAI). No OA group (NOA) consisted of knees that were grade 0 or 1 on Kellgren and Lawrence grade (K/L) both at baseline and 48 months later, and pre-radiographic-OA group (PROA) consisted of knees that were grade 0 or 1 at baseline but grade ≥2 48 months later. Baseline MR images were evaluated in terms of ME and osteophyte formation at the posterior condylar notch. ME was evaluated both by meniscus subluxation index (MSI) indicating the ratio of the extruded width of the medial meniscus to the width of medial meniscal body and by the medial radial displacement (MRD) indicating actual extruded width. The size of the osteophyte was assessed using a semi-quantitative whole-organ magnetic resonance imaging score (WORMS). The predictive accuracy of KOA was assessed by the area under the receiver operating characteristic curve (AUC) and optimal cutoff was determined for each parameter. RESULTS: The AUC for MSI was 0.654 (0.561-0.748: 95% CI) and the cutoff value was determined as 17%. That for MRD was 0.677 (0.584-0.770) and the cutoff value was 2.2 mm. The AUC for the WORMS score at the posterior condylar notch was 0.667 (0.579-0.756) and the cutoff value was 2. CONCLUSIONS: Similar predicting capacity of KOA development was found both in ME and osteophyte formation at the posterior condylar notch. Using these simple parameter, mas-screening for KOA development would be possible.

Single vs. repeated matrix metalloproteinase-13 knockdown with intra-articular short interfering RNA administration in a murine osteoarthritis model.

Purpose: Our aims were 1) to estimate the duration of short interfering RNA (siRNA) effect on matrix metalloproteinase-13 (Mmp-13) levels by a single intra-articular injection using a mouse knee osteoarthritis (OA) model and 2) to test whether repeated injections results in any additional suppressive effect on cartilage degradation compared to a single injection. Materials and Methods: OA was induced in 9 weeks old male C57BL/6 mice by destabilization of medial meniscus (DMM). Chemically modified siRNA targeted for Mmp-13 was injected into the knee joint at 1 week post-DMM surgery. Control group of knees received that for non-targeted genes. Synovial tissue was collected to measure Mmp-13 expression levels by quantitative polymerase chain reaction (qPCR) at 2, 3, and 6 weeks after surgery in each group. To test the effect of multiple injections, we created four experiment groups according to the number of injections. Histological assessment of articular cartilage was performed at 8 weeks post-DMM surgery. Results: In the Mmp-13 siRNA-treated group, expression levels of Mmp-13 mRNA were decreased by 40% compared to the control group at 2 weeks after surgery (p = 0.04), before returning to baseline at 3 weeks after surgery. A significant improvement in the histological score was observed in all Mmp-13 siRNA-treated groups compared to the control group (p < 0.05). However, no significant differences were seen between the single and multiple injection group. Conclusions: Our results suggested that the duration of siRNA effect in the knee joint lasts for at least 1 week, and that no further benefit is achieved by multiple injections.

Mapping of a responsible region for sex reversal upstream of Sox9 by production of mice with serial deletion in a genomic locus.

Sox9 plays critical roles in testis formation. By mapping four familial cases of disorders of sexual development, a 32.5 kb sequence located far upstream of SOX9 was previously identified as being a commonly deleted region and named the XY sex reversal region (XYSR). To narrow down a responsible sequence in XYSR, we generated mutant mice with a series of deletions in XYSR by application of the CRISPR/Cas9 system, using a mixture of sgRNAs targeting several kilobase (kb) intervals in the region. When the whole XYSR corresponding sequence in mice was deleted in XY karyotype individuals, the mutation resulted in female offspring, suggesting that an expression mechanism of SOX9/Sox9 through XYSR is conserved in human and mouse. Male-to-female sex reversal was found in mice with a 4.8 kb deletion. We identified a sequence conserved among humans, mice, and opossum, the deletion of which (783 bp) in mice resulted in male-to-female sex reversal. The sequence includes a recently reported critical gonad enhancer for Sox9. Although it cannot be concluded that the human sequence is responsible for XYSR, it is likely. This method is applicable for fine mapping of responsible sequences for disease-causing deletions especially with regard to rare diseases.

Total Synthesis of (+)-Pyrenolide D.

An efficient approach to stereoselective construction of a spiro-γ-lactone core structure via BF3-promoted formal [3 + 2] annulation of aldehydo-aldose derivatives with γ-methylene-γ-butyrolactone has been developed. The spiro-γ-lactone derivative was then used in an efficient total synthesis of (+)-pyrenolide D. The developed chemistry paves the way for total synthesis of structurally diverse natural products containing spiro-lactone cores.

Peptidyl arginine deiminase 2 (Padi2) is expressed in Sertoli cells in a specific manner and regulated by SOX9 during testicular development.

Peptidyl arginine deiminases (PADIs) are enzymes that change the charge of proteins through citrullination. We recently found Padi2 was expressed exclusively in fetal Sertoli cells. In this study, we analyzed the transcriptional regulation of Padi2 and the role of PADI2 in testicular development. We showed SOX9 positively regulated Padi2 transcription and FOXL2 antagonized it in TM3 cells, a model of Sertoli cells. The responsive region to SOX9 and FOXL2 was identified within the Padi2 sequence by reporter assay. In fetal testes from Sox9 knockout (AMH-Cre:Sox9flox/flox) mice, Padi2 expression was greatly reduced, indicating SOX9 regulates Padi2 in vivo. In vitro analysis using siRNA suggested PADI2 modified transcriptional regulation by SOX9. However, Padi2-/- XY mice were fertile and showed no apparent reproductive anomalies. Although, PADI2 is known as an epigenetic transcriptional regulator through H3 citrullination, no significant difference in H3 citrullination between wildtype and Padi2-/- XY gonads was observed. These results suggest Padi2 is a novel gene involved in testis development that is specifically expressed in Sertoli cells through the regulation by SOX9 and FOXL2 and PADI2 supports regulation of target genes by SOX9. Analysis of the Padi2-/- XY phenotype suggested a redundant factor compensated for PADI2 function in testicular development.