Short-segment spinal fusion for chronic low back pain with bone marrow edema adjacent to the vertebral endplate in adult spinal deformity.

PURPOSE: Corrective long spinal fusion is a widely accepted surgical method for patients with adult spinal deformities. However, instrumented long fusion is associated with a significant risk of complications. Therefore, we aimed to assess the success of short-segment spinal fusion, particularly for bone marrow edema (BME) adjacent to the vertebral endplate, in patients with low back pain (LBP) and spinal deformity. METHODS: A prospective study was performed at multiple hospitals wherein we monitored patients with spinal deformities and accompanying LBP. Patients aged ≥ 50 years with a minimum LBP severity score of 40 mm on the visual analog scale (VAS) were included in the study. We also included patients with lumbar BME on magnetic resonance imaging. Short spinal fusion was performed on segments with BME. Clinical evaluations of LBP on VAS and Oswestry Disability Index (ODI), and radiological parameters for sagittal vertical axis (SVA), pelvic incidence (PI), lumbar lordosis (LL) and pelvic tilt (PT) were carried out. RESULTS: Overall, 35 patients (22 men and 13 women), with a mean age of 66.7 years and a mean follow-up period of 32 months, were included in the study. The mean VAS and ODI scores were 72.4 mm and 49.0% before surgery and 25.5 mm and 29.9% at the final follow-up, respectively; these parameters significantly improved after surgery. The SVA, PI-LL, and PT scores were 70.1 mm, 20.9°, and 22.8° before surgery and 85.4 mm, 13.8°, and 22.7° at the final follow-up, respectively. The spinal alignment parameters did not change significantly after surgery. CONCLUSIONS: Short-segment spinal fusion is effective for treating LBP and spinal deformity with BME adjacent to the vertebral endplate without spinal correction.

Balancing ability of patients with lumbar spinal canal stenosis.

PURPOSE: To determine the relationship between postural sway and the severity of lumbar spinal canal stenosis as well as the effect on postoperative improvement. METHODS: Stabilometry was performed before and 6 months after surgery in 52 patients (29 men and 23 women; mean age, 74.1 ± 7.8 years) who underwent decompression surgery for lumbar spinal canal stenosis. The environmental area (EA; the area surrounding the circumference of the stabilogram) and locus length per EA (L/EA) were evaluated. The patients were divided into moderate (n = 22) and severe (n = 30) groups according to the severity of canal stenosis. Patient characteristics and parameters were compared between the groups before and after surgery, including the visual analog scale (VAS) score for leg pain, Oswestry Disability Index (ODI), EA, and L/EA. In addition, factors affecting EA and L/EA were evaluated using multiple regression analysis. RESULTS: Age (p = 0.031), preoperative EA (p < 0.001), preoperative L/EA (p = 0.032), and sagittal vertical axis (p = 0.033) were significantly different between groups. The VAS score and ODI significantly improved postoperatively in both groups (p < 0.001). The EA significantly improved postoperatively only in the severe group (p < 0.001), whereas the L/EA did not significantly improve in either group. Multiple regression analysis showed that only the severity of canal stenosis was significantly associated with preoperative EA (p = 0.030), whereas age (p = 0.040) and severity of canal stenosis (p = 0.030) were significantly associated with preoperative L/EA. Diabetes was significantly associated with postoperative EA (p = 0.046) and L/EA (p = 0.030). CONCLUSION: The severity of canal stenosis affected abnormal postural sway, which improved after decompression surgery.

A New Lignan (Polonilignan) and Inhibitors of Nitric Oxide Production from Penicillium polonicum, an Endophytic Fungi of Piper nigrum.

Inhibiting nitric oxide (NO) or its production is found to be of therapeutic benefit. To discover natural small molecule inhibitors of NO production, a bioassay- and LC/MS-guided chemical investigation was done on the metabolites of endophytic fungus isolated from fresh Piper nigrum fruits. The isolated pure strain was identified as Penicillium polonicum by 16S rDNA sequence comparison. The culture broth extract of P. polonicum (EEPP) exhibited a significant reduction of NO production (Griess method) in LPS-stimulated RAW 264.7 cells (P<0.0001). To understand the chemical constituents of bioactive EEPP, column chromatography and p-TLC studies were carried out, which yielded eight pure compounds. They were characterised as botryosphaeridione (1), 3-(3,5-di-tert-butyl-4-hydroxy)phenylpropionic acid (2), variabilone (3), 2,4-di-tert-butylphenol (4), indole-3-carboxylic acid (5), tyrosol (6), ethyl ferulate (7) and a new lignan (8) based on the spectral analysis. The structure elucidation of the new lignan, named polonilignan (8), was based on HR-MS, 1 H- & 13 C-NMR, H-H COSY, HSQC and HMBC spectra. Compounds 2, 4, 5 and 6 showed a significant decrease (P<0.0001) in the production of NO in LPS-induced RAW 264.7 cells. Tyrosol (6) and indole-3-carboxylic acid (5) controlled nitrite release with IC50 values of 22.84 and 55.01 µM, respectively. This is the first report of (i) P. polonicum as an endophytic fungus of pepper fruits, (ii) isolation of compounds 1-8 except 6 from P. polonicum culture broth extract and (iii) NO inhibition effect of 2, 4, 5 and 6.

Prognostic factors for spontaneous spinal epidural hematoma: a multicenter case-control study.

BACKGROUND: To determine the optimal treatment for spontaneous spinal epidural hematoma (SSEH). The aim of this study was to identify factors associated with SSEH. METHODS: In 62 patients with SSEH, several patient-related parameters were analyzed as candidate factors associated with Frankel grade before treatment or at the last follow-up. These parameters were compared between patients with and without surgery. In addition, multivariate ordinal logistic regression analysis was used to identify factors significantly associated with Frankel's grade before treatment or at the last follow-up. RESULTS: There were significant differences in age, location of the hematoma, and Frankel grade before treatment and at the last follow-up between surgical and nonsurgical cases in all patients, but there were no significant differences in any of these parameters when comparing patients with pre-treatment Frankel grade C. The location of the hematoma was significantly associated with the severity of paralysis before treatment. In surgical cases, the time from onset to surgery and the location of the hematoma was significantly associated with the prognosis. When the time from onset to surgery was evaluated using the criteria of 12, 24, and 48 h, 24 and 48 h had a significant impact on the prognosis. In the analysis of nonsurgical cases, only the vertical size of the hematoma was significantly associated with prognosis. CONCLUSION: The time from onset to surgery and the location of the hematoma were prognostic factors in surgical cases, while the vertical size of the hematoma was a prognostic factor in nonsurgical cases.

Surgical outcomes of cervical myelopathy in patients with athetoid cerebral palsy.

PURPOSE: Surgical treatment for cervical myelopathy with athetoid cerebral palsy remains unestablished. Instrumented fusion is reported to have good clinical results; however, there are no data of decompression surgery for this pathology in recent years. This study aimed to assess the surgical outcomes of laminoplasty with or without posterior instrumented fusion for cervical myelopathy in patients with athetoid cerebral palsy. METHODS: A multi-centre surgical series of patients with cervical myelopathy and athetoid cerebral palsy were enrolled in this study. All patients showed symptoms and signs suggestive of cervical myelopathy and underwent laminoplasty with or without instrumented fusion. The Japanese Orthopaedic Association (JOA) score, Barthel index (BI), and changes in the C2-C7 sagittal Cobb angle in the lateral plain radiograph were analysed. RESULTS: There were 25 patients (16 men and 9 women; mean age, 54.4 ± 10.8 years) with cervical myelopathy and athetoid cerebral palsy who underwent surgical treatment. The mean follow-up period was 41.9 ± 35.6 months. Overall, the BI significantly improved after surgery, whereas the JOA score and C2-C7 angle did not improve postoperatively. The recovery rate of the JOA score in the laminoplasty group was significantly higher than that of the fusion group (P = 0.02). CONCLUSIONS: Cervical laminoplasty with or without instrumented fusion for treating cervical myelopathy due to athetoid cerebral palsy is effective in improving activities of daily living. Cervical laminoplasty may be an effective and less invasive surgical method for selective patients, especially for those with small involuntary movements and no remarkable cervical kyphosis nor instability.

Long-term outcome of targeted therapy for low back pain in elderly degenerative lumbar scoliosis.

PURPOSE: Treatment of low back pain (LBP) associated with elderly degenerative lumbar scoliosis (DLS) remains controversial. We have developed percutaneous intervertebral-vacuum polymethylmethacrylate injection (PIPI) targeting to the intervertebral vacuum as a minimally invasive surgery. The present study compared the long-term clinical outcomes of PIPI to that of nonoperative treatment. METHODS: Patients with de novo DLS, aged ≥ 65 years, who had LBP with visual analog scale (VAS) of ≥ 50 for ≥ 6 months with intervertebral vacuum on computed tomography and bone marrow edema (BME) on magnetic resonance imaging were included. The clinical outcomes were evaluated using VAS and the Oswestry Disability Index (ODI) at baseline, 1, 6, 12, 24 months, and at the final follow-up. The course of BME was also evaluated. RESULTS: One hundred and one patients underwent PIPI and 61 received nonoperative treatment. The mean follow-up duration after PIPI and nonoperative treatment was 63.7 ± 32.4 and 43.9 ± 20.9 months, respectively. VAS and ODI after PIPI were significantly improved compared to post-nonoperative treatment. BME decreased substantially in the PIPI group and it was significantly correlated with VAS and ODI improvement. Following PIPI, LBP recurred in 28 patients (35%). LBP recurrence was identified at the same level of PIPI in 10 patients, at the adjacent level of PIPI in 11 patients, and at the non-adjacent level of PIPI in seven patients. Eighteen patients underwent additional PIPIs, and both VAS and ODI were significantly improved after additional PIPIs. CONCLUSION: Bone marrow lesions of the endplate are strongly associated with the presence of LBP. PIPI can be considered as an effective, safe and repeatable treatment for LBP in elderly DLS patients.

Third DWF1 paralog in Solanaceae, sterol Δ24-isomerase, branches withanolide biosynthesis from the general phytosterol pathway.

A large part of chemodiversity of plant triterpenes is due to the modification of their side chains. Reduction or isomerization of double bonds in the side chains is often an important step for the diversification of triterpenes, although the enzymes involved are not fully understood. Withanolides are a large group of structurally diverse C28 steroidal lactones derived from 24-methylenecholesterol. These compounds are found in the Indian medicinal plant Withania somnifera, also known as ashwagandha, and other members of the Solanaceae. The pathway for withanolide biosynthesis is unknown, preventing sustainable production via white biotechnology and downstream pharmaceutical usages. In the present study, based on genome and transcriptome data we have identified a key enzyme in the biosynthesis of withanolides: a DWF1 paralog encoding a sterol Δ24-isomerase (24ISO). 24ISO originated from DWF1 after two subsequent duplication events in Solanoideae plants. Withanolides and 24ISO appear only in the medicinal plants in the Solanoideae, not in crop plants such as potato and tomato, indicating negative selection during domestication. 24ISO is a unique isomerase enzyme evolved from a reductase and as such has maintained the FAD-binding oxidoreductase structure and requirement for NADPH. Using phylogenetic, metabolomic, and gene expression analysis in combination with heterologous expression and virus-induced gene silencing, we showed that 24ISO catalyzes the conversion of 24-methylenecholesterol to 24-methyldesmosterol. We propose that this catalytic step is the committing step in withanolide biosynthesis, opening up elucidation of the whole pathway and future larger-scale sustainable production of withanolides and related compounds with pharmacological properties.

Halodule pinifolia (Seagrass) attenuated lipopolysaccharide-, carrageenan-, and crystal-induced secretion of pro-inflammatory cytokines: mechanism and chemistry.

OBJECTIVES: The study aimed to explore the anti-inflammatory effect, underlying mechanism, and chemistry of Halodule pinifolia extract. METHODS: The ethyl acetate (EHP) and methanol (MHP) extracts of Halodule pinifolia were screened for pro-inflammatory cytokine inhibition effect under various in vitro (LPSand crystal-induced inflammation) and in vivo models (LPS-induced endotoxaemia model, carrageenan-induced paw oedema model, and oxalate-induced renal nephropathy model of inflammation). The effect of EHP on the expression of inflammatory markers using western blot analysis (in vitro) was investigated. Chemical constituents of bioactive EHP were isolated through chromatography and characterised using NMR spectroscopy. Furthermore, EHP was standardised for rosmarinic acid, vanillic acid, and ethyl protocatechuate using HPLC. Also, total phytosterols, phenolic, and flavonoid content of EHP were determined by UV spectroscopy. KEY FINDINGS: EHP was comparatively more effective than MHP in inhibiting cytokines secretions under LPS-induced in vitro models. Furthermore, EHP was screened under endotoxaemia in vivo model, EHP (250 mg/kg) reduced plasma IL-6, TNF-α, and IL-1ß levels by 88.3%, 78.2%, and 74.5%, respectively. In the carrageenan-induced oedema model, EHP (200 mg/kg) reduced paw volume and release of TNF-α (69.3%) and IL-1ß (43.1%). EHP (200 mg/kg) further controlled renal nephropathy by inhibiting plasma IL-1ß and BUN levels. Also, a significant reduction of mRNA expressions of TNF-α and IL-1ß and KIM-1 in renal tissues was observed. Through western blot, EHP was identified to regulate the expression of pro-form as well as mature-form of IL-1ß and caspase-1. EHP constituted rosmarinic acid (RA), vanillic acid (VA), ethyl protocatechuate (EP), sitosterol, stigmasterol, campesterol, and dihydrobrassicasterol. It was determined that 4.6 mg/g of RA, 2.92 mg/g of VA, 0.76 mg/g of EP, 21.7 mg/g of total phenolics, 29.8 mg/g of total flavonoids, and 48.2 mg/g of total phytosterols were present in dry EHP. The presence of anti-inflammatory constituents such as RA, VA, and PE in EHP corroborated the in vitro and in vivo anti-inflammatory activity of EHP. CONCLUSION: The anti-inflammatory property of EHP and its action through attenuation of pan-cytokines suggest that it can be developed into an oral pharmaceutical drug.

The third national Japanese antimicrobial susceptibility pattern surveillance program: Bacterial isolates from complicated urinary tract infection patients.

The antimicrobial susceptibility patterns of bacterial pathogens isolated from patients with complicated urinary tract infections were analyzed using national surveillance data. The data consisted of 881 bacterial strains from eight clinically relevant species. The data were collected for the third national surveillance project from January 2015 to March 2016 by the Japanese Society of Chemotherapy, the Japanese Association for Infectious Disease, and the Japanese Society of Clinical Microbiology. Surveillance was undertaken with the cooperation of 41 medical institutions throughout Japan. Fluoroquinolone required a MIC90 of 2-64 mg/L to inhibit the 325 Escherichia coli strains tested and the proportion of levofloxacin resistant E. coli strains increased to 38.5% from 29.6% in 2011 and 28.6% in 2008. The proportion of levofloxacin resistant strains of Pseudomonas aeruginosa and Enterococcus faecalis decreased from previous reports and the proportion of multidrug-resistant P. aeruginosa and carbapenem-resistant Enterobacteriaceae remained low. Among methicillin-resistant Staphylococcus aureus (MRSA) strains, strains with reduced susceptibility to vancomycin (minimum inhibitory concentration, 2 µg/mL) increased to 14.7% from 5.5%. Bacterial strains that produced extended-spectrum ß-lactamase included E. coli (79 of 325 strains, 24.3%), Klebsiella pneumoniae (9 of 177 strains, 7.7%), and Proteus mirabilis (6 of 55 strains, 10.9%). The proportion of extended-spectrum ß-lactamase producing E. coli and K. pneumoniae strains increased from previous surveillance reports.

New synthetic coumarinolignans as attenuators of pro-inflammatory cytokines in LPS-induced sepsis and carrageenan-induced paw oedema models.

OBJECTIVES: The aim of the study was to explore the inhibition efficacy of new synthetic coumarinolignans (SCLs) against the secretion of pro-inflammatory cytokines in two in vivo models of inflammation. METHODS: Four SCLs 1-4 were screened for their pro-inflammatory cytokine inhibitory potential through oral administration at a dose of 50 mg/kg body weight in lipopolysaccharide-induced mouse endotoxaemia and carrageenan-induced mouse paw oedema models. Levels of pro-inflammatory cytokines (IL-1ß, TNFα and IL-6) in blood and paw tissue samples were estimated using ELISA. Paw oedema was measured using a plethysmometer. Results were compared with a natural coumarinolignan, cleomiscosin A (5), and the structure-activity relationship (SAR) was interpreted. RESULTS AND DISCUSSION: Compound 2 had the greatest potential in the endotoxaemia model, exhibiting 66.41%, 62.56% and 43.15% inhibition of plasma IL-1ß, TNFα and IL-6 secretions, respectively. Further dose-dependent study revealed its anti-inflammatory potential even at dose of 10 mg/kg body weight with 24.42% decline in the level of IL-1ß. Nevertheless, SCLs 1, 3 and 4 showed marked inhibitory activity with 57.54%, 51.48% and 62.46% reduction in the levels of IL-1ß, respectively. Moreover, compound 2 decreased the plasma TNFα and IL-1ß levels to 50.03% and 36.58% along with the reduction of paw oedema volume in the local inflammation induced by carrageenan. All compounds including cleomiscosin A (5) were more effective against IL-1ß. By studying SAR, the presence of dihydroxyl groups in the phenyl ring of lignans was identified to be essential for the activity. Also, esterification of lignans and presence of a 4-methyl substituent in the coumarin nucleus were found to play some role in enhancing the activity. CONCLUSION: All four SCLs, especially compound 2, have shown vast potential to emerge out as promising anti-inflammatory drugs.

Decrease of bone mineral density in Japanese patients with non-metastatic prostate cancer treated with androgen deprivation therapy.

The aim of this study was to conduct a cross-sectional survey of investigations related to the bone mineral density (BMD) of both non-metastatic prostate cancer (NMPC) patients who have not yet received androgen deprivation therapy (ADT) and patients receiving prolonged ADT in Japan. Japanese male patients with NMPC who received continuous ADT or who were planning to receive ADT were enrolled in this study. Lumbar spine and femoral neck BMD was measured using dual-energy X-ray absorptiometry (DEXA). To assess patient characteristics, we searched medical records and questionnaires to determine whether they had any factors that could possibly affect BMD. A total of 230 patients with a mean age of 76.6 ± 6.4 years were evaluated. Of these, 151 (65.7%) were receiving ADT, and 79 (34.4%) had not yet received ADT. The mean duration of ADT was 37.4 ± 30.7 months. DEXA showed that as the duration of ADT increased, lumbar spine and femoral neck BMD decreased gradually (p = 0.0005 and p = 0.0014, respectively). Stepwise regression analyses revealed that the duration of ADT was a significant variable of both lumbar spine and femoral neck BMD. Moreover, as the duration of ADT increased, the prevalence of osteoporosis increased statistically (p = 0.0002). This study showed that ADT negatively affected lumbar spine and femoral neck BMD. It also showed a progressive increase in the prevalence of osteoporosis in Japanese NMPC patients with ADT.

Structure-based design and synthesis of new 4-methylcoumarin-based lignans as pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß) inhibitors.

Suppression of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) along with nitric oxide reduction in RAW 264.7 cells by 7,8-dihydroxy-4-methylcoumarin, ethyl p-coumarate, ethyl caffeate and ethyl ferulate drove us to search structural-analogues of the aforementioned compounds through structure-based drug design. Docking studies revealed that substituted cinnamic acids and their ethyl esters (2-7c) showed higher GoldScore-fitness (GSF) and non-bonding interactions with target proteins than 7,8-dihydroxy-4-methylcoumarin (1a) and 7,8-dihydroxy-5-methylcoumarin (1b). With this background, the methylcoumarins (1a and 1b) and the cinnamic acid derivatives (2-7c) were fused in different permutations and combinations to generate sixty novel fused-cyclic coumarinolignans (FCLs) (8-13k). Docking studies on 8-13k indicated that several FCLs possess higher GSF, interesting active site interactions and distinctive π-π interactions compared to the standards (cleomiscosin A, diclofenac Na and prednisolone). Based on these findings, four novel FCLs (9d, 10d, 11d and 11e) were synthesized and tested for inhibition effect on TNF-α, IL-1ß and IL-6 expressions in LPS and oxalate crystal-induced in-vitro models. Compound 10d exhibited significant effect (P < 0.0001 at 100 µM) with an IC50 value of 8.5 µM against TNF-α. Compound 11e possessed IC50 values of 13.29 µM and 17.94 µM against IL-6 and IL-1ß, respectively. Study on SAR corroborated the requirement of C-4-methyl substituent in the coumarin moiety, dihydroxyl groups in the phenyl ring, and esterification of lignans for potent activity. Additionally, the reported excellent anti-inflammatory activity of cleomiscosin-A-glucoside was corroborated by from the higher GSF and better hydrophobic interactions than cleomsicosin A in the docking study. As an outcome, some novel and potentially active FCLs acting through NFκB and caspase 1 signaling pathways have been discovered as multiple cytokine inhibitors.

Transforaminal percutaneous endoscopic discectomy for lumbar disc herniation in athletes under the local anesthesia.

BACKGROUND: Percutaneous endoscopic discectomy (PED) has been reported to be less invasive and effective procedure for lumbar disc herniation (LDH). Damage to the back muscle is considered minimal, which is particularly important for athletes. However, the results of PED for LDH in athletes have not been reported well. The purpose of this study was to evaluate the clinical outcomes of PED for LDH in athletes. METHODS: We retrospectively analyzed 21 athlete patients with LDH who had undergone PED. All patients received athletic rehabilitation immediately after surgery. The clinical outcomes were evaluated from the visual analogue scale (VAS) for leg pain and low back pain (LBP), the Oswestry Disability Index (ODI), complications and periods of return to sport. RESULTS: There were 18 men and 3 women, and the mean age at the time of surgery was 22.9 years (range: 15-43 years). The mean VAS scores for leg pain before and after surgery were 64.3 ± 2.7 mm and 12.4 ± 1.4 mm, respectively. The mean VAS scores for LBP before and after surgery were 62.1 ± 2.2 mm and 10.5 ± 1.1 mm, respectively. The mean ODI scores before and after surgery were 31.3 ± 14.0% and 14.6 ± 7.1%, respectively. The VAS for leg pain, as well as the LBP and ODI, significantly improved after surgery. There were no complications related to the surgery. Ninety-five percent (20/21) returned to play sports at the same performance level as before the procedure by an average of 9.2 weeks after PED. CONCLUSIONS: PED is a minimally invasive and effective procedure for patients with LDH, especially in athletes. Not only the patients' leg pain but also their discogenic LBP improved. PED has the benefits of preservation of normal posterior structures and a faster return to sports.

[Investigation of Clinical Outcome and Prognosis of 2nd TUR-Bt for T1 High Grade Bladder Cancer].

Second transurethral resection of bladder tumor (TUR-Bt) is a standard treatment for high grade T1 initial, non-muscle invasive bladder cancer. In our hospital from October 2008 to April 2017, 2nd TUR-Bt was performed in 51 cases of initial T1 bladder cancer. The risk factors of residual tumors on 2nd TUR-Bt and the clinical outcome were examined retrospectively. Twenty two of the 51 cases (43.1%) had residual tumors on 2nd TUR-Bt, and upstaging was not admitted. To determine the risk factor for residual tumors in 2nd TUR-Bt, we examined gender, tumor morphology (papillary/non-papillary, pedunculated/ nonpedunculated), number of tumors (single/frequent), and tumor size (≧20 mm/＜20 mm), but none of these were significant risk factors for tumor residue. The recurrence free survival (RFS) of the 51 cases was 86.0% after 1 year, and 77.0% after 3 years. There tended to be a higher RFS in the pedunculated tumor group, pT0 group on 2nd TUR-Bt, intravesical BCG therapy group, but no statistically significant difference was observed. The progression free survival (PFS) was 90.6% after 3 years, and 87.3% after 5 years. These values were similar to those reported previously.

A Dioxygenase Catalyzes Steroid 16α-Hydroxylation in Steroidal Glycoalkaloid Biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites that are found in the Solanaceae. Potato (Solanum tuberosum) contains the SGAs α-solanine and α-chaconine, while tomato (Solanum lycopersicum) contains α-tomatine, all of which are biosynthesized from cholesterol. However, although two cytochrome P450 monooxygenases that catalyze the 22- and 26-hydroxylation of cholesterol have been identified, the 16-hydroxylase remains unknown. Feeding with deuterium-labeled cholesterol indicated that the 16α- and 16ß-hydrogen atoms of cholesterol were eliminated to form α-solanine and α-chaconine in potato, while only the 16α-hydrogen atom was eliminated in α-tomatine biosynthesis, suggesting that a single oxidation at C-16 takes place during tomato SGA biosynthesis while a two-step oxidation occurs in potato. Here, we show that a 2-oxoglutarate-dependent dioxygenase, designated as 16DOX, is involved in SGA biosynthesis. We found that the transcript of potato 16DOX (St16DOX) was expressed at high levels in the tuber sprouts, where large amounts of SGAs are accumulated. Biochemical analysis of the recombinant St16DOX protein revealed that St16DOX catalyzes the 16α-hydroxylation of hydroxycholesterols and that (22S)-22,26-dihydroxycholesterol was the best substrate among the nine compounds tested. St16DOX-silenced potato plants contained significantly lower levels of SGAs, and a detailed metabolite analysis revealed that they accumulated the glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) gene gave essentially the same results. These findings clearly indicate that 16DOX is a steroid 16α-hydroxylase that functions in the SGA biosynthetic pathway. Furthermore, St16DOX silencing did not affect potato tuber yield, indicating that 16DOX may be a suitable target for controlling toxic SGA levels in potato.

Naphthoquinone Metabolites Produced by Monacrosporium ambrosium, the Ectosymbiotic Fungus of Tea Shot-Hole Borer, Euwallacea fornicatus, in Stems of Tea, Camellia sinensis.

The tea shot-hole borer beetle (TSHB, Euwallacea fornicatus) causes serious damage in plantations of tea, Camellia sinensis var. assamica, in Sri Lanka and South India. TSHB is found in symbiotic association with the ambrosia fungus, Monacrosporium ambrosium (syn. Fusarium ambrosium), in galleries located within stems of tea bushes. M. ambrosium is known to be the sole food source of TSHB. Six naphthoquinones produced during spore germination in a laboratory culture broth of M. ambrosium were isolated and identified as dihydroanhydrojavanicin, anhydrojavanicin, javanicin, 5,8-dihydroxy-2-methyl-3-(2-oxopropyl)naphthalene-1,4-dione, anhydrofusarubin and solaniol. Chloroform extracts of tea stems with red-colored galleries occupied by TSHB contained UV active compounds similar to the above naphthoquinones. Laboratory assays demonstrated that the combined ethyl acetate extracts of the fungal culture broth and mycelium inhibited the growth of endophytic fungi Pestalotiopsis camelliae and Phoma multirostrata, which were also isolated from tea stems. Thus, pigmented naphthoquinones secreted by M. ambrosium during spore germination may prevent other fungi from invading TSHB galleries in tea stems. The antifungal nature of the naphthoquinone extract suggests that it protects the habitat of TSHB. We propose that the TSHB fungal ectosymbiont M. ambrosium provides not only the food and sterol skeleton necessary for the development of the beetle during its larval stages, but also serves as a producer of fungal inhibitors that help to preserve the purity of the fungal garden of TSHB.

Enzymatically synthesized megalo-type isomaltosaccharides enhance the barrier function of the tight junction in the intestinal epithelium.

Megalo-type isomaltosaccharides are an enzymatically synthesized foodstuff produced by transglucosylation from maltodextrin, and they contain a mid-chain length polymer of D-glucose with α-1,6-glycoside linkages. The injection of a solution of megalo-type isomaltosaccharides (1-4%(w/v), average DP = 12.6), but not oligo-type isomaltosaccharides (average DP = 3.3), into the intestinal lumen dose-dependently reduced the transport rates of tight junction permeable markers in a ligated loop of the anesthetized rat jejunum. Application of the megalosaccharide also suppressed the transport of tight junction markers and enhanced transepithelial electrical resistance (TEER) in Caco-2 cell monolayers. Cholesterol sequestration by methyl-ß-cyclodextrin in the Caco-2 monolayers abolished the effect of megalosaccharide. Treatment with anti-caveolin-1 and a caveolae inhibitor, but not clathrin-dependent endocytosis and macropinocytosis inhibitors, suppressed the increase in TEER. These results indicate that isomaltosaccharides promote the barrier function of tight junctions in the intestinal epithelium in a chain-length dependent manner and that caveolae play a role in the effect.

[Clinical Study of 2014 ISUP New Grade Group Classification for Prostate Cancer Patients Treated by Androgen Deprivation Therapy].

The 2014 International Society of Urological Pathology (ISUP) has proposed a new grade group (GG) classification for Gleason scores (GS). The usefulness of the new GG classification was investigated with 518 prostate cancer patients who underwent androgen deprivation therapy. According to the new GG classification, Stages B‒D and the new GG classification relapse-free rate for each stage were calculated using the Kaplan‒Meier method. The new GG classification revealed a significant difference for the relapse-free rate only between some groups. Analysis using the Cox proportional hazards model indicated that the risk of relapse was higher in GGs 4 and 5 than in GG 1. The usefulness about the relapse-free rate in androgen deprivation therapy of the 2014 ISUP new grade group classification a waits future examination.

Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol.

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C28- and C29-Δ(24(28))-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ(24(25))intermediate. In this study, we characterized theArDWF1gene from an expression sequence tag library ofAjuga reptansvar.atropurpureahairy roots. The gene was functionally expressed in the yeast T21 strain. Thein vivoandin vitrostudy of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-(2)H3,28-(13)C)-24-methylenecholesterol, followed by(13)C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ(24(25))intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes,Oryza sativa DWF1(OsDWF1) was similarly expressed in yeast. Anin vivoassay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in theAjugahairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.