Molecular Basis for Enzymatic Aziridine Formation via Sulfate Elimination.

Natural products containing an aziridine ring, such as mitomycin C and azinomycin B, exhibit antitumor activities by alkylating DNA via their aziridine rings; however, the biosynthetic mechanisms underlying the formation of these rings have not yet been elucidated. We herein investigated the biosynthesis of vazabitide A, the structure of which is similar to that of azinomycin B, and demonstrated that Vzb10/11, with no similarities to known enzymes, catalyzed the formation of the aziridine ring via sulfate elimination. To elucidate the detailed reaction mechanism, crystallization of Vzb10/11 and the homologous enzyme, AziU3/U2, in the biosynthesis of azinomycin B was attempted, and the structure of AziU3/U2, which had a new protein fold overall, was successfully determined. The structural analysis revealed that these enzymes adjusted the dihedral angle between the amino group and the adjacent sulfate group of the substrate to almost 180° and enhanced the nucleophilicity of the C6-amino group temporarily, facilitating the SN2-like reaction to form the aziridine ring. The present study reports for the first time the molecular basis for aziridine ring formation.

Preliminary Clinical Outcome of One-level Mobi-C Total Disc Replacement in Japanese Population.

INTRODUCTION: In 2018, the first Mobi-CⓇ total disk replacement (TDR) case was performed in Japan. In this study, we examined the preliminary clinical outcome of Mobi-CⓇ for degenerative cervical spine disease. METHODS: We examined 24 consecutive patients who underwent 1-level TDR after 2018 and followed up for more than 6 months after surgery. The evaluation criteria included age, gender, diagnosis, follow-up period, surgical level, implant size, surgery time, intraoperative bleeding volume, complications, revision surgery, imaging findings, JOA score, and various questionnaires. RESULTS: The mean age was 52.7 years, 13 males and 11 females. There were 15 cases of cervical disk herniation and 9 cases of cervical spondylosis. The mean follow-up period was 17.4 months. Surgical levels were C3/4 in 4 cases, C4/5 in 2 cases, C5/6 in 16 cases, and C6/7 in 2 cases. The mean operation time was 138.5 minutes, the amount of intraoperative bleeding was 32.1 ml, and there were no serious intraoperative complications. The range of motion of the affected level increased significantly, from 6.6 degrees preoperatively to 12.2 degrees at final follow-up. No patients required revision surgery at final follow-up, and there were no cases of heterotopic ossification or adjacent segment disease. One patient exhibited radiculopathy due to mild subsidence 1 year after surgery, and 1 had asymptomatic contact of device plates. Preoperative and final JOA scores improved from 11.7 to 15.8 points, and NRS improved from 4.3 to 1.3 points for neck pain and 4.3 to 1.7 points for arm pain. Preoperative and final NDI improved from 39.7% to 14.0%, and EQ-5D improved from 0.602 to 0.801. CONCLUSIONS: The short-term treatment outcomes of Mobi-CⓇ TDR were generally favorable. Spine surgeons should comply with guidelines when introducing this procedure and strive to adopt this new technology in Japan.

Sequential MRI Changes After Lateral Lumbar Interbody Fusion in Spondylolisthesis with Mild and Severe Lumbar Spinal Stenosis.

OBJECTIVE: We assessed the sequential magnetic resonance imaging changes of indirect neural decompression after minimally invasive lumbar lateral interbody fusion (LIF) combined with posterior percutaneous pedicle screw (PPS) fixation for degenerative spondylolisthesis (DS) according to the severity of preoperative lumbar spinal stenosis. METHODS: A total of 43 patients (mean age, 68.7 years; 16 men and 27 women) with DS who had undergone LIF and closed reduction with PPS fixation were enrolled. The intervertebral levels were divided into the moderate stenosis (MS) group (preoperative cross-sectional area [CSA] of the thecal sac >50 mm2) and severe stenosis (SS) group (CSA ≤50 mm2). The CSA, ligamentum flavum thickness, and diameter of the thecal sac at the affected level were measured on cross-sectional magnetic resonance images at baseline, immediately postoperatively, and 2 years postoperatively. RESULTS: For the 31 and 29 intervertebral levels in the MS and SS groups, the mean CSA at baseline, immediately postoperatively, and 2 years postoperatively was 76.9 mm2 and 35.8 mm2, 104.3 mm2 and 81.4 mm2, and 130.9 mm2 and 105.7 mm2, respectively. The mean ligamentum flavum thicknesses at 2 years postoperatively became thinner than that immediately after surgery in both groups (P < 0.01). The mean diameter of the thecal sac at 2 years was longer than that immediately after surgery in both groups (MS group, P < 0.05; SS group, P < 0.01) The expansion ratio of the CSA at 2 years postoperatively was significantly greater in the SS group than that in the MS group (P < 0.01). CONCLUSIONS: Sequential enlargement of the spinal canal was obtained by the thinning of the ligamentum flavum after LIF and PPS fixation in patients with DS with both mild and severe stenosis. The effect of indirect neural decompression was equivalent even in those with severe lumbar spinal stenosis.

Reconstitution of a Highly Reducing Type II PKS System Reveals 6π-Electrocyclization Is Required for o-Dialkylbenzene Biosynthesis.

Natural products containing an o-dialkylbenzene moiety exhibit a wide variety of bioactivities, including antibacterial, antifungal, antitumor, and antiangiogenic activities. However, the biosynthetic scheme of the o-dialkylbenzene moiety remains unclear. In this study, we identified the biosynthetic gene cluster (BGC) of compounds 1 and 2 in Streptomyces sp. SANK 60404, which contains a rare o-dialkylbenzene moiety, and successfully reconstituted the biosynthesis of 1 using 22 recombinant enzymes in vitro. Our study established a biosynthetic route for the o-tolyl group within the o-dialkylbenzene moiety, where the triene intermediate 3 loaded onto a unique acyl carrier protein (ACP) is elongated by a specific ketosynthase-chain length factor pair of a type II polyketide synthase system with the aid of a putative isomerase to be termed "electrocyclase" and a thioesterase-like enzyme in the BGC. The C2-elongated all-trans diketo-triene intermediate is subsequently isomerized to the 6Z configuration by the electrocyclase to allow intramolecular 6π-electrocyclization, followed by coenzyme FAD/FMN-dependent dehydrogenation. Bioinformatics analysis showed that the key genes are all conserved in BGCs of natural products containing an o-dialkylbenzene moiety, suggesting that the proposed biosynthetic scheme is a common strategy to form o-dialkylbenzenes in nature.

Guanidyl modification of the 1-azabicyclo[3.1.0]hexane ring in ficellomycin essential for its biological activity.

The 1-azabicyclo[3.1.0]hexane ring is a key moiety in natural products for biological activities against bacteria, fungi, and tumor through DNA alkylation. Ficellomycin is a dipeptide that consists of l-valine and a non-proteinogenic amino acid with the 1-azabicyclo[3.1.0]hexane ring structure. Although the biosynthetic gene cluster of ficellomycin has been identified, the biosynthetic pathway currently remains unclear. We herein report the final stage of ficellomycin biosynthesis involving ring modifications and successive dipeptide formation. After the ring is formed, the hydroxy group of the ring is converted into the guanidyl unit by three enzymes, which include an aminotransferase with a novel inter ω-ω amino-transferring activity. In the last step, the resulting 1-azabicyclo[3.1.0]hexane ring-containing amino acid is connected with l-valine by an amino acid ligase to yield ficellomycin. The present study revealed a new machinery that expands the structural and biological diversities of natural products.

Vertebroplasty with posterior spinal fusion for osteoporotic vertebral fracture using computer-assisted rod contouring system: A new minimally invasive technique.

INTRODUCTION: Surgical treatment of osteoporotic vertebral fracture (OVF) has been challenging for spine surgeons, because there are potential risks of instrumentation failure; such as screw loosening, loss of correction, or pseudarthrosis, due to bone fragility in elderly patients with several comorbidities. PRESENTATION OF CASE: A 68-year-old female presented with a severe low back pain and bilateral thigh pain. She had a history of systemic scleroderma, which was complicated by interstitial lung disease. Although she initially underwent non-surgical treatment with bracing for 7 months, her symptoms had progressively deteriorated, and her radiographs showed non-union at L1 and progressive kyphotic deformity at the thoracolumbar spine. Because an anterior approach was inadvisable due to interstitial lung disease, vertebroplasty with posterior spinal fusion was performed using percutaneous pedicle screws (PPS) at the upper most and lowest instrumented vertebra combined with sublaminar taping and computer-assisted rod contouring system. Good bony union was achieved with no screw loosening at 1-year follow-up. DISCUSSION: Various surgical procedures have been applied according to the fracture type or medical condition of the patient. Minimally invasive posterior spinal fusion would be a less invasive approach in patients with poor medical condition. PPS can prevent the excessive dissection of paravertebral muscles, and this is especially advantageous at the proximal and distal end of long constructs. A recent computer-assisted rod contouring system accurately matches each screw head resulting in reduced strength of the screw-bone interface. CONCLUSION: This technique would be beneficial in the elderly or immunocompromised patients with OVF.

Selective Thoracic Fusion for King-Moe Type II/Lenke 1C Curve in Adolescent Idiopathic Scoliosis: A Comprehensive Review of Major Concerns.

INTRODUCTION: Controversies still exist in the surgical indications and outcomes of selective thoracic fusion (STF) for a primary thoracic curve with a compensatory large lumbar curve (King-Moe type II/Lenke 1C curve) in adolescent idiopathic scoliosis (AIS). Issues of the greatest concern regarding this curve type include curve criteria that indicate STF to prevent postoperative coronal decompensation and postoperative radiographic outcomes, including curve correction, coronal balance, and thoracolumbar kyphosis, after STF. METHODS: This review comprehensively documents the issues raised in the literature regarding surgical indications and radiographic outcomes of STF for King-Moe type II/Lenke 1C curve in AIS. RESULTS: Studies suggest that radiographic curve criteria indicating STF for this curve type include the preoperative dominance of the thoracic curve to the lumbar curve in the Cobb angle and the characteristics of the lumbar curve in magnitude and flexibility. Studies warn the need for a careful clinical evaluation of the thoracic and lumbar rotational prominences. Documented radiographic outcomes of importance include the postoperative behavior of the unfused lumbar curve, coronal or sagittal decompensation after STF, and factors associated with these issues. A comprehensive review of the literature suggests that the use of a segmental pedicle screw construct and better instrumented thoracic curve correction achieve better spontaneous lumbar curve correction. Although the causes of postoperative coronal decompensation remain multifactorial, preoperative coronal decompensation to the left and an inappropriate selection of the lowest instrumented vertebra are consistently reported to be the major causative factors. CONCLUSIONS: STF has been validated in general for the treatment of King-Moe type II or Lenke 1C curve in AIS; however, controversies remain regarding the surgical indications and outcomes. Long-term impacts of residual lumbar curve, coronal decompensation, and mild thoracolumbar kyphosis on clinical outcomes after STF, along with optimal indications and strategy for STF, should further be assessed.

Comprehensive Derivatization of Thioviridamides by Heterologous Expression.

New technology for the derivatization of peptide natural products is required for drug development. Despite the recent advances in the genome sequencing technique enabling us to search for the biosynthetic genes for wide variety of natural products, the technical methods to get access to them are limited. A class of RiPPs, a recently emerged natural product family such as thioviridamide, is one of those possessing such unexplored chemical space. In this paper, we report a streamlined method to generate new thioviridamide derivatives and to assess their biological activities. Heterologous expression of 42 constructs in an engineered Streptomyces avermitilis host gave 35 designed thioviridamide derivatives, along with several unprecedented analogues. Moreover, cytotoxicity assay revealed that several derivatives showed more potent activities than those of prethioviridamide. These results indicate that this strategy can become one of the potential ways to produce supreme unnatural products.

Glutamate Dehydrogenase from Thermus thermophilus Is Activated by AMP and Leucine as a Complex with Catalytically Inactive Adenine Phosphoribosyltransferase Homolog.

Glutamate dehydrogenase (GDH) from a thermophilic bacterium, Thermus thermophilus, is composed of two heterologous subunits, GdhA and GdhB. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. In the present study, we performed a pulldown assay using recombinant T. thermophilus, producing GdhA fused with a His tag at the N terminus, and found that TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, was copurified with GdhA. When GdhA, GdhB, and APRTh were coproduced in Escherichia coli cells, they were purified as a ternary complex. The ternary complex exhibited GDH activity that was activated by leucine, as observed for the GdhA-GdhB binary complex. Furthermore, AMP activated GDH activity of the ternary complex, whereas such activation was not observed for the GdhA-GdhB binary complex. This suggests that APRTh mediates the allosteric activation of GDH by AMP. The present study demonstrates the presence of complicated regulatory mechanisms of GDH mediated by multiple compounds to control the carbon-nitrogen balance in bacterial cells.IMPORTANCE GDH, which catalyzes the synthesis and degradation of glutamate using NAD(P)(H), is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the antenna helix plays a key role to transmit the allosteric signals. In contrast, bacterial GDH was believed not to be regulated allosterically because it lacks the antenna helix. We previously reported that GDH from Thermus thermophilus (TtGDH), which is composed of two heterologous subunits, is activated by leucine. In the present study, we found that AMP activates TtGDH using a catalytically inactive APRTh as the sensory subunit. This suggests that T. thermophilus possesses a complicated regulatory mechanism of GDH to control carbon and nitrogen metabolism.

Optimized Screw Trajectory for Lumbar Cortical Bone Trajectory Pedicle Screws Based on Clinical Outcome: Evidence Favoring the Buttress Effect Theory.

BACKGROUND: Cortical bone trajectory (CBT) is a relatively new technique for pedicle screw insertion in the field of spine surgery. Previous studies have demonstrated the significantly better pullout and toggle characteristics the new method offers, and it appears to have certain advantages over the widely used traditional trajectory. The mechanism of the pullout and toggle characteristics still remains unknown. PURPOSE: To report the medium- to long-term follow-up findings of patients who underwent posterior lumbar interbody fusion or posterior lumbar fusion (PLIF/PLF) at our institution and to discuss the ideal screw trajectory when using this technique. STUDY DESIGN: Retrospective radiologic study. METHODS: Fifty-five patients who underwent PLIF/PLF for lumbar spondylosis using the new technique between January 2011 and January 2016 were included. Clinical outcome was assessed using the Japanese Orthopaedic Association scores for low back pain and visual analog scale scores. Screw loosening was evaluated via the presence of a translucent zone surrounding the pedicle screw using plain X-radiography (Xp). The screw trajectory was evaluated by measuring the rostral range (RR) and the lateral range score (LRS). The RR is the angle between the line drawn along the distal end plate of the vertebra and the line drawn along the screw on the lateral view. The LRS is the score given depending on the position of the tip of the screw seen on the Xp taken in the anteroposterior (AP) view. The pedicle ring is equally divided into the medial third (zone A), central third (zone B), and the lateral third (zone C) by four vertical lines. A score of 0 to 3 points is given depending on the position of the tip of the screw: 0, outside the pedicle ring; 1, zone A; 2, zone B; and 3, zone C. RESULTS: Bone fusion was recorded in 49 patients (49/55 patients). The total number of screws with a visible translucent zone on Xp was 26 (26/242 screws). When the screws are inserted at a more acute angle in the lateral view (low RR) or when the screws are directed more medially (low LRS), the risk of developing a translucent zone around the pedicle screw increased. CONCLUSION: With CBT, the pedicle screws are inserted from the pars interarticularis that is rich in cortical bone, strong enough to withstand the substantial forces usually encountered in the region. Our clinical data suggest that the optimal screw trajectory is one where the screw is inserted at an angle > 22.55 degrees (high RR) on the lateral view Xp and where the tip of the screw is directed toward the lateral third of the pedicle on the AP Xp. We believe the naturally occurring buttress effect that exists in the vertebra may give CBT a mechanical and anatomical advantage over the traditional screw trajectory.

[Surgical Technique for Lumbar Spinal Extraforaminal Schwannoma].

INTRODUCTION: Spinal neurinomas are frequently observed in the cauda equina, particularly within the dura mater. However, extraforaminal schwannomas(EFS), which are neither intradural nor dumbbell type, are relatively rare; hence, different surgical procedures have been employed. Here, we report 12 cases of lumbar EFS with total resection that were safely performed. METHODS AND SUBJECTS: This study was comprised of 12 patients with lumbar EFS(excluding those with neurofibromas)who were previously treated at our hospital. Using the Wiltse paraspinal approach, we inserted the microscope until it reached the tumor surface. After confirming the tumor surface, we inserted a scalpel to make an incision in a portion of the outer capsule. Then, we confirmed that the surfaces of the outer capsule and the tumor within the capsule could be dissected. Next, a thread was attached to the outer capsule and pulled upwards so that there was enough space to perform the required procedures within the capsule. Using a CUSA®, we removed the intracapsular tumor. After its removal, the outer capsule was treated carefully to ensure that the origin nerve was not damaged. RESULTS: In all cases, we successfully performed a total removal of the intracapsular tumor;in the mean postoperative observation period of 8 years, no recurrence was observed. Although temporary paralysis was observed in three patients, this improved over time. CONCLUSION: We report our experience with a surgical technique that ensured the safe removal of lumbar EFS. Satisfactory results were achieved, with no recurrences observed during the mean 8-year postoperative observation period.

Fosfomycin Biosynthesis via Transient Cytidylylation of 2-Hydroxyethylphosphonate by the Bifunctional Fom1 Enzyme.

Fosfomycin is a wide-spectrum phosphonate antibiotic that is used clinically to treat cystitis, tympanitis, etc. Its biosynthesis starts with the formation of a carbon-phosphorus bond catalyzed by the phosphoenolpyruvate phosphomutase Fom1. We identified an additional cytidylyltransferase (CyTase) domain at the Fom1 N-terminus in addition to the phosphoenolpyruvate phosphomutase domain at the Fom1 C-terminus. Here, we demonstrate that Fom1 is bifunctional and that the Fom1 CyTase domain catalyzes the cytidylylation of the 2-hydroxyethylphosphonate (HEP) intermediate to produce cytidylyl-HEP. On the basis of this new function of Fom1, we propose a revised fosfomycin biosynthetic pathway that involves the transient CMP-conjugated intermediate. The identification of a biosynthetic mechanism via such transient cytidylylation of a biosynthetic intermediate fundamentally advances the understanding of phosphonate biosynthesis in nature. The crystal structure of the cytidylyl-HEP-bound CyTase domain provides a basis for the substrate specificity and reveals unique catalytic elements not found in other members of the CyTase family.

Onset and remodeling of coronal imbalance after selective posterior thoracic fusion for Lenke 1C and 2C adolescent idiopathic scoliosis (a pilot study).

BACKGROUND: Postoperative coronal imbalance is a significant problem after selective thoracic fusion for primary thoracic and compensatory lumbar curves in adolescent idiopathic scoliosis (AIS). However, longitudinal studies on postoperative behavior of coronal balance are lacking. This multicenter retrospective study was conducted to analyze factors related to onset and remodeling of postoperative coronal imbalance after posterior thoracic fusion for Lenke 1C and 2C AIS. METHODS: Twenty-one Lenke 1C or 2C AIS patients, who underwent posterior thoracic fusion ending at L3 or above, were included with a minimum 2-year follow-up. The mean patients' age was 15.1 years at the time of surgery. Radiographic measurements were performed on Cobb angles of the main thoracic (MT) and thoracolumbar/lumbar (TLL) curves and coronal balance. Factors related to the onset of immediately postoperative coronal decompensation (IPCD) and postoperative coronal balance remodeling (PCBR), defined as an improvement of coronal balance during postoperative follow-up, were investigated using comparative and correlation analyses. RESULTS: Mean Cobb angles for the MT and TLL curves were 57.3° and 42.3° preoperatively and were corrected to 22.8° and 22.5° at final follow-up, respectively. Mean preoperative coronal balance of -3.8 mm got worse to -21.2 mm postoperatively, and regained to -12.0 mm at final follow-up. Coronal decompensation was observed in two patients preoperatively, in ten patients immediately postoperatively, and in three patients at final follow-up. The preoperative coronal balance and lowest instrumented vertebra (LIV) selection relative to stable vertebra (SV) were significantly different between patients with IPCD and those without. PCBR had significantly negative correlation with immediately postoperative coronal balance. CONCLUSIONS: IPCD after posterior thoracic fusion for Lenke 1C and 2C AIS was frequent and associated with preoperative coronal balance and LIV selection. However, most patients with IPCD regained coronal balance through PCBR, which was significantly associated with immediately postoperative coronal balance. A fixation more distal to SV shifted the coronal balance further to the left postoperatively.

Characterization of bafilomycin biosynthesis in Kitasatospora setae KM-6054 and comparative analysis of gene clusters in Actinomycetales microorganisms.

Bafilomycins A1, C1 and B1 (setamycin) produced by Kitasatospora setae KM-6054 belong to the plecomacrolide family, which exhibit antibacterial, antifungal, antineoplastic and immunosuppressive activities. An analysis of gene clusters from K. setae KM-6054 governing the biosynthesis of bafilomycins revealed that it contains five large open reading frames (ORFs) encoding the multifunctional polypeptides of bafilomycin polyketide synthases (PKSs). These clustered PKS genes, which are responsible for bafilomycin biosynthesis, together encode 11 homologous sets of enzyme activities, each catalyzing a specific round of polyketide chain elongation. The region contains an additional 13 ORFs spanning a distance of 73 287 bp, some of which encode polypeptides governing other key steps in bafilomycin biosynthesis. Five ORFs, BfmB, BfmC, BfmD, BfmE and BfmF, were involved in the formation of methoxymalonyl-acyl carrier protein (ACP). Two possible regulatory genes, bfmR and bfmH, were found downstream of the above genes. A gene-knockout analysis revealed that BfmR was only a transcriptional regulator for the transcription of bafilomycin biosynthetic genes. Two genes, bfmI and bfmJ, were found downstream of bfmH. An analysis of these gene-disruption mutants in addition to an enzymatic analysis of BfmI and BfmJ revealed that BfmJ activated fumarate and BfmI functioned as a catalyst to form a fumaryl ester at the C21 hydroxyl residue of bafilomycin A1. A comparative analysis of bafilomycin gene clusters in K. setae KM-6054, Streptomyces lohii JCM 14114 and Streptomyces griseus DSM 2608 revealed that each ORF of both gene clusters in two Streptomyces strains were quite similar to each other. However, each ORF of gene cluster in K. setae KM-6054 was of lower similarity to that of corresponding ORF in the two Streptomyces species.

The hematopoietic cell-specific transcription factor PU.1 is critical for expression of CD11c.

PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family, which plays an important role in the development of dendritic cells (DCs). CD11c (encoded by Itgax) is well established as a characteristic marker of hematopoietic lineages including DCs. In the present study, we analyzed the role of PU.1 (encoded by Spi-1) in the expression of CD11c. When small interfering RNA (siRNA) for Spi-1 was introduced into bone marrow-derived DCs (BMDCs), the mRNA level and cell surface expression of CD11c were dramatically reduced. Using reporter assays, the TTCC sequence at -56/-53 was identified to be critical for PU.1-mediated activation of the promoter. An EMSA showed that PU.1 directly bound to this region. ChIP assays demonstrated that a significant amount of PU.1 bound to this region on chromosomal DNA in BMDCs, which was decreased in LPS-stimulated BMDCs in accordance with the reduced levels of mRNAs of Itgax and Spi-1, and the histone acetylation degree. Enforced expression of exogenous PU.1 induced the expression of the CD11c protein on the cell surface of mast cells, whereas control transfectants rarely expressed CD11c. Quantitative RT-PCR also showed that the expression of a transcription factor Irf4, which is a partner molecule of PU.1, was reduced in PU.1-knocked down BMDCs. IRF4 transactivated the Itgax gene in a synergistic manner with PU.1. Taken together, these results indicate that PU.1 functions as a positive regulator of CD11c gene expression by directly binding to the Itgax promoter and through transactivation of the Irf4 gene.

Multi-phasic bi-directional chemotactic responses of the growth cone.

The nerve growth cone is bi-directionally attracted and repelled by the same cue molecules depending on the situations, while other non-neural chemotactic cells usually show uni-directional attraction or repulsion toward their specific cue molecules. However, how the growth cone differs from other non-neural cells remains unclear. Toward this question, we developed a theory for describing chemotactic response based on a mathematical model of intracellular signaling of activator and inhibitor. Our theory was first able to clarify the conditions of attraction and repulsion, which are determined by balance between activator and inhibitor, and the conditions of uni- and bi-directional responses, which are determined by dose-response profiles of activator and inhibitor to the guidance cue. With biologically realistic sigmoidal dose-responses, our model predicted tri-phasic turning response depending on intracellular Ca2+ level, which was then experimentally confirmed by growth cone turning assays and Ca2+ imaging. Furthermore, we took a reverse-engineering analysis to identify balanced regulation between CaMKII (activator) and PP1 (inhibitor) and then the model performance was validated by reproducing turning assays with inhibitions of CaMKII and PP1. Thus, our study implies that the balance between activator and inhibitor underlies the multi-phasic bi-directional turning response of the growth cone.

Postoperative behavior of thoracolumbar/lumbar curve and coronal balance after posterior thoracic fusion for Lenke 1C and 2C adolescent idiopathic scoliosis.

BACKGROUND: Controversy still exists around surgical strategies for Lenke type 1C and 2C curves with primary thoracic and compensatory lumbar curves in adolescent idiopathic scoliosis (AIS). The benefit of selective thoracic fusion (STF) for these curve types is spontaneous lumbar curve correction while saving more mobile lumbar segments. However, a risk of postoperative coronal decompensation after STF has also been reported. This multicenter retrospective study was conducted to evaluate postoperative behavior of thoracolumbar/lumbar (TLL) curve and coronal balance after posterior thoracic fusion for Lenke 1C and 2C AIS. METHODS: Twenty-four Lenke 1C and 2C AIS patients who underwent posterior thoracic fusion were included. The mean age of patients was 15.7 years old at time of surgery. Constructs used for surgery in all cases were pedicle screw constructs ending at L3 or above. Radiographic measurements were performed on Cobb angles of the main thoracic and TLL curves and coronal balance. Factors related to final Cobb angle of TLL curve and postoperative change of coronal balance were investigated. RESULTS: Mean Cobb angles for main thoracic and TLL curves were 59.0° and 43.9° preoperatively, and were corrected to 21.5° and 22.0° at final follow-up, respectively. Mean coronal balance was -5.6 mm preoperatively and was corrected to -14.6 mm at final follow-up. Final Cobb angle of TLL curve was significantly correlated with immediate postoperative Cobb angle of main thoracic curve and tilt of lowest instrumented vertebra (LIV). Postoperative change of coronal balance was significantly correlated with selection of LIV relative to stable vertebra. CONCLUSION: Spontaneous correction of TLL curve occurred consistently by correcting the main thoracic curve and making the LIV more horizontal after posterior thoracic fusion for Lenke 1C and 2C AIS. The more distal fixation to stable vertebra resulted in coronal balance shifting more to the left postoperatively.

Two ATP-binding cassette transporters involved in (S)-2-aminoethyl-cysteine uptake in thermus thermophilus.

Thermus thermophilus exhibits hypersensitivity to a lysine analog, (S)-2-aminoethyl-cysteine (AEC). Cosmid libraries were constructed using genomes from two AEC-resistant mutants, AT10 and AT14, and the cosmids that conferred AEC resistance on the wild-type strain were isolated. When the cosmid library for mutant AT14 was screened, two independent cosmids, conferring partial AEC resistance to the wild type, were obtained. Two cosmids carried a common genomic region from TTC0795 to TTC0810. This region contains genes encoding an ATP-binding cassette (ABC) transporter consisting of TTC0806/TTC0795, using TTC0807 as the periplasmic substrate-binding protein. Sequencing revealed that AT14 carries mutations in TTC0795 and TTC0969, causing decreases in the thermostability of the products. TTC0969 encodes the nucleotide-binding protein of a different ABC transporter consisting of TTC0967/TTC0968/TTC0969/TTC0970 using TTC0966 as the periplasmic substrate-binding protein. By similar screening for cosmids constructed for the mutant AT10, mutations were found at TTC0807 and TTC0969. Mutation in either of the transporter components gave partial resistance to AEC in the wild-type strain, while mutations of both transporters conferred complete AEC resistance. This result indicates that both transporters are involved in AEC uptake in T. thermophilus. To elucidate the mechanism of AEC uptake, crystal structures of TTC0807 were determined in several substrate-binding forms. The structures revealed that TTC0807 recognizes various basic amino acids by changing the side-chain conformation of Glu19, which interacts with the side-chain amino groups of the substrates.

Diagnosis and surgical strategy for sacral meningeal cysts with check-valve mechanism: technical note.

OBJECTIVE: There is agreement that symptomatic sacral meningeal cysts with a check-valve mechanism and/or large cysts representing space-occupying lesions should be treated surgically. This study investigated factors indicating a need for surgical intervention and surgical techniques for sacral meningeal cysts with a check-valve mechanism. METHODS: In ten patients presenting with sciatica and neurological deficits, myelography, computed tomography (CT) myelography, and magnetic resonance imaging (MR imaging) detected sacral meningeal cysts with a check-valve mechanism. One patient had two primary cysts. Ten cysts were type 2 and one cyst was type 1. Nine of the ten patients had not undergone previous surgery, while the remaining case involved recurrent cyst. For the seven patients with normal (i.e., not huge or recurrent) type 2 cysts and no previous surgery (eight cysts), suture after collapse of the cyst wall was performed. For the recurrent type 2 cyst, duraplasty and suture with collapse of the cyst wall were performed to eliminate the check-valve mechanism. For the remaining type 2 cyst, a primary root was sacrificed because of the huge size of the cyst. For the type 1 cyst, the neck of the cyst was ligated. RESULTS: In all cases, chief complaints disappeared immediately postoperatively and no deterioration of clinical symptoms has been seen after a mean follow-up of 27 months. CONCLUSIONS: The presence or absence of a check-valve mechanism is very important in determining the need for surgical intervention for sacral meningeal cysts.

Transcriptional regulation of the mouse CD11c promoter by AP-1 complex with JunD and Fra2 in dendritic cells.

CD11c, a member of the ß(2) integrin family of adhesion molecule, is expressed on the surface of myeloid lineages and activated lymphoid cells and forms a heterodimeric receptor with CD18. We analyzed the mouse CD11c promoter structure to elucidate the transcriptional regulation in dendritic cells (DCs). By reporter assay, the -84/-65 region was identified to be essential for activity of the mouse CD11c promoter in the mouse bone marrow-derived (BM) DCs and monocyte cell line RAW264.7. An electrophoretic mobility shift assay using a number of antibodies against transcription factors revealed that the target region was recognized by a complex including JunD and Fra2, which are transcription factors belonging to the AP-1 family. The direct interaction of JunD and Fra2 with the CD11c promoter was further confirmed by a chromatin immunoprecipitation assay using CD11c-positive cells purified from BMDCs. Finally, mouse JunD and/or Fra2 siRNA was introduced into BMDCs to evaluate the involvement of these factors against CD11c transcription and found that Fra2 siRNA reduced cell surface expression level of CD11c. These results indicate that AP-1 composed with JunD and Fra2 protein plays a primary role in enhancing the transcription level of the CD11c gene in DC.