Multi-pose-based convolutional neural network model for diagnosis of patients with central lumbar spinal stenosis.

Although the role of plain radiographs in diagnosing lumbar spinal stenosis (LSS) has declined in importance since the advent of magnetic resonance imaging (MRI), diagnostic ability of plain radiographs has improved dramatically when combined with deep learning. Previously, we developed a convolutional neural network (CNN) model using a radiograph for diagnosing LSS. In this study, we aimed to improve and generalize the performance of CNN models and overcome the limitation of the single-pose-based CNN (SP-CNN) model using multi-pose radiographs. Individuals with severe or no LSS, confirmed using MRI, were enrolled. Lateral radiographs of patients in three postures were collected. We developed a multi-pose-based CNN (MP-CNN) model using the encoders of the three SP-CNN model (extension, flexion, and neutral postures). We compared the validation results of the MP-CNN model using four algorithms pretrained with ImageNet. The MP-CNN model underwent additional internal and external validations to measure generalization performance. The ResNet50-based MP-CNN model achieved the largest area under the receiver operating characteristic curve (AUROC) of 91.4% (95% confidence interval [CI] 90.9-91.8%) for internal validation. The AUROC of the MP-CNN model were 91.3% (95% CI 90.7-91.9%) and 79.5% (95% CI 78.2-80.8%) for the extra-internal and external validation, respectively. The MP-CNN based heatmap offered a logical decision-making direction through optimized visualization. This model holds potential as a screening tool for LSS diagnosis, offering an explainable rationale for its prediction.

Screening patients requiring secondary lumbar surgery for degenerative lumbar spine diseases: a nationwide sample cohort study.

This study aims to identify healthcare costs indicators predicting secondary surgery for degenerative lumbar spine disease (DLSD), which significantly impacts healthcare budgets. Analyzing data from the National Health Insurance Service-National Sample Cohort (NHIS-NSC) database of Republic of Korea (ROK), the study included 3881 patients who had surgery for lumbar disc herniation (LDH), lumbar spinal stenosis without spondylolisthesis (LSS without SPL), lumbar spinal stenosis with spondylolisthesis (LSS with SPL), and spondylolysis (SP) from 2006 to 2008. Patients were categorized into two groups: those undergoing secondary surgery (S-group) and those not (NS-group). Surgical and interim costs were compared, with S-group having higher secondary surgery costs ($1829.59 vs $1618.40 in NS-group, P = 0.002) and higher interim costs ($30.03; 1.86% of initial surgery costs vs $16.09; 0.99% of initial surgery costs in NS-group, P < 0.0001). The same trend was observed in LDH, LSS without SPL, and LSS with SPL (P < 0.0001). Monitoring interim costs trends post-initial surgery can effectively identify patients requiring secondary surgery.

The iterative implementation of a comprehensive enhanced recovery after surgery protocol in all spinal surgery in Korea: a comparative analysis of clinical outcomes and medical costs between primary spinal tumors and degenerative spinal diseases.

OBJECTIVE: Most studies on the enhanced recovery after surgery (ERAS) protocol in spine surgery have focused on patients with degenerative spinal diseases (DSDs), resulting in a lack of evidence for a comprehensive ERAS protocol applicable to patients with primary spine tumors (PSTs) and other spinal diseases. The authors had developed and gradually adopted components of the comprehensive ERAS protocol for all spine surgical procedures from 2003 to 2011, and then the current ERAS protocol was fully implemented in 2012. This study aimed to evaluate the impact and the applicability of the comprehensive ERAS protocol across all spine surgical procedures and to compare outcomes between the PST and DSD groups. METHODS: Adult spine surgical procedures were conducted from 2003 to 2021 at the Seoul National University Hospital Spine Center and data were retrospectively reviewed. The author divided the study periods into the developing ERAS (2003-2011) and post-current ERAS (2012-2021) periods, and outcomes were compared between the two periods. Surgical procedures for metastatic cancer, infection, and trauma were excluded. Interrupted time series analysis (ITSA) was used to assess the impact of the ERAS protocol on medical costs and clinical outcomes, including length of stay (LOS) and rates of 30-day readmission, reoperation, and surgical site infection (SSI). Subgroup analyses were conducted on the PST and DSD groups in terms of LOS and medical costs. RESULTS: The study included 7143 surgical procedures, comprising 1494 for PSTs, 5340 for DSDs, and 309 for other spinal diseases. After ERAS protocol implementation, spine surgical procedures showed significant reductions in LOS and medical costs by 22% (p = 0.008) and 22% (p < 0.001), respectively. The DSD group demonstrated a 16% (p < 0.001) reduction in LOS, whereas the PST group achieved a 28% (p < 0.001) reduction, noting a more pronounced LOS reduction in PST surgical procedures (p = 0.003). Medical costs decreased by 23% (p < 0.001) in the DSD group and 12% (p = 0.054) in the PST group, with a larger cost reduction for DSD surgical procedures (p = 0.021). No statistically significant differences were found in the rates of 30-day readmission, reoperation, and SSI between the developing and post-current ERAS implementation periods (p = 0.65, p = 0.59, and p = 0.52, respectively). CONCLUSIONS: Comprehensive ERAS protocol implementation significantly reduced LOS and medical costs in all spine surgical procedures, while maintaining comparable 30-day readmission, reoperation, and SSI rates. These findings suggest that the ERAS protocol is equally applicable to all spine surgical procedures, with a more pronounced effect on reducing LOS in the PST group and on reducing medical costs in the DSD group.

The precise midline myelotomy through anatomical posterior median septum by dissecting dorsal column in microsurgical resection of ependymoma (2-dimensional operative video).

Although resection is the gold standard treatment for spinal ependymoma, permanent neurological deterioration has been reported postoperatively in 20%-27% of patients. Despite thorough dissection of the tumor from its surroundings, conventional longitudinally directed midline myelotomy can lead to injury to the dorsal column, possibly due to deformation of the posterior median septum as the tumor grows. To address this issue, the authors have been performing precise midline myelotomy through the anatomical posterior median septum by directly dissecting the dorsal column. This video presents the principles and application of this technique.

Surgical treatment of spondylolisthesis by oblique lumbar interbody fusion and transpedicular screw fixation: Comparison between conventional double position versus navigation-assisted single lateral position.

BACKGROUND AND OBJECTIVES: Oblique lumbar interbody fusion (OLIF) procedures involve anterior insertion of interbody cage in lateral position. Following OLIF, insertion of pedicle screws and rod system is performed in a prone position (OLIF-con). The location of the cage is important for restoration of lumbar lordosis and indirect decompression. However, inserting the cage at the desired location is difficult without reduction of spondylolisthesis, and reduction after insertion of interbody cage may limit the amount of reduction. Recent introduction of spinal navigation enabled both surgical procedures in one lateral position (OLIF-one). Therefore, reduction of spondylolisthesis can be performed prior to insertion of interbody cage. The objective of this study was to compare the reduction of spondylolisthesis and the placement of cage between OLIF-one and OLIF-con. METHODS: We retrospectively reviewed 72 consecutive patients with spondylolisthesis for this study; 30 patients underwent OLIF-one and 42 underwent OLIF-con. Spinal navigation system was used for OLIF-one. In OLIF-one, the interbody cage was inserted after reducing spondylolisthesis, whereas in OLIF-con, the cage was inserted before reduction. The following parameters were measured on X-rays: pre- and postoperative spondylolisthesis slippage, reduction degree, and the location of the cage in the disc space. RESULTS: Both groups showed significant improvement in back and leg pains (p < .05). Transient motor or sensory changes occurred in three patients after OLIF-con and in two patients after OLIF-one. Pre- and postoperative slips were 26.3±7.7% and 6.6±6.2% in OLIF-one, and 23.1±7.0% and 7.4±5.8% in OLIF-con. The reduction of slippage was 74.4±6.3% after OLIF-one and 65.4±5.7% after OLIF-con, with a significant difference between the two groups (p = .04). The cage was located at 34.2±8.9% after OLIF-one and at 42.8±10.3% after OLIF-con, with a significant difference between the two groups (p = .004). CONCLUSION: Switching the sequence of surgical procedures with OLIF-one facilitated both the reduction of spondylolisthesis and the placement of the cage at the desired location.

Impact of C3 laminectomy on cervical sagittal alignment in cervical laminoplasty: a prospective, randomized controlled trial comparing clinical and radiological outcomes between C3 laminectomy with C4-C6 laminoplasty and C3-C6 laminoplasty.

BACKGROUND CONTEXT: C3 laminectomy in cervical laminoplasty is a modified laminoplasty technique that can preserve the semispinalis cervicis muscle attached to the C2 spinous process. Several previous studies have shown that this technique can lead to better outcomes of postoperative axial neck pain and C2-C3 range of motion (ROM) than conventional cervical laminoplasty. However, there is still a lack of understanding of total and proportional postoperative cervical sagittal alignment outcomes. PURPOSE: To assess the effects of C3 laminectomy in cervical laminoplasty on postoperative cervical alignment and clinical outcomes. DESIGN: A single-center, patient-blinded, randomized controlled trial. PATIENT SAMPLE: We included consecutive 126 patients diagnosed with cervical spondylotic myelopathy (CSM) or ossification of posterior longitudinal ligament (OPLL) who were scheduled for cervical laminoplasty from March 2017 to January 2020. OUTCOME MEASURES: The primary outcome measures were C2-C7 Cobb angle (CA) and neck disability index (NDI). Secondary outcomes measures included other clinical outcomes and radiographic parameters including segmental Cobb angle and presence of C2-C3 interlaminar fusion. METHODS: Patients were randomly allocated to either the C3 laminectomy with C4-C6 laminoplasty group (LN group) or the C3-C6 laminoplasty group (LP group) at a 1:1 ratio. Laminoplasty was performed using a unilateral open-door technique and stabilized with titanium mini plates. A linear mixed model analysis was employed to examine the longitudinal data from postoperative 1-year through 3-year. Additional analysis between three types of cervical sagittal alignment morphology was done. RESULTS: Among 122 patients who were randomly allocated to one of two groups (LN group, n=61; LP group, n=61), modified intent-to-treat analysis was done for 109 patients (LN group, n=51, LP group, n=58) who had available at least a year of postoperative data. Postoperative C2-C7 CA was not significantly different between the two groups. However, NDI was significantly different between the two groups (12.8±1.0 in the LN group vs 8.6±1.0 in LP group, p=.005), which exceeded the minimum clinically important difference (MCID). The postoperative C2-C3 CA was significantly greater in the LN group (7.1±0.5° in LN group vs 3.2±0.5° in LP group, p<.001) while C4-C7 CA was significantly smaller in the LN group (3.9±0.8° in LN group vs 7.7±0.7° in LP group, p<.001) with greater cSVA in the LN group (31.6±1.4 mm in LN group vs 25.5±1.3 mm in LP group at postoperative 3-year, p=.002). Postoperative Euro-Quality of Life-5 Dimension (EQ-5D), numerical rating scores for neck pain (NRS-N) were significantly better in the LP group than in the LN group (all p<.05) and only EQ-5D surpassed the MCID. The C2-C3 fusion rate was significantly different between the LN group (9.8%) and the LP group (44.8%) (p<.001). The LN group showed a higher prevalence of a specific cervical alignment morphology characterized by a sigmoid shape with proximal lordosis and distal kyphosis (S curve). This S curve demonstrated significantly unfavorable outcomes across multiple outcome variables. CONCLUSION: The impact of C3 laminectomy in cervical laminoplasty on postoperative kyphosis among patients with CSM or OPLL did not significantly differ from that of C3-C6 laminoplasty. However, C3 laminectomy in cervical laminoplasty might result in an unfavorable clinical outcome with an unbalanced cervical sagittal alignment characterized by a sigmoid shape with proximal lordosis and distal kyphosis.

Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates.

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNAArg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNAArg, and a 3'-proximal segment of the tRNAArg moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe3+-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.

Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex.

Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N1- and N3-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications.

Surgical Outcomes of Thalamic Tumors in Children: The Importance of Diffusion Tensor Imaging, Neuro-Navigation and Intraoperative Neurophysiological Monitoring.

BACKGROUND: Recently, modern technology such as diffusion tensor imaging (DTI), neuro-navigation and intraoperative neurophysiological monitoring (IOM) have been actively adopted for the treatment of thalamic tumors. We evaluated surgical outcomes and efficacy of the aforementioned technologies for the treatment of pediatric thalamic tumors. METHODS: We retrospectively reviewed clinical data from 37 children with thalamic tumors between 2004 and 2017. There were 44 operations (27 tumor resections, 17 biopsies). DTI was employed in 17 cases, neuro-navigation in 23 cases and IOM in 14 cases. All diagnoses were revised according to the 2016 World Health Organization Classification of Tumors of the Central Nervous System. Progression-free survival (PFS) and overall survival (OS) rates were calculated, and relevant prognostic factors were analyzed. The median follow-up duration was 19 months. RESULTS: Fifteen cases were gross total resections (GTR), 6 subtotal resections (STR), and 6 partial resections (PR). Neurological status did not worsen after 22 tumor resections. There were statistically significant differences in terms of the extent of resection between the groups with DTI, neuro-navigation and IOM (n=12, GTR or STR=12) and the group without at least one of the three techniques (n=15, GTR or STR=9, p=0.020). The mean PFS was 87.2±38.0 months, and the mean OS 90.7±36.1 months. The 5-year PFS was 37%, and the 5-year OS 47%. The histological grade (p≤0.001) and adjuvant therapy (done vs. not done, p=0.016) were significantly related to longer PFS. The histological grade (p=0.002) and the extent of removal (GTR/STR vs. PR/biopsy, p=0.047) were significantly related to longer OS. CONCLUSION: Maximal surgical resection was achieved with acceptable morbidity in children with thalamic tumors by employing DTI, neuro-navigation and IOM. Maximal tumor resection was a relevant clinical factor affecting OS; therefore, it should be considered the initial therapeutic option for pediatric thalamic tumors.

The C-terminal region of ATG101 bridges ULK1 and PtdIns3K complex in autophagy initiation.

The initiation of macroautophagy/autophagy is tightly regulated by the upstream ULK1 kinase complex, which affects many downstream factors including the PtdIns3K complex. The phosphorylation of the right position at the right time on downstream molecules is governed by proper complex formation. One component of the ULK1 complex, ATG101, known as an accessory protein, is a stabilizer of ATG13 in cells. The WF finger region of ATG101 plays an important role in the recruitment of WIPI1 (WD repeat domain, phosphoinositide interacting protein 1) and ZFYVE1 (zinc finger FYVE-type containing 1). Here, we report that the C-terminal region identified in the structure of the human ATG101-ATG13HORMA complex is responsible for the binding of the PtdIns3K complex. This region adopts a ß-strand conformation in free ATG101, but either an α-helix or random coil in our ATG101-ATG13HORMA complex, which protrudes from the core and interacts with other molecules. The C-terminal deletion of ATG101 shows a significant defect in the interaction with PtdIns3K components and subsequently impairs autophagosome formation. This result clearly presents an additional role of ATG101 for bridging the ULK1 and PtdIns3K complexes in the mammalian autophagy process. Abbreviations: ATG: autophagy related; BECN1: beclin 1; GFP: green fluorescent protein; HORMA: Hop1p/Rev7p/MAD2; HsATG13HORMA: HORMA domain of ATG13 from Homo sapiens; KO: knockout; MAD2: mitotic arrest deficient 2 like 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K: phosphatidylinositol 3-kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SAXS: small-angle X-ray scattering; ScAtg13HORMA: HORMA domain of Atg13 from Sccharomyces cerevisiae; SEC-SAXS: size-exclusion chromatography with small-angle X-ray scattering; SpAtg13HORMA: HORMA domain of Atg13 from Schizosaccharomyces pombe; SQSTM1/p62: sequestosome 1; ULK1: unc51-like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; WIPI1: WD repeat domain: phosphoinositide interacting 1; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

Intracranial neurenteric cyst arising at the suprasellar cistern with extension to middle cranial fossa.

INTRODUCTION: Intracranial neurenteric cysts are rare and are not often found in the supratentorial space. We present a case of an intracranial neurenteric cyst that arose at the suprasellar cistern with extension to the middle cranial fossa. HISTORY: A 3-year-old girl presented with seizure. Neuroimaging showed a huge extra-axial cyst involving the suprasellar cistern and left middle cranial fossa. Under the impression of an arachnoid cyst, she underwent fenestration of the cyst with basal cisterns and ventricle. The pathologic diagnosis was neurenteric cyst. Four years after the surgery, MRI showed no recurrence but did show poorly developed myelination of the left temporal white matter. Three years after surgery, seizures recurred. We consider epilepsy surgery in case of uncontrolled seizure. DISCUSSION: We discuss the unique case of supratentorially located neurenteric cyst that is just treated by fenestration. A close follow-up is needed for children with intracranial neurenteric cysts to surveil not only recurrence of lesion but also normal brain development.

A novel conformation of the LC3-interacting region motif revealed by the structure of a complex between LC3B and RavZ.

LC3-family member proteins play a critical role in autophagy, a cellular process responsible for the degradation of massive cellular components including intracellular pathogens. A variety of molecules involved in the autophagic pathway engage in specific interactions with a unique sequence motif referred to as the LIR (LC3-interacting region) motif. Although identification of conserved structural features of LIR motifs in complex with LC3-family members has established a canonical LIR motif, atypical conformations of LIR motifs have recently been revealed. Here, we determined the three-dimensional crystal structures of LC3B in complex with three different LIR motifs of RavZ from Legionella pneumophila, an intracellular pathogen that can manipulate the host autophagy system. The tandem LIR motifs located in the N-terminal region of RavZ adopt a novel ß-sheet conformation and thus provide specific ionic interactions with LC3B in addition to canonical hydrophobic plugged-in interactions. Consequently, these motifs possess higher binding affinity to LC3-family members than canonical LIR motifs, although the tandem repeats can only bind to one LC3 molecule. These findings broaden our understanding of the functional repertoire of LIR motifs in autophagy.

ACCORD: an assessment tool to determine the orientation of homodimeric coiled-coils.

The coiled-coil (CC) domain is a very important structural unit of proteins that plays critical roles in various biological functions. The major oligomeric state of CCs is a dimer, which can be either parallel or antiparallel. The orientation of each α-helix in a CC domain is critical for the molecular function of CC-containing proteins, but cannot be determined easily by sequence-based prediction. We developed a biochemical method for assessing differences between parallel and antiparallel CC homodimers and named it ACCORD (Assessment tool for homodimeric Coiled-Coil ORientation Decision). To validate this technique, we applied it to 15 different CC proteins with known structures, and the ACCORD results identified these proteins well, especially with long CCs. Furthermore, ACCORD was able to accurately determine the orientation of a CC domain of unknown directionality that was subsequently confirmed by X-ray crystallography and small angle X-ray scattering. Thus, ACCORD can be used as a tool to determine CC directionality to supplement the results of in silico prediction.

Swapping of interaction partners with ATG5 for autophagosome maturation.

Autophagy is a tightly regulated lysosome-mediated catabolic process in eukaryotes that maintains cellular homeostasis. A distinguishable feature of autophagy is the formation of double-membrane structures, autophagosome, which envelopes the intracellular cargoes and finally degrades them by fusion with lysosomes. So far, many structures of Atg proteins working on the autophagosome formation have been reported, however those involved in autophagosome maturation, a fusion with lysosome, are relatively unknown. One of the molecules in autophagosome maturation, TECPR1, has been identified and recently, structural studies on both ATG5-TECPR1 and ATG5-ATG16L1 complexes revealed that TECPR1 and ATG16L1 share the same binding site on ATG5. These results, in combination with supporting biochemical and cellular biological data, provide an insight into a model for swapping ATG5 partners for autophagosome maturation.

Insights into autophagosome maturation revealed by the structures of ATG5 with its interacting partners.

Autophagy is a bulky catabolic process that responds to nutrient homeostasis and extracellular stress signals and is a conserved mechanism in all eukaryotes. When autophagy is induced, cellular components are sequestered within an autophagosome and finally degraded by subsequent fusion with a lysosome. During this process, the ATG12-ATG5 conjugate requires 2 different binding partners, ATG16L1 for autophagosome elongation and TECPR1 for lysosomal fusion. In our current study, we describe the crystal structures of human ATG5 in complex with an N-terminal domain of ATG16L1 as well as an internal AIR domain of TECPR1. Both binding partners exhibit a similar α-helical structure containing a conserved binding motif termed AFIM. Furthermore, we characterize the critical role of the C-terminal unstructured region of the AIR domain of TECPR1. These findings are further confirmed by biochemical and cell biological analyses. These results provide new insights into the molecular details of the autophagosome maturation process, from its elongation to its fusion with a lysosome.

Structural basis for recognition of autophagic receptor NDP52 by the sugar receptor galectin-8.

Infectious bacteria are cleared from mammalian cells by host autophagy in combination with other upstream cellular components, such as the autophagic receptor NDP52 and sugar receptor galectin-8. However, the detailed molecular basis of the interaction between these two receptors remains to be elucidated. Here, we report the biochemical characterization of both NDP52 and galectin-8 as well as the crystal structure of galectin-8 complexed with an NDP52 peptide. The unexpected observation of nicotinamide adenine dinucleotide located at the carbohydrate-binding site expands our knowledge of the sugar-binding specificity of galectin-8. The NDP52-galectin-8 complex structure explains the key determinants for recognition on both receptors and defines a special orientation of N- and C-terminal carbohydrate recognition domains of galectin-8. Dimeric NDP52 forms a ternary complex with two monomeric galectin-8 molecules as well as two LC3C molecules. These results lay the groundwork for understanding how host cells target bacterial pathogens for autophagy.

Structure of the autophagic E2 enzyme Atg10.

Autophagy is a regulated degradation pathway that plays a critical role in all eukaryotic life cycles. One interesting feature of the core autophagic process, autophagosome formation, is similar to ubiquitination. One of two autophagic E2 enzymes, Atg10, interacts with Atg7 to receive Atg12, a ubiquitin-like molecule, and is also involved in the Atg12-Atg5 conjugation reaction. To date, no information on the interaction between Atg10 and Atg7 has been reported, although structural information is available pertaining to the individual components. Here, the crystal structure of Atg10 from Saccharomyces cerevisiae is described at 2.7 Å resolution. A significant improvement of the diffraction limit by heavy-atom derivatization was essential for structure determination. The core fold of yeast Atg10 is well conserved compared with those of Atg3 and other E2 enzymes. In contrast to other E2 enzymes, however, the autophagic E2 enzymes Atg3 and Atg10 possess insertion regions in the middle of the core fold and may be involved in protein function. The missing segment, which was termed the `FR-region', in Atg10 may be important for interaction with the E1 enzyme Atg7. This study provides a framework for understanding the E2 conjugation reaction in autophagy.

High glucose inhibits gene expression of tyrosyl-tRNA synthetase in osteoblast cells.

It has been suggested that bone metabolism disorders are one of the major complications of diabetes mellitus. However, the exact mechanisms as to how diabetes affects bone metabolism are yet to be determined. In the present study, we have searched for high glucose regulated genes in osteoblast-like UMR-106 cells. UMR-106 cells were treated with normal glucose (5.5 mM), high glucose (16.5 mM or 30.5 mM) and mannitol (16.5 mM) as a hyperosmotic control. Following the isolation of total RNA, GeneFishing differential display-PCR (DDPCR) was carried out and followed by cloning, sequencing and searching in a gene bank data base to identify the high glucose induced gene(s). Through the DD-PCR technique which employs Annealing Control Primer, or ACP, it has been found that expression of a PCR product was significantly decreased by high glucose treatment: it was identified as tyrosyl-tRNA synthetase. Furthermore, reverse transcriptase PCR analysis confirmed that high glucose significantly decreases mRNA expression of tyrosyl-tRNA synthetase, whereas mannitol treatment does not cause any change in such expression. These results suggest that high glucose may play a significant role in the protein synthesis process of osteoblast cells by decreasing expression of tyrosyl-tRNA synthetase. In a Western blot analysis, the protein expression of tyrosyl-tRNA synthetase was also decreased by high glucose treatment. Taken together, these results suggest that high glucose could affect bone metabolism by regulating the expression of tyrosyl-tRNA synthetase genes.