Structural basis for plant lutein biosynthesis from α-carotene.

Two cytochrome P450 enzymes, CYP97A3 and CYP97C1, catalyze hydroxylations of the ß- and ε-rings of α-carotene to produce lutein. Chirality is introduced at the C-3 atom of both rings, and the reactions are both pro-3R-stereospecific. We determined the crystal structures of CYP97A3 in substrate-free and complex forms with a nonnatural substrate and the structure of CYP97C1 in a detergent-bound form. The structures of CYP97A3 in different states show the substrate channel and the structure of CYP97C1 bound with octylthioglucoside confirms the binding site for the carotenoid substrate. Biochemical assays confirm that the ferredoxin-NADP+ reductase (FNR)-ferredoxin pair is used as the redox partner. Details of the pro-3R stereospecificity are revealed in the retinal-bound CYP97A3 structure. Further analysis indicates that the CYP97B clan bears similarity to the ß-ring-specific CYP97A clan. Overall, our research describes the molecular basis for the last steps of lutein biosynthesis.

CircFOXK2 Promotes the Progression of Osteoarthritis by Regulating the miR-4640-5p/NOTCH2 Axis.

OBJECTIVE: Osteoarthritis (OA) is the most common age-related chronic and disabling joint disease, frequently causing pain and disability in the adult population. Given that there are no proven disease-modifying drugs for OA, it is urgent to gain a deeper understanding of OA pathogenesis. This study intended to uncover the circFOXK2 regulation in OA. METHODS: Firstly, in vitro OA cell model was constructed by treating murine chondrocytes with interleukin (IL)-1ß. Then, a series of functional assays were conducted to evaluate the effect of circFOXK2 on OA progression in murine chondrocytes. Bioinformatics analysis and mechanism investigations were performed to investigate the competitive endogenous RNA (ceRNA) network of circFOXK2 in OA. RESULTS: CircFOXK2 is overexpressed in IL-1ß-treated chondrocyte. We confirmed the cyclic structure and cytoplasmic distribution of circFOXK2. Functionally, circFOXK2 promotes chondrocyte apoptosis and extracellular matrix (ECM) degradation but inhibiting chondrocyte proliferation. Mechanically, circFOXK2 competitively binds to microRNA-4640-5p (miR-4640-5p) to enhance NOTCH2 expression in OA, affecting OA progression. Besides, circFOXK2 could motivate the Notch pathway to accelerate OA progression. CONCLUSION: CircFOXK2/miR-4640-5p/NOTCH2 axis stimulates the Notch pathway to promote the transcription of inflammatory cytokines (IL33, IL17F and IL6), consequently facilitating OA progression in murine chondrocytes.

Arabidopsis cryptochrome 1 functions in nitrogen regulation of flowering.

The phenomenon of delayed flowering after the application of nitrogen (N) fertilizer has long been known in agriculture, but the detailed molecular basis for this phenomenon is largely unclear. Here we used a modified method of suppression-subtractive hybridization to identify two key factors involved in N-regulated flowering time control in Arabidopsis thaliana, namely ferredoxin-NADP(+)-oxidoreductase and the blue-light receptor cryptochrome 1 (CRY1). The expression of both genes is induced by low N levels, and their loss-of-function mutants are insensitive to altered N concentration. Low-N conditions increase both NADPH/NADP(+) and ATP/AMP ratios, which in turn affect adenosine monophosphate-activated protein kinase (AMPK) activity. Moreover, our results show that the AMPK activity and nuclear localization are rhythmic and inversely correlated with nuclear CRY1 protein abundance. Low-N conditions increase but high-N conditions decrease the expression of several key components of the central oscillator (e.g., CCA1, LHY, and TOC1) and the flowering output genes (e.g., GI and CO). Taken together, our results suggest that N signaling functions as a modulator of nuclear CRY1 protein abundance, as well as the input signal for the central circadian clock to interfere with the normal flowering process.

Tetrahydrofolate Modulates Floral Transition through Epigenetic Silencing.

Folates, termed from tetrahydrofolate (THF) and its derivatives, function as coenzymes in one-carbon transfer reactions and play a central role in synthesis of nucleotides and amino acids. Dysfunction of cellular folate metabolism leads to serious defects in plant development; however, the molecular mechanisms of folate-mediated cellular modifications and physiological responses in plants are still largely unclear. Here, we reported that THF controls flowering time by adjusting DNA methylation-regulated gene expression in Arabidopsis (Arabidopsis thaliana). Wild-type seedlings supplied with THF as well as the high endogenous THF content mutant dihydrofolate synthetase folypoly-Glu synthetase homolog B exhibited significant up-regulation of the flowering repressor of Flowering Wageningen and thereby delaying floral transition in a dose-dependent manner. Genome-wide transcripts and DNA methylation profiling revealed that THF reduces DNA methylation so as to manipulate gene expression activity. Moreover, in accompaniment with elevated cellular ratios between monoglutamylated and polyglutamylated folates under increased THF levels, the content of S-adenosylhomo-Cys, a competitive inhibitor of methyltransferases, was obviously higher, indicating that enhanced THF accumulation may disturb cellular homeostasis of the concerted reactions between folate polyglutamylation and folate-dependent DNA methylation. In addition, we found that the loss-of-function mutant of CG DNA methyltransferase MET1 displayed much less responsiveness to THF-associated flowering time alteration. Taken together, our studies revealed a novel regulatory role of THF on epigenetic silencing, which will shed lights on the understanding of interrelations in folate homeostasis, epigenetic variation, and flowering control in plants.

Nitric oxide negatively regulates AKT1-mediated potassium uptake through modulating vitamin B6 homeostasis in Arabidopsis.

Nitric oxide (NO), an active signaling molecule in plants, is involved in numerous physiological processes and adaptive responses to environmental stresses. Under high-salt conditions, plants accumulate NO quickly, and reorganize Na(+) and K(+) contents. However, the molecular connection between NO and ion homeostasis is largely unknown. Here, we report that NO lowers K(+) channel AKT1-mediated plant K(+) uptake by modulating vitamin B6 biosynthesis. In a screen for Arabidopsis NO-hypersensitive mutants, we isolated sno1 (sensitive to nitric oxide 1), which is allelic to the previously noted mutant sos4 (salt overly sensitive 4) that has impaired Na(+) and K(+) contents and overproduces pyridoxal 5'-phosphate (PLP), an active form of vitamin B6. We showed that NO increased PLP and decreased K(+) levels in plant. NO induced SNO1 gene expression and enzyme activity, indicating that NO-triggered PLP accumulation mainly occurs through SNO1-mediated vitamin B6 salvage biosynthetic pathway. Furthermore, we demonstrated that PLP significantly repressed the activity of K(+) channel AKT1 in the Xenopus oocyte system and Arabidopsis root protoplasts. Together, our results suggest that NO decreases K(+) absorption by promoting the synthesis of vitamin B6 PLP, which further represses the activity of K(+) channel AKT1 in Arabidopsis. These findings reveal a previously unidentified pivotal role of NO in modulating the homeostasis of vitamin B6 and potassium nutrition in plants, and shed light on the mechanism of NO in plant acclimation to environmental changes.

[Improved unilateral puncture PVP based on 3D printing technology for the treatment of osteoporotic vertebral compression fracture].

OBJECTIVE: To investigate the clinical effect of unilateral percutaneous vertebroplasty (PVP) combined with 3D printing technology for the treatment of thoracolumbar osteoporotic compression fracture. METHODS: A total of 77 patients with thoracolumbar osteoporotic compression fractures from October 2020 to April 2022 were included in the study, all of which were vertebral body compression fractures caused by trauma. According to different treatment methods, they were divided into experimental group and control group. Thirty-two patients used 3D printing technology to improve unilateral transpedicle puncture vertebroplasty in the experimental group, there were 5 males and 27 females, aged from 63 to 91 years old with an average of (77.59±8.75) years old. Forty-five patients were treated with traditional bilateral pedicle puncture vertebroplasty, including 7 males and 38 females, aged from 60 to 88 years old with an average of(74.89±7.37) years old. Operation time, intraoperative C-arm X-ray times, anesthetic dosage, bone cement injection amount, bone cement diffusion good and good rate, complications, vertebral height, kyphotic angle (Cobb angle), visual analogue scale(VAS), Oswestry disability index (ODI) and other indicators were recorded before and after surgery, and statistically analyzed. RESULTS: All patients were followed up for 6 to 23 months, with preoperative imaging studies, confirmed for thoracolumbar osteoporosis compression fractures, two groups of patients with postoperative complications, no special two groups of patients' age, gender, body mass index (BMI), time were injured, the injured vertebral distribution had no statistical difference(P>0.05), comparable data. Two groups of patients with bone cement injection, bone cement dispersion rate, preoperative and postoperative vertebral body height, protruding after spine angle(Cobb angle), VAS, ODI had no statistical difference(P>0.05). The operative time, intraoperative fluoroscopy times and anesthetic dosage were statistically different between the two groups(P<0.05). Compared with the traditional bilateral puncture group, the modified unilateral puncture group combined with 3D printing technology had shorter operation time, fewer intraoperative fluoroscopy times and less anesthetic dosage. The height of anterior vertebral edge, kyphosis angle (Cobb angle), VAS score and ODI of the affected vertebrae were statistically different between two groups at each time point after surgery(P<0.05). CONCLUSION: In the treatment of thoracolumbar osteoporotic compression fractures, 3D printing technology is used to improve unilateral puncture PVP, which is convenient and simple, less trauma, short operation time, fewer fluoroscopy times, satisfactory distribution of bone cement, vertebral height recovery and kyphotic Angle correction, and good functional improvement.

Production and structural characterization of the cytochrome P450 enzymes in carotene ring hydroxylation.

The superfamily of cytochrome P450 (CYP) enzymes participate in diverse metabolic reactions. Although structural characterization of prokaryotic CYPs has been performed extensively, structural studies for eukaryotic, especially plant, CYPs remain scanty. Members of CYP family 97 are hydroxylases devoted to carotene ring hydroxylation. Specifically, members of the CYP97A and CYP97C subfamilies work synergistically to hydroxylate the ß- and ɛ-rings of α-carotene to form lutein. The stereospecific reactions introduce an R-chiral center at the C-3 position of each cyclohexene ring. The Arabidopsis thaliana genome encodes CYP97A3, CYP97B3, and CYP97C1. Recently, we determined crystal structures of CYP97A3 in unliganded and retinaldehyde-bound forms and of CYP97C1 in complex with detergent octylthioglucoside. These structures showed how the active site favors pro-R-hydroxylation. This chapter summarizes the steps for production of CYP97A3, CYP97B3, and CYP97C1. The in vitro assays to test hydroxylase activity and ligand binding affinity are also described, including the steps for production of the redox partner. We also review the details for crystallization and structural determination of CYP97A3 and CYP97C1. Characterization of the active site by high-resolution structure analysis provides clues to substrate channeling, which awaits further investigation.

[Development and clinical application of a new transverse process retractor based on computer-aided design and 3D printing technology].

OBJECTIVE: To manufacture a new type of transverse process retractor by using computer-aided design(CAD) combined with 3D printing technology and investigate its clinical application effect. METHODS: A new type of transverse protrusion retractor was developed by CAD combined with 3D printing technology. From September 2018 to September 2019, the new transverse process retractor was applied in clinic. Sixty patients with lumbar single segment lesions who needed treatment by pedicle screw fixation, bone grafting and interbody fusion were divided into new transverse process retractor group and control group, with 30 cases in each group. There were 14 males and 16 females in new type transverse process retractor group, the age was (68.0±4.3) years old on average; lesion segment of 8 cases were L3,4, 9 cases were L4,5, 13 cases were L5S1;5 cases of lumbar disc herniation, 20 cases of lumbar spinal stenosis, 5 cases of degenerative lumbar spondylolisthesis;new transverse process retractor was used to pedicle screw placement. While there were 15 males and 15 females in control group, with an average age of (69.2±4.5) years old;lesion segment of 8 cases were L3,4, 10 cases were L4,5, 12 cases were L5S1;5 cases of lumbar disc herniation, 21 cases of lumbar spinal stenosis, 4 cases of degenerative lumbar spondylolisthesis;the traditional lamina retractor was used for soft tissue pulling and finished pedicle screw placement by freehand. The length of surgical incision, the time required for inserting a single screw, fluoroscopy times, the times of adjusting the positioning needle or screw in insertion process, and the visual analogue scale (VAS) of surgical incision 72 hours after operation were compared between two groups. RESULTS: Using CAD and 3D printing technology, a new type of transverse protrusion retractor was developed quickly. The length of surgical incision, the time required for inserting a single screw, fluoroscopy time, and the times of adjusting the positioning needle or screw in insertion process in new transverse process retractor group were less than those in control group(P<0.05). There was no significant difference in VAS of lumbar incision pain at 72 hours after operation between two groups(P>0.05). CONCLUSION: Using CAD combined with 3D printing technology to develop a new transverse protrusion retractor has the advantages of convenient design, short development cycle and low cost. It provides a new idea for the research and development of new medical devices. The new transverse process retractor has the advantages of easy operation, reliable fixation, less damage to paravertebral muscle, convenient pedicle screw placement, reducing fluoroscopy time and so on.

Individualized 3D printed navigation template-assisted atlantoaxial pedicle screws vs. free-hand screws for the treatment of upper cervical fractures.

Objective: This study aims to compare the efficacy and safety of freehand atlantoaxial pedicle screws against custom 3D printed navigation template screws in the treatment of upper cervical fractures. Methods: In our institution from 2010 to 2020, a retrospective cohort analysis of 23 patients with upper cervical fractures was done. These patients were separated into two groups: group A (N = 12), which received customized 3D printed navigation template-assisted screws with virtual reality techniques, and group B (N = 11), which received freehand screws assisted by intraoperative fluoroscopy. Every patient was monitored for more than 1 year. The two groups were contrasted in terms of screw implant accuracy, cervical spine Japanese Orthopaedic Association (JOA) score, American Spinal Injury Association (ASIA) score, visual analogue scale (VAS) score, surgical time, fluoroscopy times, and intraoperative blood loss. Results: A total of 88 atlantoaxial pedicle screws in all, 46 in group A and 42 in group B, were implanted. In group A, the screw insertion accuracy rate was 95.7%, compared to 80.0% in group B (P < 0.05). When compared to group B, group A had shorter surgery times, less blood loss, fewer fluoroscopies, a higher short-term JOA score, and overt pain reduction (P < 0.05). However, there was no discernible difference between the two groups' VAS scores, long-term JOA scores, or ASIA scores (sensory and motor), at the most recent follow-up. Conclusion: Individualized 3D printed guide leads to significant improvement in the screw safety, efficacy, and accuracy, which may be a promising strategy for the treatment of upper cervical fractures.

[Effectiveness of synchronous unilateral percutaneous kyphoplasty in the treatment of double noncontiguous thoracolumbar osteoporotic vertebral compression fractures].

OBJECTIVE: To investigate the effectiveness of synchronous unilateral percutaneous kyphoplasty (PKP) in the treatment of double noncontiguous thoracolumbar osteoporotic vertebral compression fracture (OVCF). METHODS: Between December 2018 and September 2020, 27 patients with double noncontiguous thoracolumbar OVCF were treated by synchronous unilateral PKP. There were 11 males and 16 females, with an average age of 75.4 years (range, 66-92 years). The fractures were caused by falls in 22 cases and sprains in 5 cases. The time from injury to hospital admission was 0.5-7.0 days, with an average of 2.1 days. The fractured vertebrae located at T 9 in 2 cases, T 10 in 3 cases, T 11 in 10 cases, T 12 in 15 cases, L 1 in 12 cases, L 2 in 6 cases, L 3 in 4 cases, and L 4 in 2 cases. The volume of bone cement injected into each vertebral body, operation time, and intraoperative fluoroscopy times were recorded. Anteroposterior and lateral X-ray films of thoracolumbar spine were taken to observe the anterior height of the injured vertebra, the Cobb angle of kyphosis, and the diffusion and good distribution rate of bone cement in the thoracolumbar spine. Visual analogue scale (VAS) score and Oswestry disability index (ODI) were used to evaluate the pain and functional improvement. RESULTS: All operations completed successfully. The operation time was 34-70 minutes, with an average of 45.4 minutes. The intraoperative fluoroscopy was 21- 60 times, with an average of 38.6 times. The volume of bone cement injected into each vertebral body was 2-9 mL, with an average of 4.3 mL. All patients were followed up 6-21 months, with an average of 11.3 months. X-ray film reexamination showed that the anterior height of the injured vertebra and Cobb angle at each time point after operation were significantly improved than those before operation ( P<0.05), and there was no significant difference between different time points after operation ( P>0.05). The distribution of bone cement was excellent in 40 vertebral bodies, good in 13 vertebral bodies, and poor in 1 vertebral body, and the excellent and good rate was 98.1% (53/54). The pain of all patients significantly relieved or disappeared, and the function improved. The VAS score and ODI at each time point after operation were significantly lower than those before operation ( P<0.05), and there was no significant difference between different time points after operation ( P>0.05). CONCLUSION: For the double noncontiguous thoracolumbar OVCF, the synchronous unilateral PKP has the advantages of simple puncture, less trauma, less intraoperative fluoroscopy, shorter operation time, satisfactory distribution of bone cement, etc. It can restore the height of the vertebral body, correct the kyphotic angle, significantly alleviate the pain, and improve the function.

[Clinical application of three-dimensional printing technique combined with thoracic pedicle screw track detector in thoracic pedicle screw placement].

OBJECTIVE: To investigate the clinical application of three-dimensional (3D) printing technique combined with a new type of thoracic pedicle screw track detector in thoracic pedicle screw placement. METHODS: According to the characteristics of thoracic pedicle and common clinical screw placement methods, a new type of thoracic pedicle screw track detector was independently developed and designed. The clinical data of 30 patients with thoracic vertebrae related diseases who underwent posterior thoracic pedicle screw fixation between March 2017 and January 2020 were retrospectively analysed. Among them, there were 18 males and 12 females with an average age of 56.3 years (range, 32-76 years). There was 1 case of thoracic disc herniation, 4 cases of thoracic canal stenosis, 2 cases of ossification of posterior longitudinal ligament of thoracic vertebra, 16 cases of thoracic trauma, 2 cases of thoracic infection, and 5 cases of thoracic canal occupation. Three-dimensional CT of the thoracic vertebra was routinely performed preoperatively, and the model of the patient's thoracic vertebra was reconstructed and printed out. With the assistance of the model, preoperative simulation was performed with the combination of the new type thoracic pedicle screw track detector, and detected no nails after critical cortical damage. During operation, one side was randomly selected to use traditional hand screws placement (control group), and the other side was selected to use 3D printing technique combined with new type thoracic pedicle screw track detector to assist thoracic pedicle screws placement (observation group). The single screw placement time, adjustment times of single screw, and blood loss during screw placement were compared between the two groups. The accuracy of screw placement in the two groups was evaluated according to postoperative CT imaging data. RESULTS: The single screw placement time, adjustment times of single screw, and blood loss during screw placement in the observation group were significantly less than those in the control group ( P<0.05). Postoperative CT examination showed that the observation group had 87 screws of grade 1, 3 screws of grade 2, and the acceptable screw placement rate was 100% (90/90); the control group had 76 screws of grade 1, 2 screws of grade 2, 11 screws of grade 3, and 1 screw of grade 4, and the acceptable screw placement rate was 86.7% (78/90); showing significant difference in screw placement between the two groups ( χ 2=12.875, P=0.001). All patients were followed up 6-18 months, with an average of 11.3 months. There was no complication of vascular, nerve, spinal cord, or visceral injury, and screws or rods broken, and no patient was revised. CONCLUSION: The 3D printing technique combined with the new type of thoracic pedicle screw track detector assisted thoracic pedicle screw placement is convenient, and significantly improves the accuracy and safety of intraoperative screw placement, and overall success rate of the surgery.

Surgical treatment for upper cervical deformity with atlantoaxial joint dislocation using individualized 3D printing occipitocervical fusion instrument: A case report and literature review.

ABSTRACT: To introduce a novel technique of using individualized 3D printing occipitocervical fusion instrument (3D-OCF) for the treatment of upper cervical deformity with atlantoaxial joint dislocation.The surgery for deformity of the craniocervical junction area is a challenge in the field of spine. If the surgical deviation is too large to injure the spinal cord or vertebral artery, it will cause catastrophic damage to the patient. Therefore, it is controversial whether these patients should undergo surgical treatment. We provide a novel surgical approach for the challenging upper cervical surgery through 3D-OCF and a typical patient.We present a 54-year-old female patient, who suffered from dizziness and numbness in her limbs for 8 months. After the patient was admitted, we performed the three-dimensional CT scan, modeled using Mimics software 17.0, and designed customized occipitocervical fusion instrument. Besides, we repeatedly perform simulated surgery based on 3D-printed models before surgery.The operative time was 142 minutes and the intraoperative blood loss was 700 mL. X-ray showed reduction of atlantoaxial dislocation and accurate position of internal fixation. The patient's symptoms were significantly relieved: the sensation of dizziness and numbness of limbs was obviously relieved, and the sense of banding in chest, abdomen, and ankle was disappeared. At the last follow-up, imaging showed that 3D-OCF had bone-integration and Syringomyelia was disappeared. The patient's cervical JOA (Japanese Orthopaedic Association) score increased from 10 points to 17 points.Individualized 3D-OCF can improve the safety and accuracy of upper cervical surgery, reduce the operative time and the number of fluoroscopy. Our study provides a novel surgical approach for the challenging upper cervical surgery.

[Experimental study of individualized 3D printing-guided template combined with thoracolumbar pedicle screw placement for the treatment of ankylosing spondylitis].

OBJECTIVE: To evaluate accuracy and safety of individualized 3D printing guided template for thoracolumbar pedicle screw placement in patients with ankylosing spondylitis. METHODS: From January 2016 to September 2019, thoracolumbar spine three-dimensional CT data of 8 patients with ankylosing spondylitis were included, Mimics 17.0 and ideaMaker computer software were applied to design thoracolumbar pedicle screw guided template of patients with AS, physical model of all patients (T10-L2)were printed by 3D printer, 2 parts in each patient, and divided into guide-plate-assisted screw group (experimental group) and free-hand nail group (control group). Thoracolumbar pedicle screws of both groups were placed by the same spinal surgeon. The accuracy of pedicle screw placement between two groups were evaluated according to results of postoperative CT, the accuracy of the fixation of thoracolumbar pedicle screw was divided into 4 grades, grade 0 and 1 screws were acceptable nails, grade 2 and 3 screws were unacceptable nails. The diameter and length of pedicle screws, the distance between entry point and posterior median line designed by preoperative 3D printing were compared with actual use in operation. RESULTS: Twenty three blocks of thoracolumbar 3D printing screw of ankylosing spondylitis guided templates were designed and printed in guide-plate-assisted screw group, 46 screws were inserted and 44 screws were accepted. The time of implanting a screw into thoracolumbar pedicle was (4.20±1.15) min, the frequency of X-ray was (5.00±1.25) times and the average adjustment times of screw and Kirschner needle during screw placement was (1.76±1.32) times. In the control group, 46 nails were placed by traditional surgical method and 30 screws were accepted. The time of implanting a screw into thoracolumbar pedicle was (14.67±2.23) min, the frequency of X-ray fluoroscopy was (14.46±2.21) times and the average times of Kirschner needle adjustment was (4.76±3.39) times. The success rates between experimental group and control group were 95.65%(44 / 46) and 56.22%(30 / 46) respectively, and had statistical difference (χ2=13.538, P<0.05). There was no significant difference in diameter, length of pedicle screws and the distance of posterior median line between virtual designed by 3D printing before operation and actual situation in opertaion (P>0.05). The operation time of inserting a single screw, the times of X-ray fluoroscopy, and the average times of adjustment screw and Kirschner needle in experimental group were significant less than those in control group(P<0.01). CONCLUSION: The personalized guide template assisted the thoracolumbar fixation designed by 3D printing could significantly improve safety, accuracy and efficiency of surgery, especially suitable for thoracolumbar vertebral bodies requiring posterior pedicle screw fixation for fracture or dislocation with AS.

[3D printing technology assisted the preoperative planning and application value in adult kyphoscoliosis deformity].

OBJECTIVE: To explore the application value of 3D printing technology in preoperative surgery plan and intraoperative auxiliary operation for adult kyphoscoliosis deformity. METHODS: The clinical data of 12 adult patients with kyphoscoliosis deformity treated from September 2017 to January 2019 were retrospectively analyzed. There were 3 males and 9 females, aged from 21 to 63 years old with an average of (47.67±13.32) years old. Among them, 4 cases were congenital kyphoscoliosis, 2 cases were old tuberculosis thoracolumbar kyphosis ; 2 cases were idiopathic kyphoscoliosis, 4 cases were degenerative kyphoscoliosis. The CT scan data of the patient's spine was imported into Mimics17.0 software to establish the three dimensional model of the spine, and the spine model was produced by 3D printer. Using the spine model simulated operation, preoperative surgery program planning and formulated a precise surgery, and further analysed postoperative imaging parameters improvement. All the patients were followed up for more than 1 year. Before and after operation and at the last follow-up, the scoliosis Cobb angle, maximum kyphosis Cobb angle, and coronal plane balance (distance between C 7 plumbline and center sacral vertical line, C7PL-CSVL), sagittal plane balance (sagittal vertical axis, SVA), pelvic parameters and other related imaging parameters were measured to further evaluate its orthopedic effect. RESULTS: Twelve patients with spine deformity were treated with different osteotomy and internal fixation fusion methods under the guidance of a 1∶1 spine model (pedicle screw placement of 4 patients with severe deformity were assisted by pedicle screw guide plates), nail placement and osteotomy have good effects, no major tissue damage such as blood vessels, nerves and spinal cord during and after surgery, no complications such as cerebrospinal fluid leakage and infection. Preoperative Cobb angle of scoliosis was (56.5±22.5) °, Cobb angle of kyphosis was (65.2±19.5) °, C7 PL-CSVL was (45.8±16.9) mm, SVA was (48.7±25.4) mm. Postoperative at 4 weeks, Cobb angle of scoliosis was (20.8±11.5) °, and Cobb angle of kyphosis was (22.0±6.6) °, with correction rates of (65.1±9.7)% and (64.6± 10.6)%, respectively ; C7 PL-CSVL was (22.3±8.9) mm, and SVA was (23.3±13.1) mm, all of which were significantly improved compared with preoperative results. The mean follow-up time was (18.5±7.9) months in 12 patients. At the last follow-up, the Cobb angles of scoliosis and kyphosis were (22.2±10.8) ° and (23.6±7.7) °, respectively, C7 PL-CSVL was (23.5±10.8) mm, and SVA was (24.7±12.5) mm. The results were statistically significant compared preoperative (P<0.05). There was no significant difference at the postoperative at 4 weeks and the last follow-up (P>0.05). CONCLUSION: The 3D print model can visually and clearly show the vertebral morphology and structure of adult kyphoscolisis and its spatial relationship with the adjacent vertebrae, blood vessels, and nerves, which provides a good and intuitive stereoscopic anatomical structure observation for the individualization of the surgical plan. Pre-simulation of operations to determine the internal fixation, fusion segment and osteotomy orthopedic way, may to provide a reference for actual clinical surgery, and can improve the accuracy and safety of surgery.

[Determination of painful vertebrae treated by kyphoplasty in multiple-level vertebral compression fractures].

OBJECTIVE: To explore how to determine painful vertebrae treated by kyphoplasty in multiple-level osteoporotic vertebral compression fractures and clinical outcome. METHODS: From October 2002 to June 2005, 51 consecutive procedures with kyphoplasty were performed on 35 patients with multiple-level osteoporotic vertebral compression fractures. There were 51 painful vertebrae among 120 vertebral compression fractures. The painful vertebra was determined by the signal intensity changes in MR images, combined with radiography and local percussion pain before operation. Only painful vertebrae were treated by kyphoplasty. Preoperative, postoperative and final follow-up visual analog scale (VAS) and radiographic findings such as vertebral height and Cobb angle were recorded and analyzed. RESULTS: All patients tolerated the procedure well with immediate relief of their back pain after kyphoplasty and they can walk at 1-3 days after the procedure. There were 3 vertebrae (3/51) occurred asymptomatic extravasation of cement. 31 cases were followed up for mean 16.2 months (range 6-44 months). VAS reduced from preoperative 8.7 to final follow-up 2.1 (P <0. 01). At final follow-up the vertebral height had a recovery rate of 59.17%, and the mean Cobb angle was improved 10.1 degrees. There was a significant improvement between preoperative and final follow-up values (P < 0.01). CONCLUSIONS: The painful vertebra can be determined by signal intensity changes in MR series images in multiple-level osteoporotic vertebral compression fractures. Selecting painful vertebrae to be treated by kyphoplasty can make patients with multiple-level VCFs gain an excellent result.

Structure of the Arabidopsis thaliana NADPH-cytochrome P450 reductase 2 (ATR2) provides insight into its function.

Members of the cytochrome P450 family catalyze a variety of mono-oxygenase reactions, and for the eukaryotic membrane-bound members, NADPH is typically used as the reducing agent. The flavoprotein NADPH-cytochrome P450 reductase (CPR) enables electron transfer from NADPH to cytochrome P450 via its flavin cofactors. ATR2 is one of the two authentic CPR genes in the genome of the model plant Arabidopsis thaliana, and its product has been physiologically and kinetically characterized. Here, we report the 2.3 Å structure of Arabidopsis thaliana NADPH-cytochrome P450 reductase 2 (ATR2) and find that the position of the two flavin cofactors differs from that of other known CPR structures. Mutation of residues related to possible interflavin electron transfer retains the reductase activity of ATR2, which suggests a direct electron transfer pathway between the flavins. In contrast, mutation of a single residue (R708) mediating interdomain interaction abolishes this activity. Because this residue is only conserved in plant CPRs, we speculate a plant-specific working mechanism as observed in ATR2. DATABASE: Atomic coordinates and structure factors of ATR2 are available in the Protein Data Bank under the accession code 5GXU.

Individualized 3D printing navigation template for pedicle screw fixation in upper cervical spine.

PURPOSE: Pedicle screw fixation in the upper cervical spine is a difficult and high-risk procedure. The screw is difficult to place rapidly and accurately, and can lead to serious injury of spinal cord or vertebral artery. The aim of this study was to design an individualized 3D printing navigation template for pedicle screw fixation in the upper cervical spine. METHODS: Using CT thin slices data, we employed computer software to design the navigation template for pedicle screw fixation in the upper cervical spine (atlas and axis). The upper cervical spine models and navigation templates were produced by 3D printer with equal proportion, two sets for each case. In one set (Test group), pedicle screws fixation were guided by the navigation template; in the second set (Control group), the screws were fixed under fluoroscopy. According to the degree of pedicle cortex perforation and whether the screw needed to be refitted, the fixation effects were divided into 3 types: Type I, screw is fully located within the vertebral pedicle; Type II, degree of pedicle cortex perforation is <1 mm, but with good internal fixation stability and no need to renovate; Type III, degree of pedicle cortex perforation is >1 mm or with the poor internal fixation stability and in need of renovation. Type I and Type II were acceptable placements; Type III placements were unacceptable. RESULTS: A total of 19 upper cervical spine and 19 navigation templates were printed, and 37 pedicle screws were fixed in each group. Type I screw-placements in the test group totaled 32; Type II totaled 3; and Type III totaled 2; with an acceptable rate of 94.60%. Type I screw placements in the control group totaled 23; Type II totaled 3; and Type III totaled 11, with an acceptable rate of 70.27%. The acceptability rate in test group was higher than the rate in control group. The operation time and fluoroscopic frequency for each screw were decreased, compared with control group. CONCLUSION: The individualized 3D printing navigation template for pedicle screw fixation is easy and safe, with a high success rate in the upper cervical spine surgery.

[The relationship between lumbosacral transitional vertebra and the lumbar disc herniation].

OBJECTIVE: To investigate the relationship between lumbosacral transitional vertebra and the lumbar disc herniation (LDH). METHODS: The X-ray photographs of lumbar vertebra were retrospectively studied of patients with and without LDH confirmed by surgery, furthermore, the differential incidence of LDH between the two groups and the relationship between transitional vertebra and the position of disc herniation were statistically analyzed. RESULTS: The incidence of lumbosacral transitional vertebra was 18.3% in the control group, 52.7% in the LDH group, the difference was statistically significant. In the group with single lumbosacral transitional vertebra, there was 75.9% of LDH occurred on the same side of the transitional vertebra, 81.8% of which occurred at the upper one disc of the transitional vertebra; whereas 17.2% of LDH on the opposite side of the transitional vertebra, 80.0% of which occurred at the lower one disc of the transitional vertebra. CONCLUSIONS: There is a closer relationship between lumbosacral transitional vertebra and the LDH, and the lumbosacral transitional vertebra is one of the important factors in the emergence of LDH.

Crystal structure of the zinc-bound HhoA protease from Synechocystis sp. PCC 6803.

The high temperature requirement A (HtrA) proteases are oligomeric serine proteases essential for protein quality control. HtrA homolog A (HhoA) from the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 assembles into a proteolytically active hexamer. Herein, we present the crystal structure of the hexameric HhoA in complex with the copurified peptide. Our data indicate the presence of three methionines in close proximity to the peptide-binding site of the PDZ domain. Unexpectedly, we observed that a zinc ion is accommodated within the central channel formed by a HhoA trimer. However, neither calcium nor magnesium showed affinity for HhoA. The role of the zinc ion in HhoA was tested in an in vitro proteolytic assay against the nonspecific substrate ß-casein and was found to be inhibitory. Our findings provide insights into the regulation of HhoA by a redox-related mechanism involving methionine residues and by zinc ion-binding within the central channel.

Structural basis for copper/silver binding by the Synechocystis metallochaperone CopM.

Copper homeostasis integrates multiple processes from sensing to storage and efflux out of the cell. CopM is a cyanobacterial metallochaperone, the gene for which is located upstream of a two-component system for copper resistance, but the molecular basis for copper recognition by this four-helical bundle protein is unknown. Here, crystal structures of CopM in apo, copper-bound and silver-bound forms are reported. Monovalent copper/silver ions are buried within the bundle core; divalent copper ions are found on the surface of the bundle. The monovalent copper/silver-binding site is constituted by two consecutive histidines and is conserved in a previously functionally unknown protein family. The structural analyses show two conformational states and suggest that flexibility in the first α-helix is related to the metallochaperone function. These results also reveal functional diversity from a protein family with a simple four-helical fold.