Structural basis for FGF hormone signalling.

α/ßKlotho coreceptors simultaneously engage fibroblast growth factor (FGF) hormones (FGF19, FGF21 and FGF23)1,2 and their cognate cell-surface FGF receptors (FGFR1-4) thereby stabilizing the endocrine FGF-FGFR complex3-6. However, these hormones still require heparan sulfate (HS) proteoglycan as an additional coreceptor to induce FGFR dimerization/activation and hence elicit their essential metabolic activities6. To reveal the molecular mechanism underpinning the coreceptor role of HS, we solved cryo-electron microscopy structures of three distinct 1:2:1:1 FGF23-FGFR-αKlotho-HS quaternary complexes featuring the 'c' splice isoforms of FGFR1 (FGFR1c), FGFR3 (FGFR3c) or FGFR4 as the receptor component. These structures, supported by cell-based receptor complementation and heterodimerization experiments, reveal that a single HS chain enables FGF23 and its primary FGFR within a 1:1:1 FGF23-FGFR-αKlotho ternary complex to jointly recruit a lone secondary FGFR molecule leading to asymmetric receptor dimerization and activation. However, αKlotho does not directly participate in recruiting the secondary receptor/dimerization. We also show that the asymmetric mode of receptor dimerization is applicable to paracrine FGFs that signal solely in an HS-dependent fashion. Our structural and biochemical data overturn the current symmetric FGFR dimerization paradigm and provide blueprints for rational discovery of modulators of FGF signalling2 as therapeutics for human metabolic diseases and cancer.

Multivalent Recruitment of Human Argonaute by GW182.

In miRNA-mediated gene silencing, the physical interaction between human Argonaute (hAgo) and GW182 (hGW182) is essential for facilitating the downstream silencing of the targeted mRNA. GW182 can interact with hAgo via three of the GW/WG repeats in its Argonaute-binding domain: motif-1, motif-2, and the hook motif. The structure of hAgo1 in complex with the hook motif of hGW182 reveals a "gate"-like interaction that is critical for GW182 docking into one of hAgo1's tryptophan-binding pockets. We show that hAgo1 and hAgo2 have a single GW182-binding site and that miRNA binding increases hAgo's affinity to GW182. With target binding occurring rapidly, this ensures that only mature RISC would be recruited for silencing. Finally, we show that hGW182 can recruit up to three copies of hAgo via its three GW motifs. This may explain the observed cooperativity in miRNA-mediated gene silencing.

Mechanism of replication-coupled DNA interstrand crosslink repair.

DNA interstrand crosslinks (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a cell-free system based on Xenopus egg extracts that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the crosslink. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymerase zeta. Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication.

How cervical curvature changes after ACAF and ACDF: a radiological retrospective study.

To assess changes in cervical curvature and demonstrate the feasibility of using ACAF technology to restore lordosis, imaging data from patients undergoing multilevel ACAF and ACDF surgeries were retrospectively analyzed. Forty-seven patients receiving multilevel ACAF and ACDF were included in the study. Total cervical curvature and anterior, middle, and posterior column curvature, spinal canal diameter, cervical range of motion, and surgical complications were measured and analyzed by non-parametric or chi-square tests before and after surgery. The Spearman correlation between imaging data was analyzed. Compared with ACDF, the operation time of the ACAF group was longer, the cervical motion was larger, the median and change value of the middle column curvature was larger, and the change value of the posterior column curvature was smaller (P < 0.05). The postoperative differences in cervical lordosis angle, vertebral canal diameter, and middle and posterior column curvature in the ACAF group were significantly greater than those in the ACDF group (p = 0.015). The expansion of vertebral canal diameter was significantly correlated with the difference in curvature between the middle and posterior columns (r = 0.523, P < 0.01), and the curvature of the anterior column was correlated with that of the middle and posterior columns (P < 0.05). The curvature change of the anterior column is closely related to the curvature change of the middle column and the posterior column. Compared with ACDF, ACAF expands the diameter and volume of the spinal canal by increasing the curvature of the middle column and reducing the anterior movement of the posterior column.

Anterior controllable antidisplacement and fusion surgery for the treatment of extensive cervico-thoracic ossification of posterior longitudinal ligament with severe myelopathy: case report and literature review.

OPLL generally occurs in the cervical spine and involves no more than three vertebral segments, while extensive OPLL that involves the cervico-thoracic spine and spans over multiple segments is rare. Surgically it is difficult to achieve a satisfactory clinical outcome without surgical complications via the traditional anterior or posterior approaches. We report the first application of Anterior controllable antidisplacement and fusion (ACAF) in treating extensive cervico-thoracic OPLL. A 45-year old patient experienced severe walking disturbance, bladder and bowel dysfunction for 5 months after a fall. His preoperative Japanese Orthopedic Association (JOA) score was 8 of 17. Preoperative CT and MRI demonstrated a K-line (-) and mixed-type extensive OPLL from C2 to T2, causing significant cord compression. After ACAF surgery, neurological symptoms improved immediately without postoperative complications. Postoperative CT and MRI scanning showed restoration of spinal canal cross section and cord decompression. At 6 months he was able to stand and walk again without assistance and urinary bladder and bowel function returned to normal completely. At 15 months his JOA score was 14 of 17. ACAF surgery provides a promising alternative for the treatment of extensive cervico-thoracic OPLL.

Vericiguat Modulates Osteoclast Differentiation and Bone Resorption via a Balance between VASP and NF-κB Pathways.

Bone homeostasis has been a dynamic equilibrium between osteoclasts (OCs) and osteoblasts (OBs). However, excessive activation of OCs could disturb the bone homeostasis. As a result, effective medical interventions for patients are greatly demanding. NO/guanylate cyclase (GC)/cGMP signaling cascade has been previously reported to regulate bone metabolism, and GC plays a significantly critical role. Vericiguat, as a novel oral soluble guanylate cyclase (sGC) stimulator, has been firstly reported in 2020 to treat patients with heart failure. Nevertheless, the biological effects of Vericiguat on the function of OCs have not yet been explored. In this present study, we found that Vericiguat with the concentration between 0 and 8 µM was noncytotoxic to bone marrow-derived monocyte-macrophage lineage (BMMs). Vericiguat could enhance the differentiation of OCs at concentration of 500 nM, whereas it inhibited OC differentiation at 8 µM. In addition, Vericiguat also showed dual effects on OC fusion and bone resorption in a dose-dependent manner. Furthermore, a molecular assay suggested that the dual regulatory effects of Vericiguat on OCs were mediated by the bidirectional activation of the IκB-α/NF-κB signaling pathway. Taken together, our present study demonstrated the dual effects of Vericiguat on the formation of functional OCs. The regulatory effects of Vericiguat on OCs were achieved by the bidirectional modulation of the IκB-α/NF-κB signaling pathway, and a potential balance between the IκB-α/NF-κB signaling pathway and sGC/cGMP/VASP may exist.

Urodynamic evaluation of bladder function in patients with urinary incontinence secondary to congenital tethered cord syndrome after homogeneous spinal-shortening axial decompression procedure.

OBJECTIVE: To evaluate the effect of homogeneous spinal-shortening axial decompression procedure (HSAD) on bladder function in patients with spina bifida tethered syndrome. METHODS AND MATERIALS: Patients with tethered spinal cord syndrome were collected prospectively, and all patients were treated with lumbar HSAD. Patients' urodynamic evaluation mainly included detrusor function, sphincter function, sphincter coordination (Ig TLR, ratio of tension and loose of urethral sphincter), and bladder compliance. Meanwhile, all patients were followed up with ICI-Q-SF, SF-12, and Rantala scores. RESULTS: Twenty-four patients were included, with the average age of 27 ± 16 years. At the final follow-up, patients' detrusor function, sphincter function, sphincter coordination, and bladder compliance, were all improved dramatically (all P < 0.01). The preoperative SF-12 score, ICQ, and Rantala score were [52.16 ± 5.64, 14.11 ± 5.25, 7.84 ± 4.87], whereas the postoperative mean was [33.53 ± 3.53, 9.05 ± 4.89, 15 ± 3.77] (P < 0.01, respectively). According to objective evaluation, 16.7% of them recovered to normal. According to the subjective evaluation, 25% of the patients returned to normal. Only one patient (4.2%) deteriorated. Limitations include none-randomized controlled design and limited patient samples. CONCLUSIONS: The HSAD can significantly restore the bladder function in patients with long-term urinary incontinence.

Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function.

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA.

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.

Long noncoding RNA AC092155 facilitates osteogenic differentiation of adipose-derived stem cells through the miR-143-3p/STMN1 axis.

BACKGROUND: Numerous studies have demonstrated that long noncoding RNAs (lncRNAs) induce osteogenesis in adipose-derived stem cells (ADSCs). This study aimed to explore the role of lncRNAs AC092155 in promoting osteogenic differentiation of ADSCs. METHODS: MicroRNA (miRNA) and lncRNA sequencing were performed in ADSCs that underwent normal or osteogenic induction. Differentially expressed miRNAs and lncRNAs were identified using R software. The relative expression levels of lncRNA AC092155, miR-143-3p, and STMN1 during the process of osteogenic induction were determined by real-time polymerase chain reaction (RT-PCR). ADSCs were then transfected with agomiR-143-3p and pcDNA3.1-sh-lncRNA AC092155. Alkaline phosphatase (ALP) and alizarin red staining (ARS) were used to confirm the regulatory function of the lncRNA AC092155/miR-143-3p/STMN1 axis in osteogenic differentiation of ADSCs. RESULTS: lncRNA AC092155 was significantly upregulated in ADSCs following induction in the osteogenic medium. lncRNA AC092155 and STMN1 mimics increase the markers of osteogenic differentiation in the early and late phases, which was reflected in increased ALP activity as well as the higher deposition of calcium nodules. An miR-143-3p mimic showed the opposite effect. Luciferase reporter gene analysis demonstrated that lncRNA AC092155 directly targets miR-143-3p. Moreover, the lncRNA AC092155/miR-143-3p/STMN1 regulatory axis was found to activate the Wnt/ß-catenin signaling pathway. CONCLUSIONS: lncRNA AC092155 contributes to the osteogenic differentiation of ADSCs. The lncRNA AC092155/miR-143-3p/STMN1 axis may be a new therapeutic target for bone-related diseases.

An open-label randomized multi-Centre study to evaluate anterior controllable Antedisplacement and fusion versus posterior Laminoplasty in patients with cervical ossification of the posterior longitudinal ligament: study design and analysis plan (STAR).

BACKGROUND: In treating patients with cervical ossification of the posterior longitudinal ligament (COPLL), a novel surgery technique - anterior controllable antedisplacement and fusion (ACAF) suggested promising clinical benefits in recent exploratory studies. METHODS: This is a multicentre, randomized, open-label, parallel-group, active controlled trial that will compare the clinical benefits of ACAF versus conventional posterior laminoplasty (LAMP) in severe COPLL patients. A total of 164 patients will be enrolled and randomized in a 1:1 ratio to either ACAF or LAMP group. The primary efficacy measure is cervical- Japanese Orthopaedic Association (C-JOA) recovery rate at 12 months post operation, which is to be derived by Hirabayashi's method from JOA data (range, 0 [worst] to 17 [normal condition]). Other important secondary efficacy endpoints include visual analogue scale (VAS) pain score (range, 0 [no pain] to 10 [most severe]), 10-item neck disability index (NDI, a total range of 0 to 50 points, the highest index the worst) and 6-level Nurick disability grade (range, 0 [mild] to 5 [severe]). Safety endpoints including adverse events, perioperative complications, and adverse events of special interest will also be assessed in this study. Full analysis set for baseline and efficacy data analyses according to the intention-to-treat principle will be established as the primary analysis population. Analysis of covariance (ANCOVA) will be used to analyze the C-JOA recovery rate, with random stratification factors (if appropriate) and the treatment group as fixed factors, and the baseline level of C-JOA score as covariate. DISCUSSION: This study is designed to demonstrate the clinical benefits of ACAF as compared to conventional LAMP in COPLL patients. It will provide clinical evidence that the novel surgery technique - ACAF might be more favorable in treating patients with severe cervical ossification of the posterior longitudinal ligament. (Words: 290). TRIAL REGISTRATION: ClinicalTrials.gov number, NCT04968028 .

Expression of Nogo-A in dorsal root ganglion in rats with cauda equina injury.

OBJECTIVE: To investigate the expression of Nogo-A in dorsal root ganglion (DRG) in rats with cauda equina injury and the therapeutic effects of blocking Nogo-A and its receptor. METHODS AND MATERIALS: Fifty-eight male Sprague-Dawley rats were divided randomly into either the sham operation group (n = 24) or the cauda equina compression (CEC) control group (n = 34). Behavioral, histological, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were conducted to assess the establishment of the model. The dynamic expression change of Nogo-A was evaluated using real time-qPCR. Immunofluorescence was used to evaluate the expression of Nogo-A in the DRG and cauda equina. Furthermore, 20 male Sprague-Dawley rats were equally divided into 4 groups, including the sham group, the CEC group, the NEP1-40 (the NgR antagonist peptide) treatment group, and the JTE-013 (the S1PR2 antagonist) treatment group. Behavioral assessments and western blotting were used to evaluate the therapeutic effect of cauda equina injury via blocking Nogo-A and its receptor. RESULTS: Tactile allodynia and heat hyperalgesia in the CEC model developed as soon as 1 day after surgery and recovered to normal at 7 days, which was followed by the downregulation of Nogo-A in DRG neurons. However, the locomotor function impairment in the CEC model showed a different prognosis from the sensory function, which was consistent with the expression change of Nogo-A in the spinal cord. Immunofluorescence results also demonstrated that Nogo A-positive/NF200-negative neurons and axons increased in the DRG and cauda equina 7 days after surgery. Surprisingly, Schwann cells, which myelinate axons in the PNS, also expressed considerable amounts of Nogo-A. Then, after blocking the Nogo-A/NgR signaling pathway by NEP1-40, significant improvement of mechanical allodynia was identified in the first 2 days after the surgery. Western blotting suggested the NEP1-40 treatment group had lower expression of cleaved caspase-3 than the CEC and JTE-013 treatment group. CONCLUSION: Neuronal Nogo-A in the DRG may be involved in regeneration and play a protective role in the CEC model. Whereas Nogo-A, released from the injured axons or expressed by Schwann cells, may act as an inhibiting factor in the process of CEC repairment. Thus, blocking the Nogo-A/NgR signaling pathway can alleviate mechanical allodynia by apoptosis inhibition.

Analysis of the spinal cord angle for severe cervical ossification of the posterior longitudinal ligament: comparison between anterior controllable antedisplacement and fusion (ACAF) and posterior laminectomy.

OBJECTIVE: To investigate the changes of spinal cord angle between anterior controllable antedisplacement and fusion (ACAF) and posterior laminectomy in treating severe ossification of the posterior longitudinal ligament (OPLL). PATIENTS AND METHODS: Seventy-one patients with cervical OPLL were enrolled. Patients in this study were divided into group A and group P. Japanese Orthopaedic Association (JOA) score was utilized to evaluate the neurological function. Radiological assessments included the spinal cord angle, Cobb angle, and area of the spinal cord. Surgery-related complications were also recorded. RESULTS: At the final follow-up, patients in group A had better recovery of local and whole cord angle, and the area of the cord than those in group P (all p < 0.05). A strong correlation between the change of local cord angle and the recovery of the spinal cord area was observed (r = - 0.867, p < 0.05). In addition, patients in group P had worse Cobb angle (9.15° ± 1.10°) than in group A (18.58° ± 0.73°) (p < 0.05). The final mean JOA score and its improvement rate were better in the group A than in group P (p < 0.05). During the follow-up, 15.15% patients in group P experienced surgery-related complications and 7.89% in group A. CONCLUSION: This present study revealed that ACAF can achieve better recovery of the expansion of the spinal cord, spinal cord alignment, and Cobb angle, with better postoperative JOA score and less complications, compared with posterior laminectomy in treating severe cervical OPLL. These slides can be retrieved under Electronic Supplementary Material.

Reliability and validity of multi-shot DWI in diagnosis of cervical spondylotic myelopathy: a study based on 3-T MRI.

OBJECTIVE: To investigate whether multi-shot diffusion-weighted imaging (ms-DWI) could be applied in diagnosis and quantitative evaluation of cervical spondylotic myelopathy (CSM). METHODS: Thirty-three normal volunteers and 78 patients with CSM were included in this study. The apparent diffusion coefficient (ADC) values were measured at C2-C7 levels on sagittal section ADC map. The intraclass correlation coefficient (ICC) and Bland and Altman plot and Spearman coefficient were used to quantify the reproducibility of test and retest and inter-rater reliability. Pearson correlations were calculated to compare lADC and rADC versus mJOA and NDI scores. Receiver operating characteristic curve and the area under the curve (AUC) were applied to evaluate the diagnostic reliability. RESULTS: There was no statistically significant difference between ADC values obtained from normal volunteers at C2-C7 levels (P < 0.05). The ICC and spearman coefficient of lADC and rADC indicated excellent test-retest and inter-rater reliability. The mean lADC and rADC were significantly higher from patients than that from volunteers (all P < 0.01). The lADC had moderate to good correlations with mJOA and NDI (all P < 0.0001). Moreover, rADC had good to excellent correlations with mJOA and NDI (all P < 0.0001). Comparing AUCs, rADC was significantly superior in diagnosis which participants were CSM than lADC (P = 0.0118). CONCLUSION: The ms-DWI could be applied in diagnosis and quantitive assessment of CSM according to lADC and rADC. A new parameter, rADC, could be served as a diagnostic indice for CSM, which may quantitively reflect the severity of CSM. These slides can be retrieved under Electronic Supplementary Material.

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation.

The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5'-3' through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.

A modified procedure of single-level transforaminal lumbar interbody fusion reduces immediate post-operative symptoms: a prospective case-controlled study based on two hundred and four cases.

STUDY DESIGN: This is a prospective case-controlled study. PURPOSE: The purpose of this study is to investigate the effect of a modified transforaminal lumbar interbody fusion (TLIF) on the immediate post-operative symptoms in patients with lumbar disc herniation (LDH) accompanied with stenosis. METHODS: A total of 204 LDH patients with single-level TLIF were enrolled. According to the sequence of the placement of rods and cage, patients were divided into group R (rod-prior-to-cage) and group C (cage-prior-to-rod). Neurological function was evaluated by the Japanese Orthopedic Association (JOA) score. Radiological assessment includes height of intervertebral space (HIS), foraminal height (FH), foraminal area (FA), and segmental lordosis (SL). Change of original symptoms (pain/numb) and new-onset symptoms (pain/numb) after surgery were also recorded. RESULTS: Patients in group R had less change of HIS at L3/4, L4/5, and L5/S1 levels compared with pre-operation (all p > 0.05), whereas group C had larger change (all p < 0.05). No statistical difference was found in FH between the two groups before and after surgery at L3/4, L4/5, and L5/S1, respectively (all p > 0.05). In terms of FA, patients in group R had better improvement after surgery than those in group C at L3/4 and L4/5 (both p < 0.05). Patients in both groups acquired good improvement of neurological function. However, there were fewer patients in group R who experienced post-operative leg pain or numb compared with those in group C (p < 0.05). CONCLUSION: The modified open TLIF can significantly reduce the incidence of immediate post-operative symptoms for patients with single-level lumbar disc herniation via installation of rods prior to insertion of cage and the "neural standard" should serve as the goal of decompression for spine surgeons to restore disc/foraminal height and to minimize nerve distraction.

ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice.

Osteogenic differentiation refers to the process of bone formation and remodeling, which is controlled by complex molecular mechanisms. Activin A receptor type I (ACVR1) is reported to be associated with osteogenic differentiation. However, the underlying molecular mechanism remains elusive. Therefore, this study evaluates the function of ACVR1 in osteogenic differentiation through the Wnt signaling pathway. The expression of osteocalcin (Oc) and osterix together with osteogenic differentiation and mineralization was examined in ACVR1-knockout (KO) mouse. Furthermore, the Wnt signaling pathway was inhibited in bone marrow stromal cells (BMSCs) of mice to explore the role of the Wnt signaling pathway in osteogenic differentiation by means of alkaline phosphatase (ALP) activity detection and evaluation of mineralized nodules and calcium content. Subsequently, the effect of ACVR1 on the Wnt signaling pathway was assessed by determining the expression of ACVR1, ß-catenin, glycogen synthase kinase 3 ß (GSK3ß), dickkopf-related protein 1 (DKK1), and frizzled class receptor 1 (FZD1). Both their effects on osteogenic differentiation were further evaluated by determination of Oc, osterix, and Runx2 expression. AVCR1 KO mice exhibited increased Oc and osterix expression and promoted bone resorption and formation. ACVR1-knockout was observed to activate the Wnt signaling pathway with an increase of ß-catenin and reductions in GSK3ß, DKK1, and FZD1. With the inhibited Wnt signaling pathway expression of Oc, osterix, and Runx2 was decreased, and ALP activity, mineralized nodule, and calcium content in cellular matrix were decreased as well, indicating that inactivation of the Wnt signaling pathway reduced the differentiation of BMSCs into osteoclasts. These findings indicate that ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice.

Crystal structure of PppA from Pseudomonas aeruginosa, a key regulatory component of type VI secretion systems.

The Type VI secretion system (T6SS) is a membrane protein complex related to inter-bacterial competitions and host-pathogen interactions in Pseudomonas aeruginosa. The T6SS is regulated by a great variety of regulatory mechanisms at multiple levels, including post-translational modification with threonine phosphorylation mediated by Ser/Thr protein kinase PpkA and phosphatase PppA. The T6SS is activated by PpkA via Thr phosphorylation of Fha, and PppA can antagonize PpkA. PppA is a PP2C-family protein phosphatase and plays a key role in the disassembly and reassembly of T6SS organelles. Herein, we report the first crystal structure of PppA from Pseudomonas aeruginosa, which was determined at a resolution of 2.10 Å. The overall structure consists of a bacteria PPM structural core and a flexible flap subdomain. PppA harbors a catalytic pocket containing two manganese ions which correspond to the canonical dinuclear metal center of Ser/Thr protein phosphatases including the bacterial PPM phosphatases and human PP2C. The flexibility and the diversity of the sequence of flap subdomain across the homologues might provide clues for substrates specific recognition of phosphatases.

A de novo mutation in DHD domain of SKI causing spina bifida with no craniofacial malformation or intellectual disability.

Shprintzen-Goldberg syndrome (SGS) is a rare systemic connective tissue disorder characterized by craniofacial, skeletal, and cardiovascular manifestations. It is associated with a significant risk of intellectual disability, a feature which distinguishes it from Marfan and Loeys-Dietz syndromes. SGS is mainly caused by mutations in the SKI gene, a repressor of TGF-ß activity. Most SKI mutations are found in exon 1 of the gene and are located in the R-SMAD domain, a proposed hotspot for de novo mutations. Here, we report on a de novo SKI mutation located in the DHD domain of SKI. By adding our finding to previously reported de novo SKI mutations, a new mutational hotspot in the DHD domain is proposed. Our patient presented with a lipomeningomyelocele, tethered cord, and spina bifida but with no SGS-related clinical findings apart from a marfanoid habitus and long slender fingers. Specifically, she did not have an intellectual disability, craniofacial, or cardiovascular abnormalities. By comparing the clinical findings on patients with mutations in the R-SMAD and DHD domains of SKI, we propose that mutations in those domains have different effects on TGF-ß activity during embryonic development with resulting phenotypic differences.

Computer-Based 3D Simulations to Formulate Preoperative Planning of Bridge Crane Technique for Thoracic Ossification of the Ligamentum Flavum.

BACKGROUND The bridge crane technique is a novel surgical technique for the treatment of thoracic ossification of the ligamentum flavum (TOLF), but its preoperative planning has not been studied well, which limits the safety and efficacy of surgery to some extent. The purpose of this study was to investigate the method of application and effect of computer-aided preoperative planning (CAPP) on the bridge crane technique for TOLF. MATERIAL AND METHODS This retrospective multi-center included 40 patients with TOLF who underwent the bridge crane technique from 2016 to 2018. According to the utilization of CAPP, patients were divided into Group A (with CAPP, n=21) and Group B (without CAPP, n=19). Comparisons of clinical and radiological outcomes were carried out between the 2 groups. RESULTS The patients in Group A had higher post-mJOA scores and IR of neurological function than those in Group B (p<0.05). Group A had shorter surgery time, fewer fluoroscopic images, and lower incidence of complications than Group B. In Group A, there was a high consistency of all the anatomical parameters between preoperative simulation and postoperative CT (p>0.05). In Group B, there were significant differences in 3 anatomical parameters between postoperative simulation and postoperative CT (p<0.05). In Group B, the patients with no complications had higher post-SVOR and lower SVRR and height of posterior suspension of LOC in postoperative CT than those in postoperative simulation (p<0.05). CONCLUSIONS CAPP can enable surgeons to control the decompression effect accurately and reduce the risk of related complications, which improves the safety and efficacy of surgery.