The potential therapeutic roles of dental pulp stem cells in spinal cord injury.

Spinal cord injury (SCI) can lead to serious functional disorders, which have serious impacts on patients and society. The current traditional treatments of SCI are not effective the injured spinal cord is difficult to repair and regenerate. In recent years, stem cell transplantation for the treatment of SCI has been a hot research topic. Dental pulp stem cells have strong abilities of self-renewal and multi-directional differentiation, and have been applied for tissue engineering and regenerative medicine. And dental pulp stem cells have certain advantages in neuro-regenetation, bringing new hope to biotherapy for SCI. This article reviews the characteristics of dental pulp stem cells and their research progress in the treatment of SCI.

Enabling safer, more potent oligonucleotide therapeutics with bottlebrush polymer conjugates.

Oligonucleotide therapeutics have the unique ability to address traditionally undruggable targets through various target engagement pathways. However, despite advances in chemically modified oligonucleotides and carrier-assisted delivery systems such as lipid nanoparticles and protein/peptide conjugates, the development of oligonucleotide drugs is still plagued with lackluster potency, narrow therapeutic window, poor delivery to non-liver target sites, and/or high potential for toxicity and unwanted immune system activation. In this perspective, we discuss an unconventional delivery solution based upon bottlebrush polymers, which overcomes many key challenges in oligonucleotide drug development. We address the molecular basis of the polymer's ability to enhance tissue bioavailability and drug potency, reduce side effects, and suppress anti-carrier immunity. Furthermore, we discuss the potential of the technology in advancing oligonucleotide-based therapies for non-liver targets.

Oxygen mediated oxidative couplings of flavones in alkaline water.

Catalyzed oxidative C-C bond coupling reactions play an important role in the chemical synthesis of complex natural products of medicinal importance. However, the poor functional group tolerance renders them unfit for the synthesis of naturally occurring polyphenolic flavones. We find that molecular oxygen in alkaline water acts as a hydrogen atom acceptor and oxidant in catalyst-free (without added catalyst) oxidative coupling of luteolin and other flavones. By this facile method, we achieve the synthesis of a small collection of flavone dimers and trimers including naturally occurring dicranolomin, philonotisflavone, dehydrohegoflavone, distichumtriluteolin, and cyclodistichumtriluteolin. Mechanistic studies using both experimental and computational chemistry uncover the underlying reasons for optimal pH, oxygen availability, and counter-cations that define the success of the reaction. We expect our reaction opens up a green and sustainable way to synthesize flavonoid dimers and oligomers using the readily available monomeric flavonoids isolated from biomass and exploiting their use for health care products and treatment of diseases.

A Long-Circulating Vector for Aptamers Based upon Polyphosphodiester-Backboned Molecular Brushes.

Aptamers face challenges for use outside the ideal conditions in which they are developed. These difficulties are most palpable in vivo due to nuclease activities, rapid clearance, and off-target binding. Herein, we demonstrate that a polyphosphodiester-backboned molecular brush can suppress enzymatic digestion, reduce non-specific cell uptake, enable long blood circulation, and rescue the bioactivity of a conjugated aptamer in vivo. The backbone along with the aptamer is assembled via solid-phase synthesis, followed by installation of poly(ethylene glycol) (PEG) side chains using a two-step process with near-quantitative efficiency. The synthesis allows for precise control over polymer size and architecture. Consisting entirely of building blocks that are generally recognized as safe for therapeutics, this novel molecular brush is expected to provide a highly translatable route for aptamer-based therapeutics.

Surgical treatment of spinal deformities in spinal muscular atrophy: a single-center experience from China.

PURPOSE: To report the clinical characteristics and surgical outcomes of scoliosis in patients with spinal muscular atrophy (SMA) from Mainland China. METHODS: Nineteen patients were retrospectively analyzed. Demographic, anthropometric and respiratory parameters were collected preoperatively. Surgical program was analyzed. Radiographic data were measured perioperatively. Motor status, ventilation support, sitting ability and respiratory symptoms were evaluated preoperatively and at final follow-up. RESULTS: Age at surgery was 17.08 (12.83, 20.08) years. More than 40% of patients were diagnosed with low weight. Pulmonary dysfunction was observed in all patients. All patients received posterior spinal fusion (PSF). Sacroiliac fixation with sacral-2 alar iliac technique was used in 16 patients. Major curve correction rate was 54.87 ± 16.14%. Pelvic obliquity correction rate was 63.84 ± 23.70%. T1-T12 height, space-available-for-lung ratio and thoracic transverse diameter were increased (p < 0.001). Percentage of patients capable of sitting independently increased from 26.32% preoperatively to 73.68% at final follow-up. Cumulative scores of sitting-related items in muscular dystrophy spine questionnaire improved from 19.11 ± 5.40 preoperatively to 26.21 ± 5.20 at final follow-up. Total scores of symptomatic domains in St. George's Respiratory Questionnaire decreased from 4 (2, 12) preoperatively to 1 (0, 3) at final follow-up. CONCLUSIONS: SMA patients in China always present severe scoliosis at late adolescence, accompanied with high proportion of low weight and pulmonary dysfunction. PSF is effective for the correction of scoliosis and pelvic obliquity and the improvement of thoracic morphology. Sitting ability and respiratory symptoms were improved postoperatively.

Structure-Activity Relationship (SAR) of Flavones on Their Anti-Inflammatory Activity in Murine Macrophages in Culture through the NF-κB Pathway and c-Src Kinase Receptor.

Flavones benefit human health through their anti-inflammatory activity; however, their structure-activity relationship is unclear. Herein, we selected 15 flavones with the same backbone but different substituents and systematically assessed their anti-inflammatory activities in RAW 264.7 regarding cellular-Src kinase (c-Src) affinity, suppression of IκBα phosphorylation, inhibition of nitric oxide (NO) and inducible nitric oxidase (iNOS) production, and downregulation of genes of proinflammatory cytokines interleukin 6 (IL-6), interleukin 1ß (IL-1ß), and tumor necrosis factor α (TNF-α). Overall, our results showed that the double bond between C2-C3 and C3'- and C4'-OH promoted anti-inflammatory activity, while C8- and C5'-OH and the methoxy group on C4' attenuated the overall anti-inflammatory and antioxidant activities. The hydroxyl groups at other positions exhibited more complicated functions. The two most effective flavones are 3',4'-dihydroxyflavone and luteolin with inhibitory concentration (IC50) values for inhibiting the LPS-induced nitric oxide level are 9.61 ± 1.36 and 16.90 ± 0.74 µM, respectively. Furthermore, they suppressed the production of iNOS by approximately 90% and inhibited IL-1ß and IL-6 by more than 95%. Taken together, our results established a relationship between the flavone structure and anti-inflammatory activity in vitro.

Structure and Anti-Inflammatory Activity Relationship of Ergostanes and Lanostanes in Antrodia cinnamomea.

Antrodia cinnamomea is a precious edible mushroom originating from Taiwan that has been popularly used for adjuvant hepatoprotection and anti-inflammation; however, the chemical principle for its anti-inflammatory activity has not been elucidated, which prevents the quality control of related products. Using the RAW264.7 model for the anti-inflammatory activity assay as a guide, we reported the isolation and structural elucidation of three potent anti-inflammatory compounds from isolated ergostanes (16) and lanostanes (6). Their structures were elucidated on the basis of spectroscopic data analysis including NMR and HR-QTOF-MS. Particularly, the absolute configurations of (25R)-antcin K, (25R)-antcin A, versisponic acid D, and (25R)-antcin C were determined by single crystal X-ray diffraction (XRD). The representative and most promising compound antcin A was shown to suppress pro-inflammatory biomolecule release via the down-regulation of iNOS and COX-2 expression through the NF-κB pathway while the mRNA levels of IL-1ß, TNF-α and IL-6 were also decreased. The high dependency on structural variation and activity suggests that there might be special biological targets for antcin A. Our work makes it possible to develop evidence-based dietary supplements from Antrodia cinnamomea based on anti-inflammatory constituents.

Expanding the mutation and phenotype spectrum of MYH3-associated skeletal disorders.

Pathogenic variants in MYH3 cause distal arthrogryposis type 2A and type 2B3 as well as contractures, pterygia and spondylocarpotarsal fusion syndromes types 1A and 1B. These disorders are ultra-rare and their natural course and phenotypic variability are not well described. In this study, we summarize the clinical features and genetic findings of 17 patients from 10 unrelated families with vertebral malformations caused by dominant or recessive pathogenic variants in MYH3. Twelve novel pathogenic variants in MYH3 (NM_002470.4) were identified: three of them were de novo or inherited in autosomal dominant way and nine were inherited in autosomal recessive way. The patients had vertebral segmentation anomalies accompanied with variable joint contractures, short stature and dysmorphic facial features. There was a significant phenotypic overlap between dominant and recessive MYH3-associated conditions regarding the degree of short stature as well as the number of vertebral fusions. All monoallelic variants caused significantly decreased SMAD3 phosphorylation, which is consistent with the previously proposed pathogenic mechanism of impaired canonical TGF-ß signaling. Most of the biallelic variants were predicted to be protein-truncating, while one missense variant c.4244T>G,p.(Leu1415Arg), which was inherited in an autosomal recessive way, was found to alter the phosphorylation level of p38, suggesting an inhibition of the non-canonical pathway of TGF-ß signaling. In conclusion, the identification of 12 novel pathogenic variants and overlapping phenotypes in 17 affected individuals from 10 unrelated families expands the mutation and phenotype spectrum of MYH3-associated skeletal disorders. We show that disturbances of canonical or non-canonical TGF-ß signaling pathways are involved in pathogenesis of MYH3-associated skeletal fusion (MASF) syndrome.

A genotype-first analysis in a cohort of Mullerian anomaly.

Müllerian anomaly (M.A.) is a group of congenital anatomic abnormalities caused by aberrations of the development process of the Müllerian duct. M.A. can either be isolated or be involved in Mendelian syndromes, such as Dandy-Walker syndrome, Holt-Oram syndrome and Bardet-Biedl syndrome, which are often associated with both uterus and kidney malformations. In this study, we applied a genotype-first approach to analyze the whole-exome sequencing data of 492 patients with M.A. Six potential pathogenic variants were found in five genes previously related to female urogenital deformities (PKD1, SON, SALL1, BMPR1B, ITGA8), which are partially overlapping with our patients' phenotypes. We further identified eight incidental findings in seven genes related to Mendelian syndromes without known association with reproductive anomalies (TEK, COL11A1, ANKRD11, LEMD3, DLG5, SPTB, BMP2), which represent potential phenotype expansions of these genes.

LRP5-Mediated Lipid Uptake Modulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells.

Nutritional microenvironment determines the specification of progenitor cells, and lipid availability was found to modulate osteogenesis in skeletal progenitors. Here, we investigated the implications of lipid scarcity in the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) and the role of low-density lipoprotein receptor-related protein 5 (LRP5), a co-receptor transducing canonical Wnt/beta-catenin signals, in BMSC lipid uptake during osteogenesis. The osteogenic differentiation of murine BMSCs was suppressed by lipid scarcity and partially rescued by additional fatty acid treatment with oleate. The enhancement of osteogenesis by oleate was found to be dosage-dependent, along with the enhanced activation of beta-catenin and Wnt target genes. Conditional knockout (CKO) of Lrp5 gene in murine mesenchymal lineage using Lrp5 fl/fl ;Prrx1-cre mice led to decreased bone quality and altered fat distribution in vivo. After Lrp5 ablation using adenoviral Cre-recombinase, the accumulation of lipid droplets in BMSC cytoplasm was significantly reduced, and the osteogenesis of BMSCs was suppressed. Moreover, the impaired osteogenesis due to either lipid scarcity or Lrp5 ablation could be rescued by recombinant Wnt3a protein, indicating that the osteogenesis induced by Wnt/beta-catenin signaling was independent of LRP5-mediated lipid uptake. In conclusion, lipid scarcity suppresses BMSC osteogenic differentiation. LRP5 plays a role in the uptake of lipids in BMSCs and therefore mediates osteogenic specification.

Disruptive NADSYN1 Variants Implicated in Congenital Vertebral Malformations.

Genetic perturbations in nicotinamide adenine dinucleotide de novo (NAD) synthesis pathway predispose individuals to congenital birth defects. The NADSYN1 encodes the final enzyme in the de novo NAD synthesis pathway and, therefore, plays an important role in NAD metabolism and organ embryogenesis. Biallelic mutations in the NADSYN1 gene have been reported to be causative of congenital organ defects known as VCRL syndrome (Vertebral-Cardiac-Renal-Limb syndrome). Here, we analyzed the genetic variants in NADSYN1 in an exome-sequenced cohort consisting of patients with congenital vertebral malformations (CVMs). A total number of eight variants in NADSYN1, including two truncating variants and six missense variants, were identified in nine unrelated patients. All enrolled patients presented multiple organ defects, with the involvement of either the heart, kidney, limbs, or liver, as well as intraspinal deformities. An in vitro assay using COS-7 cells demonstrated either significantly reduced protein levels or disrupted enzymatic activity of the identified variants. Our findings demonstrated that functional variants in NADSYN1 were involved in the complex genetic etiology of CVMs and provided further evidence for the causative NADSYN1 variants in congenital NAD Deficiency Disorder.

ADAMTS5 in Osteoarthritis: Biological Functions, Regulatory Network, and Potential Targeting Therapies.

ADAMTS5 is involved in the pathogenesis of OA. As the major aggrecanase-degrading articular cartilage matrix, ADAMTS5, has been regarded as a potential target for OA treatment. We here provide an updated insight on the regulation of ADAMTS5 and newly discovered therapeutic strategies for OA. Pathophysiological and molecular mechanisms underlying articular inflammation and mechanotransduction, as well as chondrocyte hypertrophy were discussed, and the role of ADAMTS5 in each biological process was reviewed, respectively. Senescence, inheritance, inflammation, and mechanical stress are involved in the overactivation of ADAMTS5, contributing to the pathogenesis of OA. Multiple molecular signaling pathways were observed to modulate ADAMTS5 expression, namely, Runx2, Fgf2, Notch, Wnt, NF-κB, YAP/TAZ, and the other inflammatory signaling pathways. Based on the fundamental understanding of ADAMTS5 in OA pathogenesis, monoclonal antibodies and small molecule inhibitors against ADAMTS5 were developed and proved to be beneficial pre-clinically both in vitro and in vivo. Recent novel RNA therapies demonstrated potentials in OA animal models. To sum up, ADAMTS5 inhibition and its signaling pathway-based modulations showed great potential in future therapeutic strategies for OA.

Anti-Inflammation Activity of Flavones and Their Structure-Activity Relationship.

Flavones are an important class of bioactive constituents in foods, and their structural diversity enables them to interact with different targets. In particular, flavones are known for their anti-inflammatory activity. Herein, we summarized commonly applied in vitro, in vivo, and clinical models in testing anti-inflammatory activity of flavones. The anti-inflammatory structure-activity relationship of flavones was systematically mapped and supported with cross comparisons of that with flavanones, flavanols, and isoflavones. Hydroxyl groups (-OH) are indispensable for the anti-inflammatory function of flavones, and -OH at the C-5 and C-4' positions enhance while -OH at the C-6, C-7, C-8, and C-3' positions attenuate their activity. Moreover, the C2-C3 single bond, -OH at the C-3 and B-ring positions undermine flavone aglycones' activity. Most of the flavone aglycones function through NF-κB, MAPK, and JNK-STAT pathways, and their possible cell binding targets are kinase, aryl hydrocarbon receptor (AhR), G-protein coupled receptors, and estrogen receptors. However, the structure and anti-inflammatory activity relationship of flavones were unclear, and further research shall be conducted to close the gap in order to guide development of evidence-based functional foods.

Heterozygous Recurrent Mutations Inducing Dysfunction of ROR2 Gene in Patients With Short Stature.

PURPOSE: ROR2, a member of the ROR family, is essential for skeletal development as a receptor of Wnt5a. The present study aims to investigate the mutational spectrum of ROR2 in children with short stature and to identify the underlying molecular mechanisms. METHODS: We retrospectively analyzed clinical phenotype and whole-exome sequencing (WES) data of 426 patients with short stature through mutation screening of ROR2. We subsequently examined the changes in protein expression and subcellular location in ROR2 caused by the mutations. The mRNA expression of downstream signaling molecules of the Wnt5a-ROR2 pathway was also examined. RESULTS: We identified 12 mutations in ROR2 in 21 patients, including 10 missense, one nonsense, and one frameshift. Among all missense variants, four recurrent missense variants [c.1675G > A(p.Gly559Ser), c.2212C > T(p.Arg738Cys), c.1930G > A(p.Asp644Asn), c.2117G > A(p.Arg706Gln)] were analyzed by experiments in vitro. The c.1675G > A mutation significantly altered the expression and the cellular localization of the ROR2 protein. The c.1675G > A mutation also caused a significantly decreased expression of c-Jun. In contrast, other missense variants did not confer any disruptive effect on the biological functions of ROR2. CONCLUSION: We expanded the mutational spectrum of ROR2 in patients with short stature. Functional experiments potentially revealed a novel molecular mechanism that the c.1675G > A mutation in ROR2 might affect the expression of downstream Wnt5a-ROR2 pathway gene by disturbing the subcellular localization and expression of the protein.

Predicting Negative Outcomes While Awaiting Dental Treatment Under General Anesthesia.

Purpose: To determine the wait time for dental treatment under general anesthesia (GA) and its impact on clinical outcomes in a pediatric population at federally qualified health centers in the United States.
Methods: Data were collected from 566 pediatric subjects who underwent dental rehabilitation under GA between July 1, 2013, and June 30, 2014. One-way analysis of variance and linear regression analyses were performed.
Results: Patients waited 110.6 days (±standard deviation: 103.9 days) between the initial and treatment visits. Regression analysis demonstrated that prolonged wait time was a significant predictor for an increased number of preoperative visits and more teeth treated than planned. Among the 25.1 percent of patients who returned for follow-up after surgery, 18.6 percent presented with pain, swelling, or broken/ displaced restorations. The Canadian diagnostic code system was associated with the American Society of Anesthesiologists classification system (P <0.001) and was not coincident with wait time.
Conclusion: Longer wait time was associated with continuous pain, more teeth treated than planned, and more frequent pre- and post-operative visits. Wait time was predictive of a higher number of preoperative visits. Initial visit pain, and extra- and intra-oral swelling were associated with the Canadian diagnostic system.

Variants Affecting the C-Terminal of CSF1R Cause Congenital Vertebral Malformation Through a Gain-of-Function Mechanism.

CSF1R encodes the colony-stimulating factor 1 receptor which regulates the proliferation, differentiation, and biological activity of monocyte/macrophage lineages. Pathogenic variants in CSF1R could lead to autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia or autosomal recessive skeletal dysplasia. In this study, we identified three heterozygous deleterious rare variants in CSF1R from a congenital vertebral malformation (CVM) cohort. All of the three variants are located within the carboxy-terminal region of CSF1R protein and could lead to an increased stability of the protein. Therefore, we established a zebrafish model overexpressing CSF1R. The zebrafish model exhibits CVM phenotypes such as hemivertebral and vertebral fusion. Furthermore, overexpression of the mutated CSF1R mRNA depleted of the carboxy-terminus led to a higher proportion of zebrafish with vertebral malformations than wild-type CSF1R mRNA did (p = 0.03452), implicating a gain-of-function effect of the C-terminal variant. In conclusion, variants affecting the C-terminal of CSF1R could cause CVM though a potential gain-of-function mechanism.

Transplantation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Improves Cartilage Repair in a Rabbit Model.

The aim of this study was to investigate the therapeutic efficacy and safety of transplanting human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the treatment of cartilage injury. First, the articular cartilage defect model in rabbits was constructed. Then, the identified hUCB-MSCs and rabbit bone marrow stem cells (rBM-MSCs) were transplanted into the bone defect, respectively, and the cartilage repair effect was observed by hematoxylin-eosin (HE) staining and immunohistochemistry. Besides, the glycosaminoglycan (GAG) content and biomechanics of the restoration area were also evaluated. In our study, hUCB-MSCs and rBM-MSCs exhibited typical MSC characteristics, with positive expressions of CD73, CD105, and CD90 and negative for CD45, CD34, CD14, and HLA-DR. After the transplantation of hUCB-MSCs and rBM-MSCs, the overall quality of cartilage tissue was significantly improved, and the recipients did not show significant side effects in general. However, the expression of matrix metalloproteinase-13 (MMP-13) in the de novo tissues of the hUCB-MSCs and rBM-MSCs groups was both increased, indicating that the novel tissues may have some potential osteoarthritic changes. In conclusion, our results suggest the therapeutic effect of hUCB-MSCs transplantation in cartilage regeneration, providing a promising future in the clinical treatment of cartilage injury.

Diagnostic management of inpatients with a positive D-dimer test: developing a new clinical decision-making rule for pulmonary embolism.

BACKGROUND: A positive D-dimer test has high sensitivity but relatively poor specificity for the diagnosis of pulmonary embolism, causing difficulty for clinicians unskilled in pulmonary embolism diagnosis in determining whether a patient with a positive D-dimer test needs to undergo computed tomographic pulmonary angiography. OBJECTIVES: We sought to develop a new clinical decision-making rule based on a positive D-dimer result to predict the probability of pulmonary embolism and to guide clinicians in making decisions regarding the need for computed tomographic pulmonary angiography. METHODS: We conducted a prospective, multicenter study in three hospitals in China. A total of 3014 inpatients with positive D-dimer results were included. In the derivation group, we built a multivariate logistic regression model and deduced a regression equation from which our score was derived. Finally, we validated the score in an independent cohort. RESULTS: Our score included nine variables (points): chest pain (1.4), chest tightness (2.3), shortness of breath (3.6), hemoptysis (3.4), heart rate ≥100 beats/min (3.6), blood gas analysis (2.9), electrocardiogram presenting a typical S1Q3T3 pattern (4.1), electrocardiogram findings (2.4), and ultrasonic cardiogram findings (3.7). The sensitivities and specificities were 100% and 86.94%, respectively, in the derivation group and 100% and 90.82%, respectively, in the validation group. Additionally, the observed and predicted proportions of patients who underwent computed tomographic pulmonary angiography were 16.82% and 10.76%, respectively, in the derivation group and 18.72% and 11.40%, respectively, in the validation group. CONCLUSIONS: The new score can categorize inpatients with a positive D-dimer test as pulmonary embolism-likely or pulmonary embolism-unlikely, thus reducing unnecessary computed tomographic pulmonary angiography examinations.

Diagnostic yield and clinical impact of exome sequencing in early-onset scoliosis (EOS).

BACKGROUND: Early-onset scoliosis (EOS), defined by an onset age of scoliosis less than 10 years, conveys significant health risk to affected children. Identification of the molecular aetiology underlying patients with EOS could provide valuable information for both clinical management and prenatal screening. METHODS: In this study, we consecutively recruited a cohort of 447 Chinese patients with operative EOS. We performed exome sequencing (ES) screening on these individuals and their available family members (totaling 670 subjects). Another cohort of 13 patients with idiopathic early-onset scoliosis (IEOS) from the USA who underwent ES was also recruited. RESULTS: After ES data processing and variant interpretation, we detected molecular diagnostic variants in 92 out of 447 (20.6%) Chinese patients with EOS, including 8 patients with molecular confirmation of their clinical diagnosis and 84 patients with molecular diagnoses of previously unrecognised diseases underlying scoliosis. One out of 13 patients with IEOS from the US cohort was molecularly diagnosed. The age at presentation, the number of organ systems involved and the Cobb angle were the three top features predictive of a molecular diagnosis. CONCLUSION: ES enabled the molecular diagnosis/classification of patients with EOS. Specific clinical features/feature pairs are able to indicate the likelihood of gaining a molecular diagnosis through ES.