Duplications involving the long range HMX1 enhancer are associated with human isolated bilateral concha-type microtia.

BACKGROUND: Microtia is a congenital anomaly of ear that ranges in severity from mild structural abnormalities to complete absence of the outer ears. Concha-type microtia is considered to be a mild form. The H6 family homeobox 1 transcription factor gene (HMX1) plays an important role in craniofacial structures development. Copy number variations (CNVs) of a downstream evolutionarily conserved enhancer region (ECR) of Hmx1 associated with ear and eye abnormalities have been reported in different animals, but not yet in human. To date, no genetic defects responsible for isolated human microtia has been reported except for mutations in HOXA2. Here we recruited five Chinese families with isolated bilateral concha-type microtia, and attempt to identify the underlying genetic causes. METHODS: Single Nucleotide polymorphism (SNP) array was performed to map the disease locus and detect CNVs on a genome scale primarily in the largest family (F1). Whole genome sequencing was performed to screen all SNVs and CNVs in the candidate disease locus. Array comparative genomic hybridization (aCGH) was then performed to detect CNVs in the other four families, F2-F5. Quantitative real-time polymerase chain reaction (qPCR) was used to validate and determine the extent of identified CNVs containing HMX1-ECR region. Precise breakpoints in F1 and F2 were identified by gap-PCR and sanger sequencing. Dual-luciferase assays were used to detect the enhancer function. qPCR assays were also used to detect HMX1-ECR CNVs in 61 patients with other types mictrotia. RESULTS: Linkage and haplotype analysis in F1 mapped the disease locus to a 1.9 Mb interval on 4p16.1 containing HMX1 and its downstream ECR region. Whole genome sequencing detected no potential pathogenic SNVs in coding regions of HMX1 or other genes within the candidate disease locus, but it detected a 94.6 Kb duplication in an intergenic region between HMX1 and CPZ. aCGH and qPCRs also revealed co-segregated duplications in intergenic region downstream of HMX1 in the other four families. The 21.8 Kb minimal overlapping region encompassing the core sequences consensus with mouse ECR of Hmx1. Luciferase assays confirmed the enhancer function in human sequences, and proved that HOXA2 could increase its enhancer activity. No CNVs were detected in HMX1-ECR regions in 61 patients with other type of microtia. CONCLUSION: Duplications involving long range HMX1 enhancers are associated with human isolated bilateral concha-type microtia. We add to evidences in human that copy number variations in HMX1-ECR associates with ear malformations, as in other species. This study also provides an additional example of functional conserved non-coding elements (CNEs) in humans.

Two Pathways Regulate Differential Expression of nAChRs Between the Orbicularis Oris and Gastrocnemius.

BACKGROUND: We previously demonstrated differential expression of nicotinic acetylcholine receptors (nAChRs) in the facial nerve-innervated orbicularis oris and somatic nerve-innervated gastrocnemius, which contribute to different sensitivities to muscle relaxants. Furthermore, the orbicularis oris exhibits less sensitivity to muscle relaxants after facial nerve injury, which is also related to upregulation of nAChRs. Here, we explored the regulatory mechanism for the different expression patterns. Because the agrin/Lrp4/MuSK/rapsyn and neuregulin1/ErbB signaling pathways are indispensable for maintaining the expression of nAChRs, we examined the activity of these two signaling pathways in gastrocnemius and orbicularis oris innervated by normal or injured facial nerves. MATERIALS AND METHODS: A quantitative analysis of these two signaling pathways was realized by immunofluorescence, and immunoprecipitation was applied to detect the level of phosphorylated MuSK in the gastrocnemius and orbicularis oris innervated by normal or injured facial nerves in adult rats. RESULTS: ErbB and the phosphorylated MuSK were expressed more in orbicularis oris than in gastrocnemius (P < 0.05). No significant difference was found in the expression of agrin/Lrp4/MuSK/rapsyn. After facial nerve injury, the level of agrin and the percentage of phosphorylated MuSK decreased significantly, although the expression levels of MuSK, rapsyn, and neuregulin1/ErbB were highly upregulated. CONCLUSIONS: Differential expression of the neuregulin1/ErbB signaling pathway may account for the different expression patterns of nAChRs at the neuromuscular junctions of the orbicularis oris and gastrocnemius. Overexpression of MuSK and rapsyn may contribute to upregulation of nAChRs after facial nerve injury.

Rational Design and Fabrication of ZnONPs Functionalized Sericin/PVA Antimicrobial Sponge.

The interests of developing antimicrobial biomaterials based on silk sericin from Bombyx mori cocoon, have been shooting up in the last decades. Sericin is a valuable natural protein owing to its hydrophilicity, biodegradability, and biocompatibility. Here, we fabricated a sponge with antibacterial capacities for potential wound dressing application. By co-blending of sericin, polyvinyl alcohol (PVA) and zinc oxide nanoparticles (ZnONPs), the ZnONPs-sericin/PVA composite sponge (ZnONPs-SP) was successfully prepared after freeze-drying. Scanning electron microscopy showed the porous structure of ZnONPs-SP. Energy dispersive spectroscopy indicated the existence of Zn in the sponge. X-ray diffractometry revealed the hexagonal wurtzite structure of ZnONPs. Fourier transform infrared spectroscopy showed the biologic coupling of ZnONPs and sericin resulted in a decrease of α-helix and random coil contents, and an increase of ß-sheet structure in the sponge. The swelling experiment suggested ZnONPs-SP has high porosity, good hydrophilicity, and water absorption capability. The plate bacterial colony counting coupled with growth curve assays demonstrated that the composite sponge has an efficiently bacteriostatic effect against Staphylococcus aureus and Escherichia coli, respectively. Furthermore, the cell compatibility analysis suggested the composite sponge has excellent cytocompatibility on NIH3T3 cells. In all, ZnONPs-SP composite sponge has significant potentials in biomaterials such as wound dressing and tissue engineering.

Modified Methods of Fabricating Helix and Antihelix in Total Auricular Reconstruction Based on Different Length of Eighth Costal Cartilage.

BACKGROUND: Attaining an ideal appearance of the reconstructed ear depends primarily on the fabricated cartilaginous ear framework. Despite the role of eighth costal cartilage, the length of the eighth rib cartilage is difficult to anticipate, and growth of the eighth cartilage is variable. The authors discussed modified methods of fabricating helix and antihelix based on different length of eighth costal cartilage. METHODS: Based on the actual length of the eighth costal cartilage, patients were divided into group A and group B. In group A, the eighth costal cartilage was divided into 2 parts. Part I was used to fashion the helix, whereas part II was used to fabricate the antihelix and superior crus. In group B, the seventh costal cartilage was cut into 3 parts. Part I and part II were used to fashion the helix, antihelix, and superior crus as group A did. Part III was assembled to the lateral part of the framework to form the inferior crus. RESULTS: A total of 56 patients underwent auricular reconstruction adopting the modified techniques between 2015 and 2016. Three cases have been selected to illustrate the favorable result achieved. They revealed that the helix, antihelix, superior crus, and inferior crus all appeared distinct and presented a favorable result of the contour of the reconstructed auricle. CONCLUSIONS: Based on different length of eighth costal cartilage, modified methods of fabricating helix and antihelix make full use of the autogenous costal cartilage, elevate anatomical details, and enhance the overall aesthetics of reconstructed ear. The great majority of patients have satisfactory surgical outcomes, demonstrating that personalized treatment is necessary.

Polydopamine-Assisted Silver Nanoparticle Self-Assembly on Sericin/Agar Film for Potential Wound Dressing Application.

Silver nanoparticles (AgNPs) are extensively applied for their broad-spectrum and excellent antibacterial ability in recent years. Polydopamine (PDA) has great advantages for synthesizing large amounts of AgNPs, as it has multiple sites for silver ion binding and phenolic hydroxyl structure to reduce silver ions to AgNPs. Here, we mixed sericin and agar solution and dried at 65 °C to prepare a sericin (SS)/Agar composite film, and then coated polydopamine (PDA) on the surface of SS/Agar film by soaking SS/Agar film into polydopamine solution, subsequently synthesizing high-density AgNPs with the assistance of PDA to yield antibacterial AgNPs-PDA- SS/Agar film. Scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) spectra indicated the successful synthesis of high-density AgNPs on the surface of PDA-SS/Agar film. PDA coating and AgNPs modification did not affect the structure of sericin and agar. Furthermore, water contact angle, water absorption and mechanical property analysis showed that AgNPs-PDA-SS/Agar film had excellent hydrophilicity and proper mechanical properties. Inhibition zone and growth curve assays suggested the prepared film had excellent and long-lasting antibacterial ability. In addition, it had excellent cytocompatibility on the fibroblast NIH/3T3 cells. The film shows great potential as a novel kind of wound dressing.

Sternal Development and Variations and Anomalies in Patients With Microtia: Evaluation Using 3-Dimensional Computed Tomography.

OBJECTIVE: The objectives of this study were to evaluate sternal development and variations in patients with microtia and to identify the incidence of congenital sternal anomalies and then to investigate the interaction between microtia and sternal anomalies. METHODS: A total of 212 consecutive patients received a preoperative 3-dimensional chest computed tomography. A retrospective study was performed with the clinical and imaging data from November 2014 to July 2015. Descriptive statistics, analysis of variance, Spearman analysis, χ test, and Fisher χ test were performed for statistics analysis. RESULTS: We evaluated the ossification centers and developmental variations in the manubrium and body, as well as the xiphoid process, manubriosternal and sternoxiphoidal fusion, and sternal anomalies. Significant variations were observed from person to person. Sternal foramen was detected in 6 male patients (2.8%). All foramina were located in the inferior part of the body. Sternal cleft was observed in 4 cases (1.9%), of which 2 were accompanied by a foramen in the distal part of the sternum. CONCLUSIONS: The development of the different components of the sternum is a process with wide variation among patients with microtia. A different distribution of mesosternal types I to II among our population age range was found, and the incidence of sternal foramina was lower in patients with microtia.

Analysis of Rib Cartilages Anomalies in Patients With Microtia.

OBJECTIVE: The objective of this study was to identify the characteristics and incidence of rib cartilages anomalies in patients with microtia and to investigate the interaction between microtia and rib cartilages anomalies. METHODS: A total of 214 consecutive patients received a preoperative 3-dimensional chest computed tomography. A retrospective study was performed with the clinical and imaging data from November 2014 to July 2015. Pearson X2 test and Spearman analysis were used to analyze the interaction between microtia and rib cartilages anomalies. RESULTS: With the 3-dimensional chest computed tomography images, a total of 62 patients (29.0%) were documented with rib cartilages anomalies including 4 patients (1.9%) with numerical variations, 13 patients (6.1%) with structural changes, and 51 patients (23.8%) with abnormal articulations. The incidence of rib anomalies (P = 0.035) varied with grades of microtia. CONCLUSIONS: The incidence of rib cartilages anomalies was high in patients with microtia. The authors observed a higher incidence of costal cartilages anomalies in patients with a more serious grade of microtia. Microtia with rib cartilages anomalies may involve a new syndrome previously undiscovered or just another extension with the very wide spectrum of microtia.

Resistance to rocuronium of rat diaphragm as compared with limb muscles.

BACKGROUND: Skeletal muscles are composed of different muscle fiber types. We investigated the different potency to rocuronium among diaphragm (DIA), extensor digitorum longus (EDL), and soleus (SOL) in vitro as well as to investigate the differences of acetylcholine receptors (AChRs) among these three typical kinds of muscles. MATERIALS AND METHODS: The isolated left hemidiaphragm nerve-muscle preparations, the EDL sciatic nerve-muscle preparations, and the SOL sciatic nerve-muscle preparations were established to evaluate the potency to rocuronium. Concentration-response curves were constructed and the values of IC50 were obtained. The density of AChRs at the end plate and the number of AChRs per unit fiber cross fiber area (CSA), AChR affinity for muscle relaxants were evaluated. RESULTS: The concentration-twitch tension curves of rocuronium were significantly different. The curves demonstrated a shift to the right of the DIA compared with the EDL and SOL (P < 0.01), whereas no significant difference was observed between EDL and SOL (P > 0.05). IC50 was significantly largest in DIA, second largest in SOL, and smallest in EDL (P < 0.05). The number of AChRs per unit fiber CSA was largest in DIA, second largest in EDL, and smallest in SOL (P < 0.01 or P < 0.05). The DIA showed the lowest affinity of the AChRs, whereas the SOL showed the highest affinity. CONCLUSIONS: The resistance to rocuronium of DIA compared with EDL and SOL was verified. The DIA was characterized by the largest number of AChRs per unit fiber CSA and the lowest affinity of the AChRs. Although compared with SOL, EDL was proved to have larger number of AChRs per unit fiber CSA and the lower affinity of the AChRs. These findings may be the mechanisms of different potency to rocuronium in DIA, EDL, and SOL. The results of the study could help to explain the relationship between different composition of muscle fibers and the potency to muscle relaxants. Extra caution should be taken in clinical practice when monitoring muscle relaxation in anesthetic management using different muscles.

Urbanization, economic agglomeration and economic growth.

Are Chinese cities too agglomerated, and are production factors continuing to flow to cities?In recent years, research on this issue has attracted more and more attention from academics, for the research on this issue, academics generally believe that urbanization has a positive role in promoting economic growth, but whether urbanization will bring economic agglomeration and what is the role of economic agglomeration on the development of the economy? The research on this issue is still insufficient, therefore, Based on the panel data of 27 provinces from 2006 to 2020, it is generally believed that urbanization has a positive role in promoting economic growth. This paper adds economic agglomeration factors and uses a spatial econometric model to test the spillover effect of urbanization growth. The main conclusions are as follows: First, urbanization and economic agglomeration have a positive effect on economic growth, and there is a spatial spillover effect. Secondly, the advancement of urbanization is closely associated with economic growth, particularly in relation to the development of economic agglomeration. When economic agglomeration is based on a single-center model, it tends to impede urbanization and economic progress. In contrast, an economic agglomeration model with multiple centers has the potential to stimulate urbanization and further enhance economic growth. Thirdly, it is worth noting that the influence of urbanization on economic growth varies across different regions. Factors such as geographical location, resource availability, and local economic conditions contribute to this regional variation.

Evaluation and clinical significance of contrast-enhanced ultrasound on changes in liver blood flow perfusion after TIPS surgery.

To investigate the clinical value of contrast-enhanced ultrasound in the prediction of hepatic encephalopathy (HE) in patients with hepatitis B cirrhosis after intrahepatic portal-systemic shunt via jugular vein. In this retrospective study, we collected data from 75 patients with hepatitis B, cirrhosis, and portal hypertension who underwent jugular intrahepatic portosystemic shunt from February 2019 to February 2022. The diagnostic instrument used was the TOSHIBA Aplio500 color Doppler ultrasound with contrast-enhanced ultrasound capabilities. The trial group comprised 20 patients with HE within 3 months postsurgery, while the control group (CG) included 55 patients without HE within the same postoperative period. All patients underwent various examinations before and within 48 hours after surgery, including observation of liver and spleen size and stent position, as well as assessment of blood flow direction in portal and hepatic veins. Subsequently, contrast-enhanced ultrasound was employed to examine and observe perfusion changes of contrast agents in hepatic veins, hepatic arteries, and portal veins (PV). Changes in PV pressure gradient, intrahepatic, and stent blood flow perfusion (BFP) were explored in both postoperative trials and CGs. The trial group exhibited higher BFP volume, PV pressure gradient difference, and percentage decrease compared to the CG. A weak positive correlation was observed between blood flow within the liver stent and PV pressure gradient difference, as well as the percentage decrease in PV pressure gradient. The correlation coefficient between blood flowing perfusion volume within the stent and the difference in PV pressure gradient was R = 0.415 (P = .000). The correlating coefficient between BFP amount within the stent and the percentage decrease in PV pressure gradient was R = 0.261 (P = .027). The area under the receiver operating characteristic curve for stent perfusion volume, difference in PV pressure gradient, and percentage decrease in PV pressure gradient was 0.691, 0.759, and 0.742, respectively. An increase in PV pressure gradient accelerates blood flow within the stent, predisposing to HE. Changes in hepatic BFP following transjugular intrahepatic portosystemic shunt can effectively predict the occurrence of HE, demonstrating significant clinical relevance.

[Clinical Significance of the Levels of Peripheral Blood Tregs and Cytokines IL-35, TGF-ß and IL-10 in Hemophilia A Patients with Fâ § Inhibitor].

OBJECTIVE: To explore the levels of regulatory T cells (Tregs) and cytokines IL-35, TGF-ß and IL-10 in peripheral blood of hemophilia A(HA) patients with FⅧ inhibitor and their clinical significance. METHODS: 43 HA patients admitted to the Hematology Department of the Affiliated Hospital of North China University of Science and Technology from October 2019 to December 2020 were selected, including 6 cases with FⅧ inhibitor and 37 cases without FⅧ inhibitor. In addition, 20 healthy males who underwent physical examinations were selected as healthy controls. Flow cytometry was used to detect the levels of CD4 + CD25 + CD127 - Tregs in peripheral blood of the HA patients and healthy controls, and ELISA assay was used to detect the expression levels of IL-35, TGF-ß and IL-10 in serum, and their differences between different groups were compared. RESULTS: Compared with the healthy control group, the level of Tregs in HA patients was decreased, and the level of Tregs in the FⅧ inhibitor positive group was the lowest, the difference was statistically significant (P <0.05). There was no significant difference in the expression level of Tregs in HA patients of different severity levels. The serum IL-35, TGF-ß, and IL-10 levels in both FⅧ inhibitor negative and positive groups were significantly lower than those in healthy control group, and those in FⅧ inhibitor positive group were significantly lower than those in FⅧ inhibitor negative group (all P <0.05). CONCLUSION: The decrease of Tregs, IL-35, TGF-ß, and IL-10 levels in HA patients may be related to the formation of FⅧ inhibitors.

Phrenic nerve stimulation protects against mechanical ventilation-induced diaphragm dysfunction in rats.

INTRODUCTION: We investigated a novel application of phrenic nerve stimulation (PNS) in diaphragm dysfunction induced by mechanical ventilation (MV). METHODS: Twenty-one Sprague-Dawley rats were assigned randomly to 3 groups: spontaneous breathing, 18-h controlled MV, and 18-h controlled MV with PNS. Upon completion of the experimental protocol, diaphragm contractility, gene expression of insulin-like growth factor-1 (IGF-1) and ubiquitin ligases, and serum IGF-1 levels were analyzed. RESULTS: Compared with the spontaneously breathing rats, impaired diaphragm contractile function, including force-related properties and force-frequency responses, were pronounced with MV. Furthermore, MV suppressed IGF-1 and induced muscle ring finger 1 mRNA expression in the diaphragm. In contrast, PNS counteracted MV-induced gene expression changes in the diaphragm and restored diaphragm function. CONCLUSIONS: PNS exerted a protective effect against MV-induced diaphragm dysfunction by counteracting altered expression of IGF-1 and ubiquitin ligase in the diaphragm.

Inheritance analysis and family history for microtia: A hospital-based study in China.

OBJECTIVES: Microtia is a congenital anomaly of the outer ear. Although genetic and environmental factors could play a role, no consensus has been established on the pathogenesis and cause of this condition. In this study, we surveyed the frequency and pattern of family history in patients with microtia in a Chinese specialty clinic population. METHODS: We evaluated data from 672 patients (mean age = 9.2, male-to-female ratio = 2.6:1) with microtia admitted to the Department of Auricular Reconstruction at the Plastic Surgery Hospital of Peking Union Medical College from December 2014 to February 2016. Family history of congenital ear anomalies across three generations was recorded. Pearson chi-square test or Fisher exact test was used to test the associations between the characteristics of microtia and hereditary features. RESULTS: A family history of auricle anomalies was identified in 202 patients (30.1%), of whom, 95 families showed vertical transmission, 14 families skipped a generation, and 120 families showed family aggregations. The incidence of family history varied with grades of microtia (P = 0.001). Patients with preauricular tags or pits (38.3%) had a higher familial incidence of microtia than those with simple microtia (24.1%) (P < 0.001). CONCLUSION: Patients with a lower grade of microtia demonstrated a higher incidence of family history. Patients with microtia had significantly more relatives with preauricular tags or pits. Microtia and preauricular tags or pits are different manifestations of the same defect, and their significant concurrency among relatives suggests that a considerable proportion of microtia is inherited and could recur with varying degrees of severity in other family members.

The reciprocal relationships between Chinese children's perception of interparental conflict, negative thinking, and depression symptoms: A cross-lagged study.

The present longitudinal study used the traditional cross-lagged panel model (CLPM) and autoregressive latent trajectory model with structured residuals (ALT-SR) to examine the relationships between perceived interparental conflict (IPC), negative thinking (NT), and depression symptoms in Chinese children. Changes in these three variables over time were also examined, as well as the trait and state aspects of the relationships between them. A sample of 516 third-grade primary students completed questionnaires about IPC, NT, and depression three times over a period of 1 year, at 6-month intervals. The CLPM findings indicated that, assuming that stability of each variable across time was controlled, Chinese children's perception of IPC significantly affected their level of depression through the mediating path of NT. After taking trait factors into account, among all the significant autoregressive and cross-lagged paths originally found in the CLPM, only one third remained significant in the ALT-SR model. More specifically, the ALT-SR model, revealed a driving effect of children's NT on perceived IPC and depression symptoms. The CLPM model although elucidated the interplay among three variables, the ALT-SR model showed little evidence of their interrelated growth across time. Taken together, these results indicate that children's perceived IPC in the long term are a stable trait, with few state-level fluctuations, and is not a significant within-person predictor of subsequent children's internalization problems. These perceptions appear to contribute more to children's general psychological tendency than do changes over time. The research is the first to test the reciprocal relationships between Chinese children's perceived IPC, NT, and depression symptoms. The findings demonstrate that previously proposed theories about the bidirectional relation between IPC and children's social adjustment, to some extent, may reflect a correlation at a trait level. Put another way, it is IPC's central tendency to be sensitive in the long term as a stable trait that is associated with their children's general tendency to show well adjustment. The study contributes to our understanding of that extend previous results and have implications for complementary theoretical and practical interventions. The complementary techniques of CLPM and ALT-SR models offer different insights into children's internalization problems, and hold promise for supporting the building of more comprehensive children's developmental theories that acknowledge the interconnectedness of different domains of mental health.

Destructive interparental conflict affects Chinese children's emotional and behavioral problems: Indirect pathways via parent-child attachment and emotional insecurity.

Background: Previous studies have demonstrated that destructive interparental conflict (IPC) is closely related to the emergence of emotional and behavioral problems in adolescents. In addition, in the family system, such conflict also affects the patent-child attachment relationship and emotional insecurity of adolescents. Objectives: This study mainly explores the relationship between destructive interparental conflict and adolescents' emotional and behavioral problems, focuses on the role of parent-child attachment and emotional insecurity, and analyzes whether this relationality plays multiple mediating roles in the influence of destructive interparental conflict on emotional and behavioral problems. Methods: Data for the study were obtained through a questionnaire survey conducted on 524 Chinese adolescents from primary and junior high school. Results: Structural equation modeling was conducted to test direct and indirect pathways between destructive interparental conflict and Chinese adolescents' emotional and behavioral problems. Destructive IPC negatively predicted parent-child attachment and parent-child attachment negatively predicted emotional and behavioral problems. Destructive Interparental conflict positively predicted emotional insecurity and emotional insecurity positively predicted emotional and behavioral problems. Discussion: The results show that: (1) Parent-child attachment negatively predicted emotional and behavioral problems, and emotional insecurity positively predicted the same. (2) Parent-child attachment and emotional insecurity act in a multiple mediating role between destructive IPC and adolescents' emotional and behavioral problems. (3) Parent-child attachment and emotional insecurity constitute two indirect pathways between destructive IPC and adolescents' emotional and behavioral problems, respectively. Conclusion: Destructive IPC can adversely affect emotional and behavioral problems among adolescents; destructive IPC plays a damaging role in their emotional security and parent-child attachment, consequently effecting emotional and behavioral problems.

Whole-exome sequencing analysis in 10 families of sporadic microtia with thoracic deformities.

BACKGROUND: Microtia is a congenital malformation of the external ear and may occur as an isolated deformity or as part of a syndrome. Our previous study found a high correlation between microtia and thoracic deformities, thus, we propose that external ear and thorax development may be regulated by certain genes in common. METHODS: We performed exome sequencing on 10 families of sporadic microtia with thoracic abnormalities. We identified mutated genes under different models of inheritance, and checked them through Mouse Genome Informatics and association analysis. RESULTS: We identified 45 rare mutations, including 9 de novo mutations, 20 heterozygous mutations, 3 homozygous mutations, and 13 hemizygous mutations, of which 2 are likely to be causative. They are de novo missense variant in PHF5A and compound heterozygous mutations in CYP26B1, of which CYP26B1 mutation is highly likely pathogenic. CONCLUSION: The results indicate that certain genes may affect both external ear and thorax development, and demonstrate the benefits of whole-exome sequencing in identifying candidate genes of microtia. This study provides a new way for genetic exploration in microtia.

Toll-Like Receptor 4 (TLR4)/Opioid Receptor Pathway Crosstalk and Impact on Opioid Analgesia, Immune Function, and Gastrointestinal Motility.

Toll-like receptor 4 (TLR4) recognizes exogenous pathogen-associated molecular patterns (PAMPs) and endogenous danger-associated molecular patterns (DAMPs) and initiates the innate immune response. Opioid receptors (µ, δ, and κ) activate inhibitory G-proteins and relieve pain. This review summarizes the following types of TLR4/opioid receptor pathway crosstalk: (a) Opioid receptor agonists non-stereoselectively activate the TLR4 signaling pathway in the central nervous system (CNS), in the absence of lipopolysaccharide (LPS). Opioids bind to TLR4, in a manner parallel to LPS, activating TLR4 signaling, which leads to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression and the production of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6. (b) Opioid receptor agonists inhibit the LPS-induced TLR4 signaling pathway in peripheral immune cells. Opioids operate as pro-inflammatory cytokines, resulting in neuroinflammation in the CNS, but they mediate immunosuppressive effects in the peripheral immune system. It is apparent that TLR4/opioid receptor pathway crosstalk varies dependent on the cell type and activating stimulus. (c) Both the TLR4 and opioid receptor pathways activate the mitogen-activated protein kinase (MAPK) pathway. This crosstalk is located downstream of the TLR4 and opioid receptor signaling pathways. Furthermore, the classic opioid receptor can also produce pro-inflammatory effects in the CNS via MAPK signaling and induce neuroinflammation. (d) Opioid receptor agonists induce the production of high mobility group box 1 (HMGB1), an endogenous TLR4 agonist, supporting intercellular (neuron-to-glia or glia-to-neuron) interactions. This review also summarizes the potential effects of TLR4/opioid receptor pathway crosstalk on opioid analgesia, immune function, and gastrointestinal motility. Opioids non-stereoselectively activate the TLR4 pathway, and together with the subsequent release of pro-inflammatory cytokines such as IL-1 by glia, this TLR4 signaling initiates the central immune signaling response and modifies opioid pharmacodynamics. The DAMP HMGB1 is associated with the development of neuropathic pain. To explain morphine-induced persistent sensitization, a positive feedback loop has been proposed; this involves an initial morphine-induced amplified release of IL-1ß and a subsequent exacerbated release of DAMPs, which increases the activation of TLR4 and the purinergic receptor P2X7R. Opioid receptor (µ, δ, and κ) agonists are involved in many aspects of immunosuppression. The intracellular TLR4/opioid receptor signaling pathway crosstalk induces the formation of the ß-arrestin-2/TNF receptor-associated factor 6 (TRAF6) complex, which contributes to morphine-induced inhibition of LPS-induced TNF-α secretion in mast cells. A possible molecular mechanism is that the TLR4 pathway initially triggers the formation of the ß-arrestin-2/TRAF6 complex, which is amplified by opioid receptor signaling, suggesting that ß-arrestin-2 acts as a functional component of the TLR4 pathway.

Identification of loss-of-function HOXA2 mutations in Chinese families with dominant bilateral microtia.

HOX genes are important regulatory genes patterning head formation, including development of the ear. Microtia is a congenital ear anomaly characterized by lacking all or part of the structures of the outer ear. To date, only four HOXA2 mutations were reported in families with autosomal-recessive or dominant microtia, with or without hearing impairment. More identified mutations are needed to confirm the correlation between genotype and phenotype. Here, we collect two Chinese families with non-syndromic bilateral microtia. Next generation sequencing identified two heterozygous nonsense HOXA2 mutations, one in each family. One mutation (c.637A > T, p.Lys213*) is newly reported, while the other one (c.703C > T,p.Gln235*) is consistent with a previous report. In mouse, Hoxa2 can bind to a long-range enhancer and regulate expression of the Hmx1 gene, which is a crucial transcription factor in eye and ear development. Using dual luciferase reporter assays, we showed that both HOXA2 mutations have impaired activation of the long-range enhancer of HMX1. In the present study, we report the first two bilateral non-syndromic microtia cases with HOXA2 mutations of Chinese origin and identify a novel mutation. Our results also provide molecular insights about how these nonsense HOXA2 mutations could affect the activation of its downstream target HMX1 by interacting with the long-range enhancer.

Alzheimer's Disease Presenilin-1 Mutation Sensitizes Neurons to Impaired Autophagy Flux and Propofol Neurotoxicity: Role of Calcium Dysregulation.

BACKGROUND: Disruption of intracellular Ca2+ homeostasis and associated autophagy dysfunction contribute to neuropathology in Alzheimer's disease (AD). OBJECTIVE: To study the effects of propofol on cell viability via its effects on intracellular Ca2+ homeostasis, and the impact of autophagy, in a neuronal model of presenilin-mutated familial AD (FAD). METHODS: We treated PC12 cells, stably transfected with either mutated presenilin-1 (L286V) or wild type (WT) controls, with propofol at different doses and durations, in the presence or absence of extracellular Ca2+, antagonists of inositol trisphosphate receptors (InsP3R, xestospongin C) and/or ryanodine receptors (RYR, dantrolene), or an inhibitor of autophagy flux (Bafilomycin). We determined cell viability, cytosolic Ca2+ concentrations ([Ca2+]c), vATPase protein expression, and lysosomal acidification. RESULTS: The propofol dose- and time-dependently decreased cell viability significantly more in L286V than WT cells, especially at the pharmacological dose (>50µM), and together with bafilomycin (40 nM). Clinically used concentrations of propofol (<20µM) tended to increase cell viability. Propofol significantly increased [Ca2+]c more in L286V than in WT cells, which was associated with decrease of vATPase expression and localization to the lysosome. Both toxicity and increased Ca2+ levels were ameliorated by inhibiting InsP3R/RYR. However, the combined inhibition of both receptors paradoxically increased [Ca2+]c, by inducing Ca2+ influx from the extracellular space, causing greater cytotoxicity. CONCLUSION: Impairment in autophagy function acts to deteriorate cell death induced by propofol in FAD neuronal cells. Cell death is ameliorated by either RYR or InsP3R antagonists on their own, but not when both are co-administered.