[Management of complex renal stones by a novel ureterorenoscope with vacuum suctioning system combined with flexible ureteroscope].

Objective: To evaluate the feasibility and safety of Sotn ureterorenoscope combined with flexible ureteroscope on managing complex renal stones. Methods: Patients treated with the Sotn ureterorenoscope combined with flexible ureteroscope between January 2010 and December 2019 were employed from the Sun Yat-sen Memorial Hospital of Sun Yat-sen University and Jiangmen Wuyi Traditional Chinese Medicine Hospital. The patients' information of age, gender, comorbidities, stone characteristics (stone size, hounsfield units, stone composition, stone location, etc.), operative time and console time, stone-free rate (SFR), and perioperative complication rate were collected. The primary outcome was defined as primary SFR in 1 month of operation, and the secondary outcome was the perioperative complication rate. The differences in preoperative and postoperative data between patients with different kinds of stones were compared. Results: A total of 347 patients were included in the study, with 220 males and 127 females. The age [M(Q1,Q3)] was 51 (42, 58) years. There were 94 patients suffered from multiple renal stones and 253 patients with staghorn renal stones. The operative time and console time age [M(Q1,Q3)] for all patients were 87 (55, 115) min and 59 (27, 75) min, respectively. The primary SFR was 81.3% [83.8% for multiple renal stones and 74.5% for staghorn renal stones (P=0.048)]. Complications occurred in 80 patients (23.1%), of which 79 cases were classified as Clavien-Dindo grade 1-2, and 1 case (0.3%) was grade 3-4. For patients with multiple renal stone, compared with the residual stone group, the complete stone-free group had smaller stone size [15.5 (12.0, 21.0) vs 22.0 (17.5, 28.1) mm, P<0.001], and lower hounsfield units [920.0 (658.0, 1 172.5) vs 1 125.0 (944.9, 1 247.5), P=0.022]. Patients with complications had longer operative time than those without complications [60.0 (38.5, 90.0) vs 75.0 (51.3, 110.0) min, P=0.022]. The SFR was higher in patients with stones size ≤ 20 mm compared to those with stones size > 20 mm (91.8% vs 67.5%, P<0.001), while the difference in complication rate was not statistically significant (P>0.05). In the staghorn renal stones group, compared with the residual stone group, the complete stone-free group had smaller stone size [35.0 (25.8, 45.3) vs 53.5 (39.3, 67.5) mm, P<0.001]. Patients with complications had larger stone size than those without complications [43.5 (34.8, 56.5) vs 36.0 (27.0, 50.0) mm, P=0.007]. Patients with stone size ≤ 40 mm had higher SFR (87.5% vs 55.3%, P<0.001) and lower complication rate(10.7% vs 31.6%, P=0.012) compared to those with stone size >40 mm. Conclusion: Sotn ureterorenoscope combined with flexible ureteroscope is an effective and safe choice for the treatment of complex renal calculi.

Mechanism of miR-122-5p regulating the activation of PI3K-Akt-mTOR signaling pathway on the cell proliferation and apoptosis of osteosarcoma cells through targeting TP53 gene.

OBJECTIVE: To explore the regulatory mechanism of microRNA-122-5p (miR-122-5) targeting tumor protein p53 (TP53) gene to mediate PI3K-Akt-mTOR signaling pathway on the proliferation and apoptosis of osteosarcoma (OS) cells. PATIENTS AND METHODS: With the collection of osteosarcoma and normal adjacent tissues, the mRNA of miR-122-5p, TP53, PTEN, PI3K, Akt, mTOR, Bim, Bax, and Bcl-2 was detected by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), followed by the detection of the protein expression by Western blot. The target relationship between miR-122-5p and TP53 gene was verified. The third generation osteosarcoma cells were divided into Blank group, miR-122-5p mimic negative control (NC) group, miR-122-5p mimic group, miR-122-5p inhibitor NC group, miR-122-5p inhibitor group, rapamycin group and miR-122-5p inhibitor + rapamycin group. Furthermore, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to detect the proliferation ability, cell cycle distribution and apoptosis of each group after transfection. RESULTS: The expression level of miR-122-5p in osteosarcoma was lower than that in normal tissues (p < 0.05), TP53, PTEN, Bim and Bax expression levels were decreased (all p < 0.05), while the expression levels of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR and Bcl-2 were highly upregulated (all p < 0.05). TP53 had the lowest expression in osteosarcoma cell line U-2OS (p < 0.05), which was selected for subsequent cell experiments. TP53 was the target gene of miR-122-5p. Compared with Blank group, miR-122-5p mimic group had increased expression of miR-122-5p (all p < 0.05); besides, there were significantly increased expression of TP53, PTEN, Bim, and Bax in miR-122-5p mimic group and rapamycin group, while remarkably decreased expression of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, and Bcl-2 (all p < 0.05), accompanied by increased proportion of cells in G0/G1 phase, decreased cell proportion in S phase, increased cell apoptosis and inhibited cell proliferation (all p < 0.05). The opposite trends were found in miR-122-5p inhibitor group relative to miR-122-5p mimic group and rapamycin group (all p < 0.05). Meanwhile, no significant difference was found in miR-122-5p inhibitor+rapamycin group when compared with that in Blank group (all p > 0.05) except for significantly decreased miR-122-5p expression (p < 0.05). CONCLUSIONS: Upregulation of miR-122-5p may inhibit the proliferation and promote the apoptosis of osteosarcoma cells by inhibiting the activation of PI3K-Akt-mTOR signaling pathway, which may be related to the targeted up-regulation of TP53 expression.

Stitching the synapse: Cross-linking mass spectrometry into resolving synaptic protein interactions.

Synaptic transmission is the predominant form of communication in the brain. It requires functionally specialized molecular machineries constituted by thousands of interacting synaptic proteins. Here, we made use of recent advances in cross-linking mass spectrometry (XL-MS) in combination with biochemical and computational approaches to reveal the architecture and assembly of synaptic protein complexes from mouse brain hippocampus and cerebellum. We obtained 11,999 unique lysine-lysine cross-links, comprising connections within and between 2362 proteins. This extensive collection was the basis to identify novel protein partners, to model protein conformational dynamics, and to delineate within and between protein interactions of main synaptic constituents, such as Camk2, the AMPA-type glutamate receptor, and associated proteins. Using XL-MS, we generated a protein interaction resource that we made easily accessible via a web-based platform (http://xlink.cncr.nl) to provide new entries into exploration of all protein interactions identified.

Proteomics of neurodegenerative diseases: analysis of human post-mortem brain.

Dementias are prevalent brain disorders in the aged population. Dementias pose major socio-medical burden, but currently there is no cure available. Novel proteomics approaches hold promise to identify alterations of the brain proteome that could provide clues on disease etiology, and identify candidate proteins to develop further as a biomarker. In this review, we focus on recent proteomics findings from brains affected with Alzheimer's Disease, Parkinson Disease Dementia, Frontotemporal Dementia, and Amyotrophic Lateral Sclerosis. These studies confirmed known cellular changes, and in addition identified novel proteins that may underlie distinct aspects of the diseases. This article is part of the special issue "Proteomics".

The Arabidopsis GORK K+-channel is phosphorylated by calcium-dependent protein kinase 21 (CPK21), which in turn is activated by 14-3-3 proteins.

Potassium (K+) is a vital ion for many processes in the plant and fine-tuned ion channels control the K+-fluxes across the plasma membrane. GORK is an outward-rectifying K+-channel with important functions in stomatal closure and in root K+-homeostasis. In this study, post-translational modification of the Arabidopsis GORK ion channel and its regulation by 14-3-3 proteins was investigated. To investigate the possible interaction between GORK and 14-3-3s an in vivo pull-down from an Arabidopsis protein extract with recombinant GORK C-terminus (GORK-C) indeed identified endogenous 14-3-3s (LAMBDA, CHI, NU) as binding partners in a phosphorylation dependent manner. However, a direct interaction between 14-3-3's and GORK-C could not be demonstrated. Since the pull-down of 14-3-3s was phosphorylation dependent, we determined GORK-C as substrate for CPK21 phosphorylation and identified three CPK21 phospho-sites in the GORK protein (T344, S518 and S649). Moreover, interaction of 14-3-3 to CPK21 strongly stimulates its kinase activity; an effect that can result in increased GORK phosphorylation and change in activity. Using the non-invasive vibrating probe technique, we measured the predominantly GORK mediated salt induced K+-efflux from wild-type, gork, cpk21, aha2 and 14-3-3 mutant roots. The mutants cpk21 and aha2 did not show statistical significant differences compared to WT. However, two (out of six) 14-3-3 isoforms, CHI and PHI, have a clear function in the salt induced K+-efflux. In conclusion, our results show that GORK can be phosphorylated by CPK21 and suggest that 14-3-3 proteins control GORK activity through binding with and activation of CPK21.

Thermal stability control system of photo-elastic interferometer in the PEM-FTs.

A drifting model for the resonant frequency and retardation amplitude of a photo-elastic modulator (PEM) in the photo-elastic modulated Fourier transform spectrometer (PEM-FTs) is presented. A multi-parameter broadband-matching driving control method is proposed to improve the thermal stability of the PEM interferometer. The automatically frequency-modulated technology of the driving signal based on digital phase-locked technology is used to track the PEM's changing resonant frequency. Simultaneously the maximum optical-path-difference of a laser's interferogram is measured to adjust the amplitude of the PEM's driving signal so that the spectral resolution is stable. In the experiment, the multi-parameter broadband-matching control method is applied to the driving control system of the PEM-FTs. Control of resonant frequency and retardation amplitude stabilizes the maximum optical-path-difference to approximately 236 µm and results in a spectral resolution of 42 cm-1. This corresponds to a relative error smaller than 2.16% (4.28 standard deviation). The experiment shows that the method can effectively stabilize the spectral resolution of the PEM-FTs.

HBsAg may reappear following reactivation in individuals with spontaneous HBsAg seroclearance 8 years previously.

HBsAg reappearance may constitute not only a risk for liver disease but also an infectious source. We aimed to determine whether HBsAg may reappear after spontaneous HBsAg seroclearance. A cohort of 2999 HBsAg-positive subjects aged 30-55 years was recruited in Guangxi, China in 2004. HBsAg was tested every 6 months from July 2004 to June 2007, then, one more time in December 2013. The results showed that spontaneous HBsAg seroclearance occurred in 41 subjects in the first 3 years, giving a 0·54% annual seroclearance rate. Thirteen of the 41 subjects were randomly tested for HBsAg in 2013. Four subjects became HBsAg positive. S gene sequences of HBV were analysed from serum collected before seroclearance and after reappearance, respectively, for subject QS840 (11 and 12 clones), subject TN98 (13 and 13 clones) and subject WX227 (10 and 8 clones). Serotype, subgenotype and amino-acid substitution pattern in each sample collected after reappearance was observed in the sample collected before HBsAg seroclearance. Nucleotide similarity between the two sequences from each subject was >99% and five sequences from subject TN98 were the same. In conclusion, following reactivation, HBsAg may reappear in individuals with spontaneous HBsAg seroclearance many years previously.

Identification of sex-specific urinary biomarkers for major depressive disorder by combined application of NMR- and GC-MS-based metabonomics.

Women are more vulnerable to major depressive disorder (MDD) than men. However, molecular biomarkers of sex differences are limited. Here we combined gas chromatography-mass spectrometry (GC-MS)- and nuclear magnetic resonance (NMR)-based metabonomics to investigate sex differences of urinary metabolite markers in MDD, and further explore their potential of diagnosing MDD. Consequently, the metabolite signatures of women and men MDD subjects were significantly different from of that in their respective healthy controls (HCs). Twenty seven women and 36 men related differentially expressed metabolites were identified in MDD. Fourteen metabolites were changed in both women and men MDD subjects. Significantly, the women-specific (m-Hydroxyphenylacetate, malonate, glycolate, hypoxanthine, isobutyrate and azelaic acid) and men-specific (tyrosine, N-acetyl-d-glucosamine, N-methylnicotinamide, indoxyl sulfate, citrate and succinate) marker panels were further identified, which could differentiate men and women MDD patients from their respective HCs with higher accuracy than previously reported sex-nonspecific marker panels. Our findings demonstrate that men and women MDD patients have distinct metabonomic signatures and sex-specific biomarkers have promising values in diagnosing MDD.

Modulation axis performs circular motion in a 45° dual-drive symmetric photoelastic modulator.

A 45° dual-drive symmetric photoelastic modulator is demonstrated. Two piezoelectric actuators are connected to a symmetric photoelastic crystal at an angle of 45°. When the amplitudes of the stress standing waves induced by the two piezoelectric actuators are equal and the phase difference between the two stress standing waves is π2, the modulation axis performs circular motion with a frequency of half of the photoelastic modulator's resonant frequency, while the retardation remains a constant that is determined at the driving voltage amplitudes. This reveals a new polarization modulation method. We have theoretically analyzed and experimentally observed the new polarization modulation, and the retardation calibration is also reported.

Neurobeachin Regulates Glutamate- and GABA-Receptor Targeting to Synapses via Distinct Pathways.

Neurotransmission and synaptic strength depend on expression of post-synaptic receptors on the cell surface. Post-translational modification of receptors, trafficking to the synapse through the secretory pathway, and subsequent insertion into the synapse involves interaction of the receptor with A-kinase anchor proteins (AKAPs) and scaffolding proteins. Neurobeachin (Nbea), a brain specific AKAP, is required for synaptic surface expression of both glutamate and GABA receptors. Here, we investigated the role of Nbea-dependent targeting of postsynaptic receptors by studying Nbea interaction with synapse-associated protein 102 (SAP102/Dlg3) and protein kinase A subunit II (PKA II). A Nbea mutant lacking the PKA binding domain showed a similar distribution as wild-type Nbea in Nbea null neurons and partially restored GABA receptor surface expression. To understand the relevance of Nbea interaction with SAP102, we analysed SAP102 null mutant mice. Nbea levels were reduced by ~80% in SAP102 null mice, but glutamatergic receptor expression was normal. A single-point mutation in the pleckstrin homology domain of Nbea (E2218R) resulted in loss of binding with SAP102. When expressed in Nbea null neurons, this mutant fully restored GABA receptor surface expression, but not glutamate receptor expression. Our results suggest that the PKA-binding domain is not essential for Nbea's role in receptor targeting and that Nbea targets glutamate and GABA receptors to the synapse via distinct molecular pathways by interacting with specific effector proteins.

Single-cell analysis of peptide expression and electrophysiology of right parietal neurons involved in male copulation behavior of a simultaneous hermaphrodite.

Male copulation is a complex behavior that requires coordinated communication between the nervous system and the peripheral reproductive organs involved in mating. In hermaphroditic animals, such as the freshwater snail Lymnaea stagnalis, this complexity increases since the animal can behave both as male and female. The performance of the sexual role as a male is coordinated via a neuronal communication regulated by many peptidergic neurons, clustered in the cerebral and pedal ganglia and dispersed in the pleural and parietal ganglia. By combining single-cell matrix-assisted laser mass spectrometry with retrograde staining and electrophysiology, we analyzed neuropeptide expression of single neurons of the right parietal ganglion and their axonal projections into the penial nerve. Based on the neuropeptide profile of these neurons, we were able to reconstruct a chemical map of the right parietal ganglion revealing a striking correlation with the earlier electrophysiological and neuroanatomical studies. Neurons can be divided into two main groups: (i) neurons that express heptapeptides and (ii) neurons that do not. The neuronal projection of the different neurons into the penial nerve reveals a pattern where (spontaneous) activity is related to branching pattern. This heterogeneity in both neurochemical anatomy and branching pattern of the parietal neurons reflects the complexity of the peptidergic neurotransmission involved in the regulation of male mating behavior in this simultaneous hermaphrodite.

Dynamic Proteomics: In Vivo Proteome-Wide Measurement of Protein Kinetics Using Metabolic Labeling.

Control of biosynthetic and catabolic rates of polymers, including proteins, stands at the center of phenotype, physiologic adaptation, and disease pathogenesis. Advances in stable isotope-labeling concepts and mass spectrometric instrumentation now allow accurate in vivo measurement of protein synthesis and turnover rates, both for targeted proteins and for unbiased screening across the proteome. We describe here the underlying principles and operational protocols for measuring protein dynamics, focusing on metabolic labeling with (2)H2O (heavy water) combined with tandem mass spectrometric analysis of mass isotopomer abundances in trypsin-generated peptides. The core principles of combinatorial analysis (mass isotopomer distribution analysis or MIDA) are reviewed in detail, including practical advantages, limitations, and technical procedures to ensure optimal kinetic results. Technical factors include heavy water labeling protocols, optimal duration of labeling, clean up and simplification of sample matrices, accurate quantitation of mass isotopomer abundances in peptides, criteria for adequacy of mass spectrometric abundance measurements, and calculation algorithms. Some applications are described, including the noninvasive "virtual biopsy" strategy for measuring molecular flux rates in tissues through measurements in body fluids. In addition, application of heavy water labeling to measure flux lipidomics is noted. In summary, the combination of stable isotope labeling, particularly from (2)H2O, with tandem mass spectrometric analysis of mass isotopomer abundances in peptides, provides a powerful approach for characterizing the dynamics of proteins across the global proteome. Many applications in research and clinical medicine have been achieved and many others can be envisioned.

Functional characterization of the PCLO p.Ser4814Ala variant associated with major depressive disorder reveals cellular but not behavioral differences.

Genome-wide association studies have suggested a role for a genetic variation in the presynaptic gene PCLO in major depressive disorder (MDD). As with many complex traits, the PCLO variant has a small contribution to the overall heritability and the association does not always replicate. One variant (rs2522833, p.Ser4814Ala) is of particular interest given that it is a common, nonsynonymous exon variant near a calcium-sensing part of PCLO. It has been suggested that the molecular effects of such variations penetrate to a variable extent in the population due to phenotypic and genotypic heterogeneity at the population level. More robust effects may be exposed by studying such variations in isolation, in a more homogeneous context. We tested this idea by modeling PCLO variation in a mouse knock-in model expressing the Pclo(SA)(/)(SA) variant. In the highly homogeneous background of inbred mice, two functional effects of the SA-variation were observed at the cellular level: increased synaptic Piccolo levels, and 30% increased excitatory synaptic transmission in cultured neurons. Other aspects of Piccolo function were unaltered: calcium-dependent phospholipid binding, synapse formation in vitro, and synaptic accumulation of synaptic vesicles. Moreover, anxiety, cognition and depressive-like behavior were normal in Pclo(SA)(/)(SA) mice. We conclude that the PCLO p.Ser4814Ala missense variant produces mild cellular phenotypes, which do not translate into behavioral phenotypes. We propose a model explaining how (subtle) cellular phenotypes do not penetrate to the mouse behavioral level but, due to genetic and phenotypic heterogeneity and non-linearity, can produce association signals in human population studies.

HCN channels are a novel therapeutic target for cognitive dysfunction in Neurofibromatosis type 1.

Cognitive impairments are a major clinical feature of the common neurogenetic disease neurofibromatosis type 1 (NF1). Previous studies have demonstrated that increased neuronal inhibition underlies the learning deficits in NF1, however, the molecular mechanism underlying this cell-type specificity has remained unknown. Here, we identify an interneuron-specific attenuation of hyperpolarization-activated cyclic nucleotide-gated (HCN) current as the cause for increased inhibition in Nf1 mutants. Mechanistically, we demonstrate that HCN1 is a novel NF1-interacting protein for which loss of NF1 results in a concomitant increase of interneuron excitability. Furthermore, the HCN channel agonist lamotrigine rescued the electrophysiological and cognitive deficits in two independent Nf1 mouse models, thereby establishing the importance of HCN channel dysfunction in NF1. Together, our results provide detailed mechanistic insights into the pathophysiology of NF1-associated cognitive defects, and identify a novel target for clinical drug development.

The effects of an oral preparation containing hyaluronic acid (Oralvisc®) on obese knee osteoarthritis patients determined by pain, function, bradykinin, leptin, inflammatory cytokines, and heavy water analyses.

The purpose of this study was to determine the effects of an oral preparation containing hyaluronic acid on osteoarthritic knee joint pain and function as well as changes in inflammatory cytokines, bradykinin, and leptin. We also used heavy water to determine the turnover rates of glycosaminoglycans in synovial fluid. This was a double-blind, randomized, placebo-controlled study of 40 subjects over a period of 3 months. Visual analog scale, Western Ontario McMaster pain, and WOMAC function scores were recorded. Serum and synovial fluid were measured by enzyme-linked immunosorbent assays for inflammatory cytokines, bradykinin, and leptin. In 20 subjects, terminal heavy water ingestion was used for spectral analyses of serum and joint fluid samples. There were statistically significant improvements in pain and function. Both serum and synovial fluid samples showed significant decreases for a majority of inflammatory cytokines, leptin, and bradykinin in the oral hyaluronic acid preparation group. Heavy water analyses revealed a significant decrease in hyaluronic acid turnover in the synovial fluid of the treatment group. A preparation containing hyaluronic acid and other glycosaminoglycans holds promise for a safe and effective agent for the treatment for patients with knee osteoarthritis and who are overweight. Further studies will be required to see whether this is a disease-modifying agent.

Higher order Arabidopsis 14-3-3 mutants show 14-3-3 involvement in primary root growth both under control and abiotic stress conditions.

Arabidopsis 14-3-3 proteins are a family of conserved proteins that interact with numerous partner proteins in a phospho-specific manner, and can affect the target proteins in a number of ways; e.g. modification of enzymatic activity. We isolated T-DNA insertion lines in six 14-3-3 genes within the non-epsilon group that phylogenetically group in three closely related gene pairs. In total, 6 single, 3 double, 12 triple, and 3 quadruple mutants were generated. The mutants were phenotyped for primary root growth on control plates: single and double mutants were indistinguishable from WT, whereas six triples and all quadruples showed a shorter primary root. In addition, length of the first epidermal cell with a visible root hair bulge (LEH) was used to determine primary root elongation on medium containing mannitol and 1-aminocyclopropane-1-carboxylic acid (ACC). This analysis showed clear differences depending on the stress and 14-3-3 gene combinations. Next to the phenotypic growth analyses, a 14-3-3 pull-down assay on roots treated with and without mannitol showed that mannitol stress strongly affects the 14-3-3 interactome. In conclusion, we show gene specificity and functional redundancy among 14-3-3 proteins in primary root elongation under control and under abiotic stress conditions and changes in the 14-3-3 interactome during the onset of stress adaptation.

Comparison of transanal haemorrhoidal dearterialisation and stapled haemorrhoidopexy in management of haemorrhoidal disease: a retrospective study and literature review.

BACKGROUND: The aim of this study was to evaluate and compare transanal haemorrhoidal dearterialisation (THD) and stapled haemorrhoidopexy [also called procedure for prolapsed haemorrhoids (PPH)] in the management of haemorrhoidal disease, in terms of short-term outcomes and efficacy. METHODS: Patients presenting with symptomatic haemorrhoids were treated with THD. Patient demographics, pre-operative data, post-operative pain scores, complications, recurrence, and patient satisfaction scores were evaluated and recorded. Patients with acute thrombosed haemorrhoids, external haemorrhoids only, or other concomitant anal diseases were excluded. These data were compared with the historical data of PPH. RESULTS: Forty consecutive patients underwent THD from February 2012 to July 2013 and were compared to 37 patients who underwent PPH taken from a medical records database. There were no significant differences in terms of demographic data, type of anaesthesia, operative time, and blood loss. Length of hospital stay, time to first post-operative bowel movement, and complications were similar between the two groups. The median pain score after THD and PPH was 1.71 and 5.00, respectively, on a scale of 0-10 (10 = worst possible pain) (p = 0.000). There was a significant improvement in bleeding and prolapse scores after THD. THD patients had an earlier return to normal daily activities (3.13 vs. 6.78 days, p = 0.001) when compared with the PPH group. Upon follow-up, patients in both groups had similar satisfaction scores, and complication and recurrence rates. CONCLUSIONS: Both THD and PPH appear to be safe procedures for haemorrhoidal disease, and they appear to have similar short-term outcomes. In particular, THD seems to be associated with a lower pain score than PPH, an earlier return to normal daily activities, and similar rates of complication and recurrence.

CCHCR1 interacts with EDC4, suggesting its localization in P-bodies.

Coiled-coil alpha-helical rod protein 1 (CCHCR1) is suggested as a candidate biomarker for psoriasis for more than a decade but its function remains poorly understood because of the inconsistent findings in the literature. CCHCR1 protein is suggested to be localized in the cytoplasm, nucleus, mitochondria, or centrosome and to regulate various cellular functions, including steroidogenesis, proliferation, differentiation, and cytoskeleton organization. In this study, we attempted to find a consensus between these findings by identifying the interaction partners of CCHCR1 using co-immunoprecipiation with a stable cell line expressing EGFP-tagged CCHCR1. Out of more than 100 co-immunoprecipitants identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the enhancer of mRNA-decapping protein 4 (EDC4), which is a processing body (P-body) component, was particularly found to be the major interacting partner of CCHCR1. Confocal imaging confirmed the localization of CCHCR1 in P-bodies and its N-terminus is required for this subcellular localization, suggesting that CCHCR1 is a novel P-body component. As P-bodies are the site for mRNA metabolism, our findings provide a molecular basis for the function of CCHCR1, any disruption of which may affect the transcriptome of the cell, and causing abnormal cell functions.

Trigeminal nerve injury-induced thrombospondin-4 up-regulation contributes to orofacial neuropathic pain states in a rat model.

BACKGROUND: Injury to the trigeminal nerve often results in the development of chronic pain states including tactile allodynia, or hypersensitivity to light touch, in orofacial area, but its underlying mechanisms are poorly understood. Peripheral nerve injury has been shown to cause up-regulation of thrombospondin-4 (TSP4) in dorsal spinal cord that correlates with neuropathic pain development. In this study, we examined whether injury-induced TSP4 is critical in mediating orofacial pain development in a rat model of chronic constriction injury to the infraorbital nerve. METHODS: Orofacial sensitivity to mechanical stimulation was examined in a unilateral infraorbital nerve ligation rat model. The levels of TSP4 in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 spinal cord (Vc/C2) from injured rats were examined at time points correlating with the initiation and peak orofacial hypersensitivity. TSP4 antisense and mismatch oligodeoxynucleotides were intrathecally injected into injured rats to see if antisense oligodeoxynucleotide treatment could reverse injury-induced TSP4 up-regulation and orofacial behavioural hypersensitivity. RESULTS: Our data indicated that trigeminal nerve injury induced TSP4 up-regulation in Vc/C2 at a time point correlated with orofacial tactile allodynia. In addition, intrathecal treatment with TSP4 antisense, but not mismatch, oligodeoxynucleotides blocked both injury-induced TSP4 up-regulation in Vc/C2 and behavioural hypersensitivity. CONCLUSIONS: Our data support that infraorbital nerve injury leads to TSP4 up-regulation in trigeminal spinal complex that contributes to orofacial neuropathic pain states. Blocking this pathway may provide an alternative approach in management of orofacial neuropathic pain states.