CircUGGT2 downregulation by METTL14-dependent m6A modification suppresses gastric cancer progression and cisplatin resistance through interaction with miR-186-3p/MAP3K9 axis.

Chemoresistance is a major therapeutic challenge in advanced gastric cancer (GC). N6-methyladenosine (m6A) RNA modification has been shown to play fundamental roles in cancer progression. However, the underlying mechanisms by which m6A modification of circRNAs contributes to GC and chemoresistance remain unknown. We found that hsa_circ_0030632 (circUGGT2) was a predominant m6A target of METTL14, and METTL14 knockdown (KD) reduced circUGGT2 m6A levels but increased its mRNA levels. The expression of circUGGT2 was markedly increased in cisplatin (DDP)-resistant GC cells. CircUGGT2 KD impaired cell growth, metastasis and DDP-resistance in vitro and in vivo, but circUGGT2 overexpression prompted these effects. Furthermore, circUGGT2 was validated to sponge miR-186-3p and upregulate MAP3K9 and could abolish METTL14-caused miR-186-3p upregulation and MAP3K9 downregulation in GC cells. circUGGT2 negatively correlated with miR-186-3p expression and harbored a poor prognosis in patients with GC. Our findings unveil that METTL14-dependent m6A modification of circUGGT2 inhibits GC progression and DDP resistance by regulating miR-186-3p/MAP3K9 axis.

METTL14-mediated m6A modification of circORC5 suppresses gastric cancer progression by regulating miR-30c-2-3p/AKT1S1 axis.

BACKGROUND: N6-methyladenosine (m6A) RNA methylation and circular RNAs (circRNAs) have been shown to act vital roles in multiple malignancies including gastric cancer (GC). However, there is little knowledge about how m6A modification of circRNAs contributes to GC progression. METHODS: The association of METTL14 expression with the clinicopathological characteristics and prognosis in patients with GC was assessed by Western blot, Immunohistochemistry and public datasets. In vitro and vivo function experiments were conducted to investigate the role of METTL14 in GC. Furthermore, m6A-circRNA epitranscriptomic microarray was utilized to identify METTL14-mediated m6A modification of circRNAs, which were validated by methylated RNA immunoprecipitation (Me-RIP), RT-qPCR and rescue experiments in GC cells. The sponge of circORC5 with miR-30c-2-3p was confirmed by luciferase gene report and RNA immunoprecipitation assays. The expression, localization and prognosis of circORC5 in GC were evaluated by fluorescence in situ hybridization. The effects of METTL14 and (or) circORC5 on miR-30c-2-3p-mediated AKT1S1 and EIF4B were estimated by RT-qPCR and Western blot analyses. RESULTS: We found that METTL14 was downregulated in GC tissue samples and its low expression acted as a prognostic factor of poor survival in patients with GC. Ectopic expression of METTL14 markedly repressed growth and invasion of GC cells in vitro and in vivo, whereas knockdown of METTL14 harbored the opposite effects. Mechanically, m6A-circRNA epitranscriptomic microarray and Me-RIP identified circORC5 as the downstream target of METTL14. Silencing of METTL14 reduced the m6A level of circORC5, but increased circORC5 expression. Moreover, circORC5 could sponge miR-30c-2-3p, and reverse METTL14-caused upregulation of miR-30c-2-3p and downregulation of AKT1S1 and EIF4B. In addition, circORC5 possessed a negative correlation with miR-30c-2-3p and indicated a poor survival in GC. CONCLUSION: Our findings demonstrate that METTL14-mediated m6A modification of circORC5 suppresses gastric cancer progression by regulating miR-30c-2-3p/AKT1S1 axis.

Is wound drainage necessary in hip arthroplasty? A meta-analysis of randomized controlled trials.

PURPOSE: The use of closed suction drainage systems for hip arthroplasty (HA) is a common practice. However, the effectiveness and safety are still questionable. Thus, the aim of this meta-analysis was to review the advantages and adverse effects of closed suction drainage systems in hip arthroplasty. METHODS: All randomized or quasi-randomized trials comparing the use of closed suction drainage systems with no drainage systems for hip arthroplasty were searched in PubMed, Medicine, EMBASE and other internet databases. We assessed the methodological quality of the studies and abstracted the relevant data independently. RESULTS: Sixteen studies involving 1,663 participants with surgical wounds comparing HA with and without the use of drainage were included in our analysis. Our results demonstrated blood transfusion was required more frequently the same as the persistent discharge in those who received drains. No significant differences in the incidence of wound hematoma, dehiscence or deep vein thrombosis were found between those allocated to drains and the non-drained wounds. Wound infection and the range of movement of the joint after surgery were similar between the two groups. CONCLUSIONS: Based on the current evidence, there is insufficient evidence to support the routine use of closed suction drainage in hip arthroplasty. At the same time, our meta-analysis study suggested that using of closed suction drainage in HA increased requirement for postoperative blood transfusion. However, there is a moderate possibility of selection bias and publication bias in this review. Because of the limited number of studies which may weaken the strength of the evidence of our results, more samples, high-quality randomized trials are needed to increase the reliability of evidences. LEVEL OF EVIDENCE: II.

The effect of tourniquet use in total knee arthroplasty: grading the evidence through an updated meta-analysis of randomized, controlled trials.

PURPOSE: Although tourniquets are widely used in total knee arthroplasty (TKA), the effectiveness and safety are still in controversy. We therefore conducted an updated meta-analysis to compare the outcomes of tourniquet-assisted to non-tourniquet-assisted TKA and provide recommendations for using tourniquet in TKA. METHODS: A systematic search of studies published through March 2013 was conducted using MEDLINE, EMBASE, OVID, and ScienceDirect. Randomized, controlled trials that assessed the influence of the use of a tourniquet in TKA and provided data on safety and clinical effects were identified. Demographic characteristics, adverse events, and clinical outcomes were manually extracted from all of the selected studies. The evidence quality levels and recommendations were assessed using the GRADE system. RESULTS: Fifteen studies encompassing 804 patients and comparing TKA with and without the use of a tourniquet met the inclusion criteria. Overall, the result of meta-analysis indicated that using a tourniquet could decrease the intraoperative blood loss but could increase the postoperative blood loss. However, there was no statistically significant difference in calculated blood loss and measured total blood loss between the tourniquet and non-tourniquet group. There was no statistically significant difference in operation time. Patients treated with a tourniquet might not have higher risks of thromboembolic complications, such as deep vein thrombosis and pulmonary thromboembolism. The overall GRADE system evidence quality was very low, which lowers our confidence in their recommendations. CONCLUSION: As a safe application, the use of a tourniquet during TKA may be effective for reducing intraoperative blood loss, but not for reducing the postoperative blood loss and actual total blood loss. However, no uniform guideline can be made based on the current evidence because of the very low evidence quality and lower GRADE recommendation strength.

Helicobacter Pylori-Enhanced hnRNPA2B1 Coordinates with PABPC1 to Promote Non-m6A Translation and Gastric Cancer Progression.

Helicobacter pylori (H. pylori) infection is the primary risk factor for the pathogenesis of gastric cancer (GC). N6-methyladenosine (m6A) plays pivotal roles in mRNA metabolism and hnRNPA2B1 as an m6A reader is shown to exert m6A-dependent mRNA stabilization in cancer. This study aims to explore the role of hnRNPA2B1 in H. pylori-associated GC and its novel molecular mechanism. Multiple datasets and tissue microarray are utilized for assessing hnRNPA2B1 expression in response to H. pylori infection and its clinical prognosis in patients with GC. The roles of hnRNPA2B1 are investigated through a variety of techniques including glucose metabolism analysis, m6A-epitranscriptomic microarray, Ribo-seq, polysome profiling, RIP-seq. In addition, hnRNPA2B1 interaction with poly(A) binding protein cytoplasmic 1 (PABPC1) is validated using mass spectrometry and co-IP. These results show that hnRNPA2B1 is upregulated in GC and correlated with poor prognosis. H. pylori infection induces hnRNPA2B1 upregulation through recruiting NF-κB to its promoter. Intriguingly, cytoplasm-anchored hnRNPA2B1 coordinated PABPC1 to stabilize its relationship with cap-binding eIF4F complex, which facilitated the translation of CIP2A, DLAT and GPX1 independent of m6A modification. In summary, hnRNPA2B1 facilitates the non-m6A translation of epigenetic mRNAs in GC progression by interacting with PABPC1-eIF4F complex and predicts poor prognosis for patients with GC.

Detachable string magnetically controlled capsule endoscopy for the noninvasive diagnosis of esophageal diseases: A prospective, blind clinical study.

BACKGROUND: Traditional esophagogastroduodenoscopy (EGD), an invasive examination method, can cause discomfort and pain in patients. In contrast, magnetically controlled capsule endoscopy (MCE), a noninvasive method, is being applied for the detection of stomach and small intestinal diseases, but its application in treating esophageal diseases is not widespread. AIM: To evaluate the safety and efficacy of detachable string MCE (ds-MCE) for the diagnosis of esophageal diseases. METHODS: Fifty patients who had been diagnosed with esophageal diseases were prospectively recruited for this clinical study and underwent ds-MCE and conventional EGD. The primary endpoints included the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of ds-MCE for patients with esophageal diseases. The secondary endpoints consisted of visualizing the esophageal and dentate lines, as well as the subjects' tolerance of the procedure. RESULTS: Using EGD as the gold standard, the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of ds-MCE for esophageal disease detection were 85.71%, 86.21%, 81.82%, 89.29%, and 86%, respectively. ds-MCE was more comfortable and convenient than EGD was, with 80% of patients feeling that ds-MCE examination was very comfortable or comfortable and 50% of patients believing that detachable string v examination was very convenient. CONCLUSION: This study revealed that ds-MCE has the same diagnostic effects as traditional EGD for esophageal diseases and is more comfortable and convenient than EGD, providing a novel noninvasive method for treating esophageal diseases.

Sinularin stabilizes FOXO3 protein to trigger prostate cancer cell intrinsic apoptosis.

Sinularin, a natural product that purified from soft coral, exhibits anti-tumor effects against various human cancers. However, the mechanisms are not well understood. In this study, we demonstrated that Sinularin inhibited the viability of human prostate cancer cells in a dose-dependent manner and displayed significant cytotoxicity only at high concentration against normal prostate epithelial cell RWPE-1. Flow cytometry assay demonstrated that Sinularin induced tumor cell apoptosis. Further investigations revealed that Sinularin exerted anti-tumor activity through intrinsic apoptotic pathway along with up-regulation of pro-apoptotic protein Bax and PUMA, inhibition of anti-apoptotic protein Bcl-2, mitochondrial membrane potential collapses, and release of mitochondrial proteins. Furthermore, we illustrated that Sinularin induced cell apoptosis via up-regulating PUMA through inhibition of FOXO3 degradation by the ubiquitin-proteasome pathway. To explore how Sinularin suppress FOXO3 ubiquitin-proteasome degradation, we tested two important protein kinases AKT and ERK that regulate FOXO3 stabilization. The results revealed that Sinularin stabilized and up-regulated FOXO3 via inhibition of AKT- and ERK1/2-mediated FOXO3 phosphorylation and subsequent ubiquitin-proteasome degradation. Our findings illustrated the potential mechanisms by which Sinularin induced cell apoptosis and Sinularin may be applied as a therapeutic agent for human prostate cancer.

TREM2 as a Prognostic Biomarker for Osteosarcoma Microenvironment Remodeling.

The tumor microenvironment (TME) acts as a crucial role in the occurrence and development of osteosarcoma (OS). Despite this, the mechanism controlling the components of immunity and stroma in the tumor microenvironment remains a mystery. To conduct this study, we download and collate transcriptome data from the TARGET database, whose full name is Therapeutically Applicable Research to Generate Effective Treatments, as well as available clinical information of OS. The CIBERSORT and ESTIMATE methodology are used to acquire the proportions of components of immunity and stroma and tumor-infiltrating immune cells (TICs). Protein-protein interaction (PPI) networks and Cox regression analysis are used to select differentially expressed genes (DEGs). A prognostic biomarker is determined by intersecting univariate COX and PPI results, which lead to the finding of Triggering receptor expressed on myeloid cells-2 (TREM2). Based on the next analysis, TREM2 expression is positively correlated with OS survival time. Immune function-related genes have enrichment in the group with high expression of TREM2, according to gene set enrichment analysis (GSEA). The percentage of TICs by CIBERSORT methodology revealed that the expression of TREM2 is positively associated with follicular helper T cells, CD8-positive T cells, and M2 macrophages and negatively correlated with plasma cells, M0 macrophages, and naive CD4-positive T cells. All results suggest a possible integral role of TREM2 in the immune-related events of TME. Therefore, TREM2 may be a potential indicator of remodeling of TME in osteosarcoma, which is useful and helpful in predicting the clinical prognostic outcome of OS patients and provide a unique perspective for immunotherapy for OS.

[Seasonal Variation of DOM Spectral Characteristics of Rivers with Different Urbanization Levels in the Three Gorges Reservoir Area].

The urbanization process of the Three Gorges Reservoir area (TGRA) shows important impacts on the formation, migration, and transportation of dissolved organic matter (DOM) in rivers. In this study, a high-urbanized river (Taohuaxi) and low-urbanized river (Pulihe) in the TGRA were selected and water samples were collected in spring (April) and summer (August) to analyze the seasonal and spatial characteristics of DOM in the rivers using UV-Vis absorption spectroscopy and three-dimensional fluorescence spectroscopy. The DOC, S275-295 (the spectral slope of the short wavelength range), SR (spectral slope ratio), BIX (recent autochthonous contribution), and Fn(280) (relative importance of protein-like substances) of the Taohuaxi and Pulihe Rivers were higher in summer (August) than in spring (April), and CDOM concentration, CDOM/DOC, SUVA254 (aromatic structure), SUVA260 (hydrophobic component), E3/E4 (proportion of fulvic acid), S350-400 (the spectral slope of the long wavelength range), and HIX (the degree of humification) were higher in spring than in summer. The overall averages of the DOC, CDOM concentration, SR, Fn (280), BIX, and FI were higher in the high-urbanized Taohuaxi River than the low-urbanized Pulihe River, whilst the CDOM/DOC, SUVA254, and SUVA260 were lower in the Taohuaxi River. With the increase of urbanization along the water flow pathways, SUVA254 and SUVA260 gradually decreased, while Fn (280) gradually increased. The humus source (FI) of the two rivers in spring and summer is mixed and includes terrigenous and biological sources (plankton and algae) but is mostly endogenous. Furthermore, DOM is characterized by a moderately recent autochthonous contribution. Our study implies a large impact of urbanization on the composition and sources of DOM.

[Absorption and Fluorescence Spectra of Dissolved Organic Matter in Rivers of the Three Gorges Reservoir Area Under the Background of Urbanization].

Studying the influences of urbanization on the composition, chemical structure, source and migration of dissolved organic matter (DOM) in rivers is of great significance for aquatic ecosystem protection and biogeochemical carbon (C) cycling. The DOM spectroscopic characteristics of the three typical rivers (Taohuaxi, Nanhe, and Puli) with different urbanization levels in the Three Gorges Reservoir area were investigated using the ultraviolet-visible (UV-Vis) absorption spectrum and three-dimensional fluorescence spectroscopy. The results demonstrated that the higher the degree of urbanization in rivers, the greater the concentrations of DOM and chromophoric dissolved organic matter (CDOM), the lower the humification degree and aromaticity of DOM, the smaller the proportion of hydrophobic components, the higher the relative level of protein-like substance concentration in DOM, and the greater the contribution of DOM from freshly autochthonous origins. The DOM of the three rivers was dominated by fulvic acids (E3/E4>3.5), and CDOM concentration was positively correlated with the concentration of DOM (P<0.01). There were significant positive correlations between the humification degree (SUVA254), aromaticity (SUVA280), and hydrophobic component (SUVA260) (P<0.01). The sources of humic substances in the DOM were mixed by terrestrial input and microbial or algae production (reflected by the fluorescence index averages of 1.7157-1.7571), and mainly from microbial sources.

Identifying Common Pathogens in Periprosthetic Joint Infection and Testing Drug-resistance Rate for Different Antibiotics: A Prospective, Single Center Study in Beijing.

OBJECTIVE: The aim of this study is to identify the common microorganisms causing PJI as well as the drug-resistant spectrum for each microorganism, to help orthopaedic surgeons to choose appropriate antibiotics. METHOD: One hundred and sixty patients who suffered from failure of primary or revision total hip or knee arthroplasty for different reasons were prospectively recruited. These patients underwent revision or re-revision total hip or knee arthroplasty in our institution between August 2013 to August 2016. The details of patients' medical history and comprehensive physical examination, as well as demographic data were recorded precisely. Routine blood test results, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), high sensitive C-reactive protein (hs-CRP), interleukin-6 (IL-6) levels, and synovial leukocyte counts were collected. Additionally, aspiration was conducted during surgery to avoid pollution unless when PJI was strongly suspected, in which case, joint puncture and aspiration were conducted before surgery. Intraoperatively, the implant-surrounding tissue and the prosthesis were collected under aseptic conditions. Postoperatively, the prosthesis, implant-surrounding tissue and synovium were sent to the laboratory immediately. The sonicate extraction (the prosthesis was sent for ultrasound sonication first), implant surrounding tissue and synovium were sent for microbiologic culture, and the implant-surrounding tissue was also sent for pathological examination. The isolated bacteria strains and drug-resistance rates for each pathogen for different antibiotics were presented. RESULTS: There were 59 PJI cases in the infectious group and 101 cases in the non-infectious group (PJI is diagnosed according to the diagnosing criteria from the Workgroup of the Musculoskeletal Infection Society). Of 69 strains of pathogens isolated, Gram-positive bacterium is the most common pathogenic bacteria causing PJI (60, 86.96%). Staphylococcus epidermidis and Staphylococcus aureus played an important role as well, followed by Gram-negative bacteria (8, 11.59%) and fungus (1, 1.45%). Penicillin (78.57%), erythromycin (66.67%) and clindamycin (44.74%) showed high antibiotic resistance rate. In addition, the second-generation cephalosporin, usually as the prophylactic antibiotic, resistance rate was high (20%) as well. Fortunately, no vancomycin-resistant bacteria were discovered in the current study. CONCLUSION: This study provides some information on the most common pathogens in our institution and the selection of antibiotics in the perioperative period in northern China. Cefuroxime and clindamycin might not be appropriate for use as prophylactic antibiotics in revision total knee or hip arthroplasty. Vancomycin is ideal for empiric antibiotic use in suspected PJI cases because of the low drug-resistance rate.

[Sericin regulates proliferation of human gastric cancer MKN45 cells through autophagic pathway].

OBJECTIVE: To investigate the effect of sericin on the proliferation of human gastric cancer MKN45 cells and explore the underlying molecular mechanism. METHODS: MKN45 cells were transfected by LC3 double fluorescent autophagic virus, and the positive cells screened by puromycin were divided into blank group, sericin group and sericin∓3-MA group. After incubation with sericin for 48 h, the cells were examined for proliferation, apoptosis and cell cycle using CCK-8 assay and flow cytometry. Cell autophagy was detected by transmission electron microscopy (TEM) and fluorescent inverted microscope, and the autophagy-related markers including LC3, p62 and Beclin proteins were detected with Western blotting. Nude mice bearing gastric cancer xenograft were treated with normal saline or sericin injections (n=5) and the changes in the tumor volume and weight were measured. RESULTS: Compared with the blank group, MKN45 cells with sericin treatment showed significantly inhibited proliferation both in vitro and in nude mice. Autophagosomes were observed in sericin-treated cells under TEM and fluorescent inverted microscope. Sericin treatment of the cells significantly increased the cell apoptosis (P<0.01), caused obvious cell cycle arrest in G2/M phase (P<0.01), up-regulated the expressions of both LC3-2 and Beclin, and down-regulated the expression of p62. The autophagy inhibitor 3-MA obviously antagonized the effects of sericin on cell apoptosis, cell cycle and autophagic protein expressions. CONCLUSION: Sericin can inhibit the proliferation of human gastric cancer MKN45 cells by regulating cell autophagy to serve as potential anti-tumor agent.

Long non-coding RNA ASncmtRNA-2 is upregulated in diabetic kidneys and high glucose-treated mesangial cells.

Diabetic nephropathy (DN) is one of the most frequent complications associated with type I and II diabetes mellitus. Kidneys from patients with DN are characterized by mesangial matrix expansion and increased thickness of the glomerular basement membrane, which are induced by reactive oxygen species (ROS) production. Previous studies have been conducted to investigate this; however, the detailed mechanism of DN progression remains to be elucidated. The present study evaluated the expression of antisense mitochondrial non-coding RNA-2 (ASncmtRNA-2) in an experimental DN model and cultured human mesangial cells. When mice that exhibited genetic type II diabetes developed DN, ASncmtRNA-2 expression was significantly increased (P=0.017) and was positively correlated with pro-fibrotic factor transforming growth factor ß1 (TGFß1) expression and its downstream gene, fibronectin. Inhibition of ROS through administration of the nitric oxide synthase inhibitor, NG-nitro-L-Arginine methylester (L-NAME), significantly reduced (P=0.022) the upregulation of ASncmtRNA-2 in DN. In cultured human renal mesangial cells (HRMCs), ASncmtRNA-2 was upregulated by high glucose stimuli in a time-dependent manner. Glucose-induced upregulation of ASncmtRNA-2 was also reduced by co-incubation of HRMCs with L-NAME. Notably, specific short hairpin RNA against ASncmtRNA-2 significantly downregulated the expression of TGFß1 in HRMCs. The present study suggests that ASncmtRNA-2 is upregulated by ROS and may promote glomerular fibrosis in DN via positively regulating the expression of pro-fibrotic factors. These findings may provide novel potential therapeutic and preventative treatments for DN.

Plating versus intramedullary fixation for mid-shaft clavicle fractures: a systemic review and meta-analysis.

Background. Plate fixation and intramedullary fixation are the most commonly used surgical treatment options for mid-shaft clavicle fractures; the latter method has demonstrated better performance in some studies. Objectives. Our aim was to critically review and summarize the literature comparing the outcomes of mid-shaft clavicle fracture treatment with plate fixation or intramedullary fixation to identify the better approach. Search Methods. Potential academic articles were identified from the Cochrane Library, MEDLINE (1966-2015.5), PubMed (1966-2015.5), EMBASE (1980-2015.5) and ScienceDirect (1966-2015.5). Gray studies were identified from the references of the included literature. Selection Criteria. Randomized controlled trials (RCTs) and non-RCTs comparing plate fixation and intramedullary fixation for mid-shaft clavicle fracture were included. Data Collection and Analysis. Two reviewers performed independent data abstraction. The I (2) statistic was used to assess heterogeneity. A fixed- or random-effects model was used for the meta-analysis. Results. Six RCTs and nine non-RCTs were retrieved, including 513 patients in the intramedullary fixation group and 521 patients in the plating group. No significant differences in terms of the union rate and shoulder function were found between the groups. Patients in the intramedullary fixation group had a shorter operative time, less blood loss, smaller wound size, and shorter union time than those in the plating group. With respect to complications, significant differences were identified for all complications and major complications (wound infection, nonunion, implant failures, transient brachial plexopathy, and pain after 6 months). Similar secondary complications (symptomatic hardware, hardware irritation, prominence, numbness, hypertrophic callus) were observed in both groups. Conclusions. Intramedullary fixation may be superior to plate fixation in the treatment of mid-shaft clavicle fractures, with similar performance in terms of the union rate and shoulder function, better operative parameters and fewer complications.

Position Emission Tomography/Single-Photon Emission Tomography Neuroimaging for Detection of Premotor Parkinson's Disease.

Premotor Parkinson's disease (PD) refers to a prodromal stage of Parkinson's disease (PD) during which nonmotor clinical features may be present. Currently, it is difficult to make an early diagnosis for premotor PD. Molecular imaging with position emission tomography (PET) or single-photon emission tomography (SPECT) offers a wide variety of tools for overcoming this difficulty. Indeed, molecular imaging techniques may play a crucial role in diagnosing, monitoring and evaluating the individuals with the risk for PD. For example, dopaminergic dysfunctions can be identified by detecting the expression of vesicular monoamine transporter (VMAT2) and aromatic amino acid decarboxylase (AADC) to evaluate the conditions of dopaminergic terminals functions in high-risk individuals of PD. This detection provides a sensitive and specific measurement of nonmotor symptoms (NMS) such as olfactory dysfunction, sleep disorders, and psychiatric symptoms in the high-risk patients, especially at the premotor phase. Molecular imaging technique is capable of detecting the dysfunction of serotonergic, noradrenergic, and cholinergic systems that are typically associated with premotor manifestations. This review discusses the importance of SPECT/PET applications in the detection of premotor markers preceding motor abnormalities with highlighting their great potential for early and accurate diagnosis of premotor symptoms of PD and its scientific significance.