By means of 24 h multi-channel intraluminal impedance and pH recording (MII/pH), patients with heartburn and normal upper gastrointestinal endoscopy findings can be classified into those with non-erosive reflux disease (NERD) and those with reflux hypersensitivity (RH). Therefore, in this study, we investigated the difference in oesophageal function tests in Chinese patients with NERD and RH.NERD patients were selected from the digestive department, Beijing Anzhen Hospital and Beijing Chao-Yang Hospital, Capital Medical University, after upper gastrointestinal endoscope, high-resolution manometry and impedance (HRiM), and MII/pH examinations between 2014 and 2016.P = 0.122); integrated relaxation pressure, 7.5 ± 4.8 and 7.9 ± 5.2 mmHg (P = 0.485); distal contractile integral, 751.9 ± 856.2 and 661.9 ± 961.7 mmHg∙s∙cm (P = 0.482); ineffective oesophageal motility rate, 49.5% and 41.3% (P = 0.241); fragmented peristalsis rate, 5.4% and 9.8% (P = 0.235); hiatal hernia rate, 9.0% and 8.6% (P = 0.938); total bolus transit time, 6.3 ± 1.3 and 6.5 ± 1.3 s (P = 0.119); complete bolus transit rate, 76.1 ± 33.0% and 73.1 ± 32.0% (P = 0.224); total acid exposure time, 6.1 ± 3.7% and 0.8 ± 0.8% (P < 0.001); total bolus exposure time, 2.5 ± 2.1% and 1.5 ± 1.1% (P < 0.001); proximal acid reflux events, 13.2 ± 10.5 and 9.7 ± 8.9 (P = 0.011); distal acid reflux events, 25.3 ± 15.8 and 13.4 ± 11.2 (P < 0.001); post-reflux swallow-induced peristaltic wave index, 25.1 ± 9.5% and 32.6 ± 15.2% (P < 0.001); and mean nocturnal baseline impedance, 1,450.2 ± 750.8 and 2,503.6 ± 964.1 ohms (P < 0.001), respectively.In total, 111 NERD patients with abnormal acid exposure, and 92 RH patients were enrolled. Values for NERD and RH were as follows: lower oesophageal sphincter pressure, 15.3 ± 8.9 and 19.3 ± 23.3 mmHg (NERD and RH patients showed similar values on HRiM. NERD patients had greater acid exposure time, bolus exposure time, proximal and distal acid reflux events, and increased impairment of chemical clearance and mucosal integrity than RH patients. NERD and RH should be classified correctly by MII/pH to provide adequate relief from related symptoms. Non-erosive reflux disease (NERD) is characterised by the absence of oesophageal mucosal damage during upper gastrointestinal endoscopy, despite the presence of the ‘classic’ symptoms of gastroesophageal reflux, such as heartburn and acid reflux . AdditioThe study received ethics approval from the local ethics board of Beijing Anzhen Hospital, Capital Medical University. All participants gave written informed consent.Chinese patients who presented with a main symptom of heartburn with a normal upper gastrointestinal endoscopic examination, and underwent high-resolution manometry and impedance (HRiM), and 24-h multi-channel intraluminal impedance and pH recording (MII/pH) in the digestive department of Beijing Anzhen Hospital and Beijing Chao-Yang Hospital between December 2014 and December 2016 were enrolled. Patients with other chronic active medical diseases  were excluded.Patients with heartburn and a normal upper gastrointestinal endoscope exam, abnormal oesophageal acid exposure, and positive or negative symptom reflux association, were diagnosed with NERD. The diagnostic criteria for RH were all of the following: (i) the presence of retrosternal symptoms, including heartburn and chest pain, (ii) normal endoscopy and absence of evidence that eosinophilic o oesophagitis was the cause of the symptoms, (iii) absence of major oesophageal motor disorders  outflow obstruction, diffuse oesophageal spasm, jackhammer oesophagus, and absent peristalsis), and (iv) evidence of triggering of symptoms by reflux events despite normal acid exposure on pH or pH-impedance monitoring (response to anti-secretory therapy does not exclude the diagnosis). The criteria had to be fulfilled for 3 months prior to the beginning of the study, with symptom onset at least 6 months prior to diagnosis with a frequency of at least twice per week .A specially designed solid-state manometry catheter  with 32 manometric sensors and four pairs of MII sensors, separated by 5-cm intervals, was used to assess oesophageal pressures and impedance with the patient in a supine position. The lower oesophageal sphincter (LES) was examined with distal circumferential manometric sensors. The catheter was positioned so that the pressure transducers were located across the upper oesophageal sphincter, oesophageal body, and LES, and the distal channels were in the stomach. Ten swallows with 5 mL normal (0.9%) saline solution were then performed at 30-s intervals.2 antagonists.The 2.1-mm outer diameter study catheter consisted of six electrode pairs for measuring intraluminal impedance  and an antimony pH sensor 5 cm above the LES . An impedance amplifier delivered an ultra-low current in a range of 1-2 kHz, with resulting current flow variations in response to intraluminal impedance changes . Signals from the six impedance channels and the pH channel were recorded at 50 samples per second. The data were stored in an ambulatory recorder and saved on a 256-MB CompactFlash card. Event markers recorded the occurrence of symptoms, times of meals, and changes in body position. The study was performed on an outpatient basis after an overnight fast with the LES located by oesophageal manometry. The patients undertook HRiM and MII/pH with a 7-day washout for proton pump inhibitors and/or HOesophageal bolus clearance can be assessed by measurement of total bolus transit time by classifying swallows as complete bolus transit  or as incomplete bolus transit , and in terms of the complete bolus transit rate. The distal contractile integral (DCI) of the distal segmental contraction is a parameter that integrates contractile pressure (mmHg), duration (s) of contraction, and the length of the smooth muscle oesophagus (cm). Distal oesophageal amplitude (DEA) is an average of the contraction amplitude at 5 and 10 cm above the LES. Integrated relaxation pressure (IRP) reports the mean EGJ pressure, which is measured with an electronic equivalent of a sleeve sensor for four continuous or non-continuous seconds of relaxation in a 10-s window following deglutitive UES relaxation. The parameters of the length of the lower oesophageal sphincter (LESL), the lower oesophageal sphincter pressure (LESP), the lower oesophageal sphincter residual pressure (LESRP), and the upper oesophageal sphincter pressure (UESP) were also measured , 8. InefA post-reflux swallow-induced peristaltic wave (PSPW) was defined as an antegrade 50% drop in impedance relative to the pre-swallow baseline originating in the most-proximal impedance site, reaching all distal impedance sites, and followed by a return to at least 50% of the baseline in the distal impedance sites (bolus exit). Post-reflux swallows that did not reach the distal impedance sites, or that occurred more than 30 s after the end of reflux episodes, were not considered. For each impedance-pH tracing, the number of refluxes followed within 30 s by a PSPW was divided by the number of total refluxes  to obtain the PSPW index .Mean nocturnal baseline impedance (MNBI) was assessed from the most distal impedance channel during a night recumbency period. Three 10-min time periods  were selected, and the mean baseline for each period was computed with the aid of the software. Time periods including swallows, refluxes, and pH drops were avoided. The mean of the three measurements was manually calculated to obtain the MNBI .There were two groups in the study: Chinese NERD patients and RH patients. The diagnosis of acid exposure was according to the results of MII/pH, presenting an abnormal upright acid exposure time (≥6.3%), recumbent acid exposure time (≥1.2%), or total acid exposure time (≥4.2%) .t-test or χ2 test. A p-value < 0.05 was considered to indicate statistical significance. All data were analysed with the SPSS software .Categorical data are described as means ± standard deviation (SD). Data were analysed using the independent-samples In total, 351 patients with heartburn for at least 6 months with a frequency of at least twice a week and without (or with ineffective) PPI treatment, and normal upper gastrointestinal endoscope examinations underwent HRM/Z and MII/pH testing between December 2013 and December 2015, 148 of whom with normal acid exposure and negative SAP and diagnosed with functional heartburn were excluded. Also, 111 patients with abnormal acid exposure were diagnosed with NERD, and 92 patients with normal acid exposure and positive SAP were diagnosed with RH; 20 patients had a history of ineffective PPI treatment (12 in the NERD group and 8 in the RH group). The group with NERD was significantly older than the group with RH. The groups showed no difference in gender.The HRiM results between NERD and RH patients are shown in Table The MII/pH results between NERD and RH patients are shown in Table The Vevey Consensus Group defined NERD as a subcategory of GERD, characterised by troublesome reflux-related symptoms in the absence of oesophageal erosions/breaks on conventional endoscopy and without recent acid-suppressive therapy . CompareIn this study, we compared oesophageal HRiM and MII/pH values between NERD and RH patients with the same Sandhill system and in the supine position. NERD and RH patients showed similar values of LESP, LESL, LESRP, IRP, UESP, DEA, and DCI. It is known that the primary determinant of GERD severity is a dysfunctional anti-reflux barrier and impaired oesophageal clearance. Anti-reflux barrier prevents reflux of gastric contents into the oesophagus, while peristalsis helps to clear the reflux, to reduce exposure to the noxious components of the gastric juice. In this study, we found NERD and FH patients showed similar rates of hiatal hernia, and similar values of TBTT and CBTR; therefore, the two groups showed similarly impaired oesophageal clearance.IEM, also known as oesophageal hypocontractility, is a manometric pattern characterised by ineffective swallows with poor bolus transit in the distal oesophagus. In the Chicago Classification (ver. 3.0), IEM is defined on Clouse plots using a DCI of <450 mmHg∙s∙cm, with > 50% ineffective swallows; IEM is highly prevalent in GERD patients , 26. In Savarino et al.  reportedRecently, impedance values for evaluation of oesophageal chemical clearance (PSPW index) and mucosal integrity (MNBI) have been proposed , 14. AftIn this study, NERD patients had significantly lower PSPW index and MNBI values, greater acid exposure time and bolus exposure time, and more proximal and distal reflux events than RH patients. Impairment of chemical clearance, as indicated by a low PSPW index, implies prolonged contact of the oesophageal mucosa with acidic and weakly acidic refluxes . A lowerOur study had some limitations. All subjects were recruited from two centres in one city using the Western normal range for MII/pH, and lacked a symptom severity score, which may have caused selection bias. The small number of patients limited the statistical power of the study. However, we are the first to compare oesophageal function tests between NERD and RH in Chinese patients.NERD and RH patients showed similar values on HRiM. NERD patients had greater acid exposure time, bolus exposure time, numbers of proximal and distal acid reflux events, and increased impairment of chemical clearance and mucosal integrity than RH patients. NERD and RH should be classified correctly by MII/pH to provide adequate relief from related symptoms.


Laminaria hyperborea is presented. Lipid extracts were fractionated into neutral lipids, free fatty acids, and polar lipids, prior to derivatization and GC-MS analysis. A total of 42 fatty acids were identified and quantified, including the n-3 fatty acids α-linolenic acid, stearidonic acid, and eicosapentaenoic acid. The fatty acid amounts are higher in blade than in stipe . The highest amounts of n-3 fatty acids are found within the neutral lipid fractions with 590.6 ug/g dry weight and 100.9 ug/g dry weight for blade and stipe, respectively. The amounts of polyunsaturated fatty acids are 3.4 times higher in blade than stipe. The blade had the highest PUFA/SFA ratio compared to stipe (1.02 versus 0.76) and the lowest n-6/n-3 ratio (0.8 versus 3.5). This study highlights the compositional differences between the lipid fractions of stipe and blade from L. hyperborea. The amount of polyunsaturated fatty acids compared to saturated- and monounsaturated fatty acids is known to influence human health. In the pharmaceutical, food, and feed industries, this can be of importance for production of different health products. A thorough analysis of the fatty acid profiles of stipe and blade from the kelp species As much as 60% of the world food energy intake is provided by the cereals wheat, rice, and corn . These cty acids –4. A proty acids . Still, ty acids ; thus, t n-3 and n-6 fatty acids [ n-6/n-3 ratio is reported to have suppressive effects on cardiovascular diseases, cancer, and inflammatory and autoimmune diseases [ n-3 PUFAs [ Laminaria hyperborea, a common species of kelp found in the northern Atlantic, has only previously been characterized with regard to fatty acids in three studies [ Laminaria hyperborea harvested on the west coast of Ireland. Variations in distribution of fatty acids between different parts of the seaweed depend on the morphology of the species as well as its physiological and ecological circumstances [Seaweed has for many years been thought to have positive effect on human health, and consumption of these marine plants has been linked to a lower incidence of cancer, hyperlipidemia, and coronary heart disease . They arty acids . A diet diseases . With a -3 PUFAs . Previou-3 PUFAs –20. More-3 PUFAs , 21–23. -3 PUFAs . The bro studies , 24, allmstances . L. hyperborea. In this study, the lipids were fractionated into free fatty acids (FFAs), acyl glycerides , and polar lipids (PLs) by using solid phase extraction. The fatty acids from each class were identified and quantified by GC-MS.Before considering the marine algae as a food source, it is important to assess its nutritional value. In this context, the aim of this study has been to provide a thorough analysis of the fatty acid profiles in stipe and blade from the macroalgaeChloroform and hexane were of Chromasolv® quality, heptane, diethyl ether, methanol, and NaCl puriss pa. quality, all from Sigma-Aldrich . The acetic acid was from Honeywell Riedel-de Haen .cis-6,9,12,15-octadecatetraenoic acid methyl ester, all-cis-8,11,14,17-eicosatetraenoic acid methyl ester, and all-cis-7,10,13,16,19-docosapentaenoic acid methyl ester. In addition, the following individual FAME standards were used: nonanoic acid methyl ester , 13-methyltetradecanoic acid methyl ester, trans-9-tetradecenoic acid methyl ester, cis-9-heptadecenoic acid methyl ester, cis-13-octadecenoic acid methyl ester, cis-9-eicosenoic acid methyl ester, and hexacosanoic acid methyl ester . Three internal standards were used , one for each lipid fraction; 1,2-dinonadecanoyl-sn-glycero-3-phosphatidylcholine for the PL fractions, nonadecanoic acid for the FFA fractions, and trinonadecanoin for the NL fractions . Nonadecanoic acid methyl ester  was added to the 37 components FAME mix for retention time identification, since C19:0 was used as internal standard in the samples.A fatty acid methyl ester (FAME) mix with 37 components  was used for identification of the FAMEs. A 21-component FAME mix , Larodan AB, Solna, Sweden) was used for identification of all-L. hyperborea was provided and identified by FMC BioPolymer AS. It was harvested off the west coast of Norway, outside Sør-Trøndelag County Municipality in October 2015. On board the trawler L. hyperborea was rinsed, crude-cut, and preserved with formalin manually. The holdfast was discarded. Once off the trawler stipe and blade were vacuum packed separately. When received at the university, stipe and blade were rinsed with water to eliminate contaminants, frozen with liquid N2 , and freeze-dried . The freeze-dried material was crushed in a QMM Micromixer and pulverized in a Laboratory Mixer 3100 (Danfoss) by G. A. Lund at Pharmatech AS, Fredrikstad, Norway. The water content of fresh seaweed was measured according to ISO 11465:1993/Cor1:1994.3 : MeOH (2/1), and 50 µL of each internal standard was added with a Hamilton® syringe. To induce phase separation, 0.9% NaCl was added after mixing (0.2 times the volume of CHCl3 : MeOH). After approximately 20 min the organic phase was transferred to a test tube . The polar phase was reextracted with CHCl3, 30–60 mL depending on amount of alga powder. The organic phases of each sample were combined and evaporated with a vacuum evaporator  at 40°C, redissolved in 1.00 mL chloroform, and transferred to vials for SPE.Four sample replicates of both stipe and blade were used and they were all treated separately during the sample preparation stages. The lipids were extracted with a modified Folch's method . In brieµL of sample was applied. The NLs  were eluted with 5 mL chloroform, then the FFAs with 5 mL diethyl ether:acetic acid (98 : 2 v/v), and lastly the PLs with 5 mL methanol. The possibility of cross contaminations between any of the three classes was checked by performing tests with standards for each lipid class. The recovery was 90% or higher. The eluates were transferred to culture tubes  and evaporated under N2 (g) at 40°C.A liquid-handling robot  was used to carry out the SPE procedure. The method used was based on previous work by Pinkart et al. 1998 and Ruiz et al. 2004 . Aminopr3-MeOH . The samples were heated for 5 min at 70°C in a water bath. After heating, 1 mL heptane was added to each sample tube before mixing on a vortex mixer. The heptane phases were transferred to vials and stored at −20°C prior to analysis by GC-MS.For formation of FAMEs the NL and PL fractions were redissolved in 2 mL of heptane, before addition of 1.5 mL of 3.3 mg/mL sodium methoxide. The sodium methoxide solution was made by dissolving metallic sodium  in methanol to a concentration of 3.3 mg/mL. The culture tubes were then shaken horizontally for 30 min at 350 rpm  and left to settle vertically for 10 min before the heptane phases were transferred to vials for storage at −20°C prior to GC-MS analysis. The FFA fractions were redissolved in 1 mL BFµm film thickness of fused silica 90% biscyanopropyl/10% phenylcyanopropyl polysiloxane stationary phase . For carrier gas, helium  was used at 1 mL/min constant flow. The EI ion source was used in positive mode, producing 70 eV electrons at 250°C. The MS was scanned in the range 40–600 m/z with 0.3 s scan time, 0.2 s interscan delay, and 0.5 s cycle time. The transfer line temperature was set at 270°C. The resolution was 1000.The analysis of the FAMEs was based on a previously published method . ShortlyµL sample. Two injections parallels were used for each sample replicate. Identification of fatty acids was performed by comparing retention times with standards as well as MS library searches. MassLynx version 4.0  and NIST 2014 Mass Spectral Library  were used. Relative response factors previously determined by Devle et al. [µg/g dry weight (DW). The GC oven had a start temperature of 65°C, held for 3 min, the temperature was then raised to 150°C (40°C/min), held for 13 min, before being increased to 151°C (2°C/min), held for 20 min, with a slow increase to 230°C (2°C/min), and held for 10 min, before a final increase to 240°C (50°C/min), and the end temperature was held for 3.7 min.A split ratio of 1/10 was used with injections of 1.0 e et al.  were emp L. hyperborea, as shown in  cis-9) and the remaining five were polyunsaturated .We have identified and quantified 42 different fatty acids inµg/g DW . Among the predominating fatty acids are important dietary n-3 fatty acids such as α-linolenic acid , stearidonic acid , and eicosapentaenoic acid  as well as two n-6 fatty acids, linoleic acid ,and arachidonic acid . How favorable L. hyperborea is for the human diet  depends on several factors, for example, content of essential FAs (LA and ALA), other important nutritional FAs , and the ratios between PUFA/SFA and n-6/n-3 fatty acids.The predominating fatty acids constitute more than 90% of the total fatty acid content in all the fractions, as shown in µg/g DW . A fattyn et al.  and also n-3 and n-6 PUFAs [ n-3 and n-6 fatty acids are found in the FFA fraction. The highest amounts are found in the NL fraction, with the exception of n-3 in blade where the amount in the PL fraction is higher than in the NL fraction . The highest amounts of the essential FAs LA and ALA were found in the NLs for the blade . However, for stipe, the highest amount of LA was found in the PL fraction (48.4 μg/mL) and highest amount of ALA was found in the NL fraction (7.3 μg/g DW). There was a lower proportion of the n-6 FA arachidonic acid in blade versus stipe . This corresponds to what was reported by Schmid and Stengel [It is known that brown seaweeds grown in temperate or subarctic areas can accumulate-6 PUFAs . In both Stengel . n-3 (DHA) as part of a healthy diet [μg/g DW). With a total content of EPA+DHA in blade at 832 μg/g DW, achieving the recommended amount by consumption of seaweed alone is highly unlikely, as the daily intake would have to be approximately 300 g dried seaweed (or 1500 g fresh seaweed). Although n-3 and n-6 PUFAs are usually easily oxidized, studies have shown that these PUFAs have exhibited high oxidative stability in seaweed lipids in dried seaweed products [The World Health Organization (WHO) recommends a daily intake of 0.25 g EPA+ C22:6thy diet . Even ththy diet . The higproducts . The reaproducts . L. hyperborea (fronds) harvested in France with 25% SFA and 22% MUFA. Mæhre et al. [Stipe and blade differ not only in content of the individual FAs but also in the amounts of SFAs, MUFAs, and PUFAs . The stie et al.  harvestee et al. . The NL L. hyperborea has an n-6/n-3 ratio of 0.8/1 in blade and 3.5/1 in stipe. The ratios vary between the lipid fractions, as seen in  n-6/n-3 ratio is 15–20/1 and this is significantly higher than ~1, which was normal during human evolution [ n-6/n-3 ratios of 2–5/1 are reported to have suppressive effects on cardiovascular, inflammatory, and autoimmune diseases [ n-6/n-3 ratios in stipe and blade, using only blade could be considered if a very low n-6/n-3 ratio is desired. Though, in this context it should also be mentioned that FAO in their 2010 report [volution . Howevervolution . n-6/n-3diseases , 38. Sin0 report  do not cWhile the same fatty acids predominate, the amounts in blade are consistently higher than those in stipe, as seen in  L. hyperborea, with maximum two fatty acids having trans configuration. Some fatty acids are found in either stipe or blade, while others are only present in certain lipid fractions  within stipe and blade. Among the predominating fatty acids are the n-3 fatty acids ALA (10.4 and 131 µg/g DW), SDA (17.2 and 394 µg/g DW), and EPA (126 and 830 µg/g DW), as well as two n-6 fatty acids: LA (96 and 296 µg/g DW) and AA (444 and 723 µg/g DW); the values in parentheses are for stipe and blade, respectively. The ratios between n-6 and n-3 fatty acids are ≤4.4/1 in all lipid fractions but especially low (≤1.3/1) in blade. The blades also presented the highest PUFA/SFA ratio. Regarding the potential of commercialization in respect of nutritional applications of L. hyperborea, blade is found to represent the most suitable material, due to higher levels of PUFAs and a low n-6/n-3 ratio.A total of 42 different fatty acids are identified and quantified in the stipe and blade of


Streptococcus mutans include acidogenicity, aciduricity, and extracellular polysaccharides (EPS) synthesis. The de novo designed antimicrobial peptide GH12 has shown bactericidal effects on S. mutans, but its interaction with virulence and regulatory systems of S. mutans remains to be elucidated. The objectives were to investigate the effects of GH12 on virulence factors of S. mutans, and further explore the function mechanisms at enzymatic and transcriptional levels. To avoid decrease in bacterial viability, we limited GH12 to subinhibitory levels. We evaluated effects of GH12 on acidogenicity of S. mutans by pH drop, lactic acid measurement and lactate dehydrogenase (LDH) assay, on aciduricity through survival rate at pH 5.0 and F1F0-ATPase assay, and on EPS synthesis using quantitative measurement, morphology observation, vertical distribution analyses and biomass calculation. Afterwards, we conducted quantitative real-time PCR to acquire the expression profile of related genes. GH12 at 1/2 MIC (4 mg/L) inhibited acid production, survival rate, EPS synthesis, and biofilm formation. The enzymatic activity of LDH and F1F0-ATPase was inhibited, and ldh, gtfBCD, vicR, liaR, and comDE genes were significantly downregulated. In conclusion, GH12 inhibited virulence factors of S. mutans, through reducing the activity of related enzymes, downregulating virulence genes, and inactivating specific regulatory systems.Cariogenic virulence factors of  Streptococcus mutans has been recognized as the principal etiological agent of dental caries [S. mutans lead to abundant acid production, acid tolerance and stable biofilm formation [S. mutans, in which lactate dehydrogenase (LDH) plays a critical role [S. mutans. Membrane-bound F1F0-ATPase works as a pump to transport protons from cells and to maintain internal pH values. This is believed as the determinant of aciduricity of this bacterium [S. mutans is its ability to produce glucosyltransferases (GTFs) to catalyze synthesis of intracellular polysaccharides (IPS) and extracellular polysaccharides (EPS) from sucrose [S. mutans could be an appealing approach to preventing dental caries.Dental caries is a transmissible infectious disease caused by cariogenic bacteria, and l caries . Cariogeormation . Productcal role . Aciduriacterium . Another sucrose . EPS, es sucrose . Hence, S. mutans is not the consequences of independent expression by several virulence genes, but the results of series of coordinate regulatory events. Regulatory systems are involved in enhancing the ecological fitness and cariogenic virulence of S. mutans [S. mutans [However, the cariogenic potential of . mutans . The Vic. mutans ,9. The C. mutans . As thes. mutans .S. mutans in vitro, including defensins, Cathelicidin LL-37, Histatin 5, Human Lactoferrin [S. mutans and enamel demineralization [de novo designed and synthesized a series of cationic, amphipathic α-helical AMPs and selected the most promising one, GH12, a 12-amino acids peptide with optimal structure and potency. Previous studies [S. mutans, was potent against growth of planktonic bacteria and had a rapid mechanism of action, killing 90% of bacteria in less than 5 min of exposure. Moreover, GH12 effectively inhibited S. mutans biofilm formation and metabolism, as well as significantly reduced the biomass of 1-day old S. mutans biofilm. Cytomembrane observation showed that GH12 had a mechanism of killing S. mutans through formation lysis and pores on the cell membrane and envelope. Meanwhile, GH12 had showed little toxic effect on the viability of human gingival fibroblasts [In recent years, studies about antimicrobial peptides (AMPs) have been extensively conducted around the world . Over thtoferrin , KSL [15toferrin , Bac8c [toferrin , and C16toferrin . Furtherlization . These f studies ,21 showeroblasts .S. mutans, but its interaction with virulence factors and regulatory systems of S. mutans remains to be elucidated. The objectives of this study were to investigate effects of GH12 on acid production, acid tolerance, EPS synthesis, and biofilm formation of S. mutans in vitro, and then further explore the function mechanism of GH12 at enzymatic and transcriptional levels. To avoid decrease in bacterial population and viability of S. mutans, we limited GH12 to subinhibitory levels. It is the first time to systematically investigate effects of AMPs on virulence and regulatory systems of S. mutans. Thereby, more information about GH12 could be obtained to further explain its anticaries potential.GH12 has shown anticaries potential because of its bactericidal effects on 2) was synthesized, identified, and purified to 98% by GL Biochem  as described before [S. mutans UA159 was obtained from the State Key Laboratory of Oral Diseases at Sichuan University  and grown in brain–heart infusion broth  anaerobically  at 37°C [Peptide GH12 , 1/4 MIC (2 mg/L) and 1/2 MIC (4 mg/L). Sterile deionized water acted as vehicle control. These tubes were incubated at 37°C anaerobically for 24 h. The absorbance at 600 nm (A600) was determined using a microplate spectrophotometer  every hour throughout 24 h of incubation. The experiments were repeated three times independently.Aliquots of overnight culture of S. mutans was assessed by the 3--2, 5-diphenyltetrazolium bromide (MTT) staining method as described [S. mutans was suspended with BHI broth containing GH12 at sub-MIC levels or sterile deionized water. Volumes of 200 μL of different suspension were inoculated into wells in a 96-well U-bottom microtiter plate, and incubated at 37°C for 24 h. After centrifugation , the supernatant was decanted. 200 μL of MTT dye (500 mg/L MTT in PBS) was added into the wells. Following incubation at 37°C for 2 h, the MTT solution was replaced with 200 μL DMSO to dissolve the formazan crystals. The absorbance at 540 nm (A540) was measured. The experiments were repeated for three times independently.The effects of GH12 at sub-MIC levels on the viability of escribed ,26 with S. mutans glycolysis was measured as described elsewhere with some modifications [S. mutans was harvested at mid-logarithmic phase, washed with salt solution (50 mM KCl + 1 mM MgCl2), and resuspended in a salt solution containing GH12 at sub-MIC levels or sterile deionized water. Initial concentration of S. mutans was adjusted to 1 × 107 CFU/mL. Glucose was added to a final concentration of 1% (w/v) and the initial pH of the mixtures was then adjusted to 7.2. The decrease in pH was monitored over a period of 120 min by Orion Dual Star, pH/ISE Benchtop . The experiments were repeated for three times independently.The effect of GH12 on ications . BrieflyS. mutans were harvested, washed twice with phosphate buffered saline (PBS) and resuspended to a final concentration of 1 × 107 CFU/mL in a 24-well plate with 1.5 mL BPW supplemented with 0.2% sucrose containing sub-MIC levels of GH12 in each well. The vehicle control contained no GH12. The plate was further incubated for 120 min at 37°C anaerobically. After removing planktonic cells by centrifugation , the supernatants were decanted to measure lactate concentrations according to the manuscript of the Lactate Assay Kit (MAK064). The absorbance at 570 nm (A570) was recorded using a microplate spectrophotometer and lactate concentrations were calculated using standard curves. The experiments were repeated for three times independently.For lactic acid measurement, the cells of 450initial) and endpoint  was recorded, and then ΔA450 was calculated to quantify the enzymatic activity. The results were expressed as the percentage of ΔA450 relative to that of the untreated control. The experiments were repeated for five times independently.The crude LDH was extracted according to Xu et al. . The cruS. mutans was evaluated by measuring the viability of bacteria after 120 min exposure of pH 5.0 [S. mutans was grown in TYEG broth until mid-logarithmic phase. After centrifugation, the cells were resuspended (1 × 107 CFU/mL) in TYEG broth buffered with 40 mM phosphate/citrate buffer (pH 5.0) containing sub-MIC levels of GH12, and incubated at 37°C for 2 h. The control mixture contained no GH12. Samples were removed before and after incubation at pH 5.0 for viable counts. The experiments were repeated for three times independently.The effect of GH12 on the acid tolerance of f pH 5.0 . S. mutaS. mutans was permeabilized according to the method described by Belli et al. [1F0-ATPase activity was determined by the amount of released inorganic phosphate in the reaction mixture of 75 μL of permeabilized cells and 3 mL of 50 mM Tris-maleate buffer (pH 6.0) with 10 mM MgSO4. When the mixture was heated to 37°C, the reaction was initiated by adding 30 μL of 0.5 M ATP (pH 6.0). After 30 min of reaction, the released phosphate was determined [i et al. . Then, ptermined , and theS. mutans was diluted with BHI broth supplemented with 1% sucrose (BHIS) and GH12 at sub-MIC levels to a final concentration of 1 × 106 CFU/mL. After anaerobic incubation , culture supernatants and planktonic cells were removed. After fixing the biofilms with methanol for 15 min and staining them with 0.1% (w/v) crystal violet for 5 min, the dye bound to the cells was resolubilized with 33% (v/v) glacial acetic acid. The absorbance at 595 nm (A595) was measured to quantify biofilm formation and the results were expressed as the percentage of A595 relative to that of the untreated control. The experiments were repeated for three times independently.The effect of GH12 at sub-MIC levels on biofilm formation was evaluated as described before . BrieflyS. mutans were produced with different sub-MIC levels of GH12 in 2 mL BHIS in 24-well plates. The water-insoluble EPS of biofilms was determined by the anthrone method [625) was monitored and the concentrations of water-insoluble EPS were calculated using standard curves. The experiments were repeated for three times independently.Biofilms of e method . BrieflyS. mutans were produced on glass coverslips in a 24-well plate under the same conditions mentioned before. 2.5 μM SYTO®9  and 2.5 μM Alexa Fluor® 647  were used according to the manufacturer (Invitrogen). Alexa Fluor® 647-labelled dextran conjugate was added at the beginning of biofilm formation, and 50 μL of SYTO®9 was used to stain bacteria after biofilm formation. The biofilms were imaged with a Leica DMIRE2 confocal laser scanning microscope  equipped with a 60 × oil immersion objective lens. The image channels were set according to the manufacturer. Each biofilm was scanned at five randomly selected positions. All three-dimensional reconstructions of biofilms were performed with Imaris 7.0.0 . The vertical distribution analyses were performed with LAS AF Lite (Leica), and the calculation of bacteria/EPS biomass was performed with COMSTAT  [For EPS staining, biofilms of ysis.dk) .S. mutans was grown in BHI broth supplemented with sub-MIC levels of GH12 until late exponential phase. The control group was not treated with GH12. The RNA isolation and purification were conducted as described before [−ΔΔCt method was used to calculate gene expression fold change, and different gene expressions were normalized to the levels of 16S rRNA gene transcripts. The experiments were repeated for three times independently.To evaluate the effect of GH12 on associated gene expressions, d before . First-sd before –34. Eachd before . The 2−ΔDifferences between the experimental group and the untreated control group were compared using one-way ANOVA and Tukey HSD tests. Statistical analyses were performed using SPSS 20.0  at a significance level of 0.05.S. mutans by growth curves and MTT assay. As shown in S. mutans. When treated by GH12 at 1/2 MIC, S. mutans exhibited an extended lag phase and lower absorbance during the logarithmic phase. At the end of 24 h, the mean absorbance of 1/2 MIC group did not significant differ from those of the non-treated control (P > 0.05). The outcome of MTT assay (S. mutans within 24 h (P > 0.05).We evaluated the effects of GH12 at sub-MIC levels on basic viability of TT assay ) also reS. mutans culture. As shown in S. mutans culture was treated with GH12 at 1/2 MIC, its glycolytic pH dropped slower, and the terminal pH was significantly higher versus that of control (P < 0.05). However, the terminal pH values were not significantly affected by GH12 at 1/4 MIC and 1/8MIC (P > 0.05). Further assays showed that 1/2 MIC of GH12 suppressed the lactic acid production , and LDoduction ) of S. mS. mutans was also inhibited by GH12 at sub-MIC levels. Firstly, S. mutans at pH 5.0 was significantly decreased in the presence of GH12 at 1/2 MIC and 1/4 MIC (P < 0.05). At the same time, GH12 exhibited inhibitory effects on the activity of the F1F0-ATPase of S. mutans. The F1F0-ATPase activity was reduced by 25.26% at 1/2 MIC of GH12 (The acidurity of  of GH12 ).Figure S. mutans to synthesize water-insoluble EPS and form biofilm. As shown in S. mutans biofilms were labelled green, and EPS were labelled red. Compared with biofilm of the untreated control, biofilms treated with GH12 at sub-MIC levels became thinner and looser, their green area was reduced and their red area was diminished. P < 0.05). P < 0.05).GH12 at sub-MIC levels disrupted the ability of ldh, atpD, gtfB, gtfC, gtfD) and TCSTS genes  of S. mutans treated with GH12 at sub-MIC levels wre determined (P < 0.05), except for atpD, which was upregulated but not significantly (P > 0.05). Notably, all GTF-associated genes  were also downregulated by 1/4 MIC of GH12, but only gtfB exhibited significant differences (P < 0.05).The expression profiles of various virulence genes  of L-K6 against S. mutans were four-fold higher than the MIC values. However, the MIC and MBC of GH12 against S. mutans were very close, suggesting that GH12 may have other potentials.Previously, we found that the classic dophilus . Accordidophilus , we concdophilus . Thus, of 8 mg/L . Notablyf 8 mg/L  deduced S. mutans essentially attribute to its clinical complications [S. mutans. Prior to our study, only that on Bac8c [S. mutans. Thus, to our knowledge, this is the first time to systematically investigate effects of AMPs on virulence and regulatory systems of S. mutans. In the present study, we found that GH12 at 1/2 MIC inhibited various cariogenic virulence factors of S. mutans, resulting in reduced acidogenicity, compromised aciduricity, declined EPS synthesis and defective ability to form biofilms. We also proved that GH12 functioned at enzymatic and transcriptional levels.It is well documented that the virulence factors of ications . Neverthon Bac8c  reportedS. mutans. LDH is one of the most important enzymes in the process. Deficiency of LDH implies that S. mutans lose cariogenic potential [S. mutans. Moreover, GH12 at 1/2 MIC suppressed the LDH at both the enzymatic and transcriptional levels in vitro. F1F0-ATPase, plays a major role in maintenance of ΔpH across the cell membrane of S. mutans, and the positive correlation between ATPase levels and acid tolerance has been proved [1F0-ATPase, which may directly lead to a rise in the cytoplasmic acidity, followed by mortal disruption of physiological processes in S. mutans. Thus, the survival rate of S. mutans at pH 5.0 was significantly reduced by GH12 at 1/2 MIC. But the expression of atpD, a gene encoding α subunit of F1F0-ATPase [1F0-ATPase, which is located at cell membrane as well. Previous studies implied that the ATPase activity does not necessarily correlate with transcription or translation levels of associated genes [gtfB) produces highly insoluble glucans, which constitute the scaffold of the EPS matrix. GTFC (encoded by gtfC) catalyzes the formation of a mixture of soluble and insoluble glucans, which shape the initial EPS layers and provide binding sites for S. mutans [S. mutans biofilm. Results of quantitative PCR confirmed that these inhibitory effects were due to the significant suppression of GTFs at the transcriptional level. Notably, GH12 at 1/4 MIC just inhibited the gene expression of gtfB significantly (P < 0.05), but it also showed strong capability to inhibit EPS synthesis and biofilm formation, which may results from that gtfB and gtfC genes are in an operon-like arrangement [Glycolysis is the main pathway to produce acid in otential ,39. The n proved ,40. GH120-ATPase , was upr0-ATPase ,42,43, wed genes . The act. mutans . In thisangement .S. mutans has developed several regulatory systems. vicR gene encodes a VicR response regulator, which is a necessary part of VicRKX TCSTS [gtfBCD [liaR gene products play an important role in biofilm formation and stress response of S. mutans [vicRKX genes [vicR and liaR may result from not only GH12 treatment, but also the interaction between the two genes. comD encodes the histidine kinase receptor to response to competence-stimulating peptide (CSP), and comE encodes an intracellular response regulator to mediate expressions of downstream genes. Activated ComE enables the production of mutacin Ⅳ and Ⅴ, as well as genetic competence [comDE was also observed in the 1/2 MIC group, and downregulation of comDE by 1/2 MIC of GH12 suppresses regulation of genetic competence and intra-species cell–cell communication. Ultimately as summarized in S. mutans, and consequently led to declines of cell persistence and disruption of biofilm formation and integrity. The inhibition of GTFs at the transcriptional level lead to reduced production of EPS and IPS. Lack of EPS disrupts both adherence of bacteria and the structure of biofilm. When exogenous substrate is depleted, S. mutans can metabolize IPS [S. mutans due to reduced preservation of IPS. Inhibition of F1F0-ATPase and small holes on the cytomembrane may directly lead to cytoplasmic acidity, which may also inhibit the normal process of glycolysis. This will in turn diminish the ATP pool, supress the activity of the proton translocator (F1F0-ATPase) and further exacerbate cytoplasmic acidity. When LDH catalyses the conversion of pyruvate to lactate under anaerobic condition, it also converts NADH to NAD+. Thus, the inhibition of LDH would also lead to increase in NADH. If not regulated by NADH oxidase and superoxide dismutase in time, for instance under excess oxidative stress, these excessive reducing equivalents will have toxic effects on S. mutans [S. mutans, which thereby leads to reduced acidogenicity, compromised aciduricity, declined EPS synthesis and defective ability to from biofilms.To cope with various environmental stress, KX TCSTS . It has  [gtfBCD . The lia. mutans . MoreoveKX genes . Thus, tmpetence . Signifilize IPS . Therefo. mutans ,52. In oldh, atpD, gtfC, gtfD, vicR, and liaR , but laS. mutans were inhibited by GH12. This saltatorial pattern has also been observed when S. mutans was treated by hydrogen peroxide, methyl viologen and chlorhexidine in studies of Bitoun et al. [S. mutans, concentration of GH12 should be maintained not lower than 1/2 MIC.However, beyond the threshold of 1/2 MIC (4 mg/L), all virulence factors of n et al. . Thus, fS. mutans in vitro, resulting in reduced acidogenicity, compromised aciduricity, declined EPS synthesis and defective ability to form biofilms through reducing the activity of related enzymes, downregulating virulence genes, and inactivating specific regulatory systems. Considering the complexity of gene regulation in S. mutans, further studies with transcriptomic and proteomic approaches should be carried out.In summary, based on our data, GH12 shows a potential to act as an alternative anticaries agent because GH12 at 1/2 MIC (4 mg/L) inhibited various cariogenic virulence factors of


Recently, we demonstrated that different running overtraining (OT) protocols with the same external load, but performed downhill (OTR/down), uphill (OTR/up), and without inclination (OTR), led to hepatic fat accumulation. As the disruption of endoplasmic reticulum (ER) homeostasis is linked to animal models of fatty liver disease, we investigated the effects of these OT models on the proteins related to ER stress  and apoptosis  in livers of C57BL/6 mice. Also, aerobic training can attenuate cardiac ER stress and improve exercise capacity. Therefore, we investigated whether the decrease in performance induced by our OT protocols is linked to ER stress and apoptosis in mouse hearts. The rodents were divided into six groups: naïve , control , trained (TR), OTR/down, OTR/up, and OTR groups. Rotarod, incremental load, exhaustive, and grip force tests were used to evaluate performance. After the grip force test, the livers and cardiac muscles  were removed and used for immunoblotting. All of the OT protocols led to similar responses in the performance parameters and displayed significantly lower hepatic ATF6beta values compared to the N group. The OTR/down group exhibited lower liver cleaved caspase-3 values compared to the CT group. However, the other proteins related to ER stress and apoptosis were not modulated. Also, the cardiac proteins related to ER stress and apoptosis were not modulated in the experimental groups. In conclusion, the OT protocols decreased the levels of hepatic ATF6beta, and the OTR/down group decreased the levels of hepatic cleaved caspase-3. Also, the decrease in performance induced by our OT models is not associated with ER stress and apoptosis in mice hearts. The endoplasmic reticulum (ER) is a dynamic organelle of the eukaryotic cells with a central role in protein and lipid biosynthesis , 2, in wWhen activated, IRE1 oligomerizes and leads to the trans-autophosphorylation of its cytosolic domain and RNase activity. Increased RNase activity cleaves the mRNA of Xbox-binding proteins (XBP1) in the cytosol, which generates spliced XBP1 mRNA encoding a basic leucine zipper (bZIP)-containing transcription factor that increases the ER-associated degradation (ERAD) components and ER chaperones . Also, IProtein kinase RNA-like endoplasmic reticulum kinase induces phosphorylation of the alpha subunit of eukaryotic translation initiation factor-2  at serine 51 . ChronicThe mechanisms above aim to protect against cell death as well as to decrease overall synthesis and increase chaperone production. However, when UPR persists, the synthesis of immature proteins is not reduced, which results in apoptosis that probably involves other mediators such as caspases 4 and 12, JNK, and CHOP –15. BourKim et al.  verifiedRegarding NFOR etiology, Smith , 23 propRecently, using immunohistochemistry staining, da Rocha et al.  observedad libitum. The experimental procedures were performed according to the Brazilian College of Animal Experimentation (COBEA) and were approved by the Ethics Committee of the University of Sao Paulo (ID 14.1.873.53.0). The rodents were randomly divided into naïve , control , trained , overtrained by downhill running , overtrained by uphill running , and overtrained by running without inclination . The mice were manipulated, trained, and overtrained in a dark room between 6:00 a.m. and 8:00 a.m.Eight-week-old male C57BL/6 mice from the Central Animal Facility of the Ribeirão Preto campus were kept in individual cages at a controlled temperature (22 ± 2°C) on a 12:12-h light–dark inversion cycle with food (Purina chow) and water ®, Ribeirão Preto, São Paulo, Brazil) for 5 days, for 10 min/day at 3 m/min. The initial intensity of the ILT was 6 m/min at 0% with increasing increments of 3 m/min every 3 min until exhaustion, defined when the rodents touched the treadmill end five times in 1 min. The animals were encouraged by physical prodding, and when they became exhausted without finishing the stage, the exhaustion velocity was corrected as proposed by Kuipers and coworkers (−1) of the ILT for the experimental groups during the evaluation weeks were (1) week 0: CT = 25.5 ± 2.8, TR = 22.0 ± 3.5, OTR/down = 20.7 ± 2.8, OTR/up = 24.2 ± 2.7, and OTR = 23.7 ± 2.5; (2) week 4: CT = 24.3 ± 2.5, TR = 27.7 ± 3.4, OTR/down = 27.3 ± 3.4, OTR/up = 27.3 ± 1.6, and OTR = 29.7 ± 0.9; (3) week 8: CT = 19.8 ± 3.3, TR = 31.0 ± 6.3, OTR/down = 15.3 ± 3.1, OTR/up = 17.1 ± 2.3, and OTR = 18.3 ± 3.4.First, the mice were adapted to treadmill running . Once anesthesia was confirmed by the loss of pedal reflexes, each mouse liver and cardiac muscle  were removed and used for immunoblotting. For the immunoblotting technique, we used 6 or 8 animals from each group for each protein.The rodents were anesthetized 36 h after the grip force test. After an overnight fast (12 h), the mice were anesthetized using an intraperitoneal injection of 2-2-2 tribromoethanol 2.5% (10–20 µL gg) for 40 min, and the supernatants were used for protein quantification using the Bradford method  were homogenized in extraction buffer  at 4°C with a Polytron PTA 20S generator , operated at maximum speed for 30 s. The homogenates were centrifuged , beta-actin (CELL4967s), Caspase-4 (CELL4050s), Caspase-12 (CELL2202s), CHOP (CELL2895s), TRAF2 (CELL4712s), phospho-TRAF2  from Cell Signaling Technology  at a dilution of 1:1,000; beta-actin (SC69879), BiP (SC33757), Caspase-3 (SC7148), eIF2alpha (SC11386), phospho-eIF2alpha , GRP94 (SC11402), PERK (SC13037), and phospho-PERK  from Santa Cruz Biotechnology  at a dilution of 1:750; IRE1 (A37073) and phospho-IRE1  from Abcam  at a dilution of 1:1,000; ATF6beta (orb155755), and phospho-Bax  from Biorbyt  at a dilution of 1:1,000.®, Lincoln, NE, USA) and quantified using the Image Studio software for C-DiGit Blot Scanner.After the membranes had been washed with TBS containing 0.1% Tween-20, they were incubated for 1 h at 4°C with secondary antibody conjugated with horseradish peroxidase. The specific immunoreactive bands were detected using chemiluminescence . Images were acquired by the C-DiGit™ Blot Scanner .The results are expressed as the mean ± SEM. While the Shapiro–Wilk Figure From week 4 to week 8, the TR group increased its ILT performance significantly compared to the OTR/down, OTR/up, and OTR groups. On the other hand, the OTR/down and OTR groups decreased their ILT performances significantly compared to the CT group. As it is known that a decrease or stagnation in performance is the only marker for the diagnosis of the NFOR state , 39–42, Figure P < 0.05) in the OTR/down (73.2%), OTR/up (67.7%), and OTR (58.6%) groups compared to the N group. Also, the OTR/down and OTR/up groups were 69.8 and 63.6% lower (P < 0.05) compared to the CT group, respectively. There was no significant difference between the experimental groups for the hepatic protein levels of CHOP, caspase-4, cleaved caspase-12, pBax (Ser184), and pTRAF2 (Ser11)  in the OTR/down (66.0%) group compared to the CT group.There was no significant difference between the experimental groups for the hepatic protein levels of BiP, pIRE1 Ser724), pPERK (Thr981), peIF2alpha (Ser52), and GRP94 , pPERK (Thr981), peIF2alpha Ser52), ATF6beta, and GRP94  experimental model of rodents—while Mikami et al.  different exercise protocols—Mikami et al.  subjectei et al.  and Huani et al.  investigi et al.  used 48 i et al.  euthanizi et al. .Although the hepatic levels of cleaved caspase-12 for the OTR/down group were 70% higher compared to the N group, the differences between these groups were not significant. Recently, Ryoo  highlighBased on the relationship between aerobic training, attenuated cardiac ER stress, and improved exercise capacity , we alsoRecently, Bourdier and coworkers  verifiedAlthough moderate or high-intensity aerobic training can reduce cardiac ER stress induced by post-myocardial infarction heart failure or intermittent hypoxia , 16, resTo date, this is the first investigation regarding the effects of OT on the hepatic and cardiac proteins related to ER stress and apoptosis. In conclusion, the running OT protocols with the same external load, but performed downhill, uphill, and without inclination, decreased the levels of hepatic ATF6beta, which may be linked to high levels of liver fat, as recently published by our research group. Also, the OTR/down protocol decreased the levels of hepatic cleaved-caspase 3. Finally, the decrease in performance induced by our OT models is not linked to ER stress and apoptosis in mice hearts. These results are summarized in Figure The experimental procedures were performed according to the Brazilian College of Animal Experimentation (COBEA) and were approved by the Ethics Committee of the University of Sao Paulo (ID 14.1.873.53.0).Conceived and designed the experiments: DC, JP, LM, ER, and AS. Performed the experiments: AP, AR, LO, GM, and LV. Analyzed the data: AP and AS. Contributed reagents/materials/analysis tools: DC, JP, LM, ER, and AS. Wrote the paper: AP and AS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Two thousand fifteen has been a winning year for Drug Eluting Stents (DES). Increase in the number of patients with cardiovascular diseases treated by Percutaneous Coronary Intervention (PCI) has resulted to a high demand for second generation DES. This current analysis aimed to compare the different types of Stent Thrombosis (ST) associated with Zotarolimus Eluting Stents (ZES) versus Everolimus Eluting Stents (EES) at 1 year follow up.Electronic databases were searched for studies comparing ZES with EES. Different types of ST reported at 1 year follow up were considered as the primary endpoints in this analysis. Odds Ratios (OR) with 95% Confidence Intervals (CIs) were used as the statistical parameters and the pooled analyses were carried out by the RevMan 5 · 3 software.P = 0.09, OR: 3.44, 95% CI: 0.82 – 14.43; P = 0.09, OR: 1.13, 95% CI: 0.43 – 2.95; P = 0.80 and OR: 2.39, 95% CI: 0.83 – 6.85; P = 0.11 respectively. Moreover, any definite or probable ST and definite/probable/possible ST were also not significantly different with OR: 1.39, 95% CI: 0.89 – 2.17; P = 0.15 and OR: 1.19, 95% CI: 0.84 – 1.70; P = 0.33 respectively. In addition, any probable ST, acute probable ST, late probable ST and possible ST were also not significantly different at 1 year follow up with OR: 1.11, 95% CI: 0.60 – 2.05; P = 0.75, OR: 0.53, 95% CI: 0.12 – 2.40; P = 0.41, OR: 1.67, 95% CI: 0.35 – 7.86; P = 0.52 and OR: 1.08, 95% CI: 0.64 – 1.82; P = 0.78 respectively.A total number of 10,512 patients were included in this analysis. No significant difference in any definite ST, acute definite ST, subacute definite ST, and late definite ST were observed between ZES and EES, at 1 year follow up with OR: 1.70, 95% CI: 0.92 – 3.16; At 1 year follow up, ZES were not associated with significantly lower or higher definite and probable ST compared to EES. In addition, no significant difference was observed in acute, subacute and late definite or probable ST. However, further trials are recommended to assess the effects of these second-generation DES during the long-term. Two thousand fifteen has been a winning year for Drug Eluting Stents (DES). Increase in the number of patients with cardiovascular diseases treated by Percutaneous Coronary Intervention (PCI) has resulted to a higher demand for second generation DES. Even if DES won the battle in terms of repeated revascularization when compared to Bare Metal Stents (BMS) , they alThe Cochrane Library, MEDLINE or PubMed database of medical research articles, and EMBASE were searched by two authors (PKB and CMY), for English publications comparing ZES with EES using the words or phrase ‘zotarolimus eluting stents and everolimus eluting stents’. To widen this search strategy, the word ‘percutaneous coronary intervention’ and the abbreviations ‘ZES, EES and PCI’ were also used. Reference lists of suitable articles were also searched for relevant trials.They were Randomized Controlled Trials (RCTs) which compared ZES with EES in patients who underwent PCI.They reported ST and other adverse outcomes as their clinical endpoints.They had a follow up period of 1 year.Studies were included if:They were non-RCTs .They did not compare ZES with EES.They did not report ST or other adverse outcomes as their clinical endpoints.They were associated with the same trial.They were repeated trials or duplicates.Studies were excluded if:Any definite ST;Acute definite ST;Subacute definite ST;Late definite ST;Any probable ST;Acute probable ST;Subacute probable ST;Late probable ST;Any definite or probable ST;Possible ST;Definite/probable or possible ST.The primary outcomes analyzed included ST defined by the Academic Research Consortium (ARC) :Any defiAll-cause death;Cardiac death;Major Adverse Cardiac Events (MACEs) which were defined as a composite of all cause death, Myocardial Infarction (MI), emergent coronary artery bypass surgery (CABG) and clinically-indicated target lesion revascularization;Stroke;Patient-oriented composite endpoint consisting of all-cause mortality, MI and any coronary revascularization;MI;Target Vessel Revascularization (TVR);Target Lesion Revascularization (TLR);Target Vessel Failure (TVF);Target Lesion Failure (TLF).The secondary outcomes analyzed included:These outcomes were followed for 1 year after PCI. Table The same two authors (PKB and CMY) who were involved in the search process, carefully reviewed the trials and assessed their methodological quality. The bias risk was assessed with reference to the Cochrane Collaboration . The sixMoreover, information and data concerning the types of study reported, the patients’ enrollment period, the total number of patients treated by ZES and EES respectively, the reported primary and secondary outcomes, the follow up periods, the number of events that occurred in the study and the control groups, and information regarding the baseline features of the patients involved in this analysis were systematically extracted. Any disagreement or confusion concerning the eligibility of trials, or concerning the inclusion of certain data were discussed between these two authors, however, if a consensus could not be reached, disagreement was finally resolved by the third author (WQH).2 statistic test. A P value of ≤ 0.05 was considered statistically significant. Moreover, an I2 value of 0% indicated no or very low heterogeneity, and an increasing percentage of I2 implied an increasing heterogeneity. In addition, a fixed effects model (I2 < 50%) and a random effects model (I2 > 50%) depending on the value of I2 obtained. Publication bias was assessed by visually observing funnel plots. Odds Ratios (OR) with 95% Confidence Intervals (CIs) were calculated and the subgroup analyses were carried out by the RevMan 5·3 software. All authors had full access to the trials and their data. Ethical or board review approval was not required for this type of research article.The Preferred Reporting Items for Systematic Reviews and Meta-Analyses  statemenFour hundred and twenty-eight (428) studies were identified from the electronic databases. Three hundred and forty-five studies were eliminated through abstracts and titles since they did not address any issue related to the idea of this research. A further 32 articles were eliminated since they replicated themselves. Fifty-one 51) full-text articles were assessed for eligibility. Seventeen (17) more articles were eliminated since they were meta-analyses (2), letters to editors (3) or they were associated with the same trial (12). Thirty-four (34) studies met most of the inclusion and exclusion criteria of this meta-analysis. However, since it involved only randomized trials, a further 28 studies were excluded because they were observational studies. Finally, 6 trials were included in this analysis  were included in this analysis. Patients were enrolled from the year 2007 to the year 2013.The baseline features of the participants have been summarized in Table According to Table Results of this analysis has been summarized in Table P = 0.09, OR: 3.44, 95% CI: 0.82 – 14.43; P = 0.09, OR: 1.13, 95% CI: 0.43 – 2.95; P = 0.80 and OR: 2.39, 95% CI: 0.83 – 6.85; P = 0.11 respectively when a fixed effects model was used. Another analysis was carried out using a random effects model to analyze the subgroup ‘any definite ST’. However, similarly, no significant difference was observed with OR: 1.53, 95% CI: 0.56 – 4.17; P = 0.41. Moreover, any definite or probable ST and definite/probable/possible ST were also not significant between these two types of second-generation DES with OR: 1.39, 95% CI: 0.89 – 2.17; P = 0.15 and OR: 1.19, 95% CI: 0.84 – 1.70; P = 0.33 respectively. Results showing definite ST and its subtypes have been illustrated in Fig. No significant difference was observed between ZES and EES when analyzing any definite ST, acute definite ST, subacute definite ST, and late definite ST observed at 1 year follow up with OR: 1.70, 95% CI: 0.92 – 3.16; P = 0.75, OR: 0.53, 95% CI: 0.12 – 2.40; P = 0.41, OR: 1.67, 95% CI: 0.35 – 7.86; P = 0.52 and OR: 1.08, 95% CI: 0.64 – 1.82; P = 0.78 respectively at 1 year following PCI  whereby comparable clinical outcomes were observed between ZES and EES . A totalMoreover, results provided by the THCRIC registry also showed no significant difference in clinical outcomes between ZES and EES during a 1 year follow up after PCI . HoweverWhen randomized trials were compared, both ZES and EES were associated with similar rates of ST and other adverse clinical outcomes as reported in the DUTCH PEER trial, and both types of stents could be recommended in the general population with coronary artery diseases. The RESOLUTE and TWENTE trials also reported comparable ST between ZES and EES further supporting the results of this current analysis. At last, even data obtained from a German DES registry, showed first and second generation DES to be clinically equivalent at least at 1 year follow-up .A recently published meta-analysis showed Dual Anti-Platelet Therapy (DAPT) use for less or equal to six months following PCI with EES or ZES not to cause any increase or decrease in ST . Even thThis study is new in the way that it involved a large number of randomized patients compared to previously published studies. By excluding patients obtained from observational cohorts, this analysis involved only good data which resulted in a low level of heterogeneity during the subgroup analysis. Moreover, previously published meta-analyses did not specifically focus on ST. This current study analyzed all possible subtypes of ST including any definite or probable ST, acute definite or probable ST, subacute definite or probable ST, late definite or probable ST, possible ST and definite/probable/possible ST. Furthermore, other adverse clinical outcomes were also analyzed in details. In addition, bias risk assessment was carried out, which was not the case in other previously published meta-analyses.Similar to other studies, this systematic review and meta-analysis also had limitations. First of all, due to the limited number of patients analyzed, the results might be affected. In addition, one trial reported ST during a follow up of only one month. However, because the other outcomes reported had a follow up of 1 year, ST reported in that particular trial were assessed along with the other trials having a follow up period of 12 months. However, the results were not affected. Another trial had a follow up period of 15 months. It was included in this analysis and was expected to partly compensate for the trial which had a follow up period of one month for ST. Furthermore, a high level of heterogeneity was observed in the subgroup analyzing subacute probable ST. Even if this was negligible since this high level of heterogeneity was present in only one subgroup, this could also contribute to the limitations observed in this study. The fact that different types of patients were included, for example a few studies involved only patients with diabetes mellitus, other studies involved patients with left main coronary artery disease and so on, this might have had an influence on the results obtained. In addition, it might be possible that the subgroup assessing ‘any definite ST’ was influenced by trials DUTCH PEER and HOST ASSURE. However, because ST with several different definitions and types were included in that particular subgroup, which might have been the cause for this difference, this particular result might be ignored. Another limitation could be the fact that this current analysis included patients who were implanted with non-resorbable polymer EES, which are older compared to recent EES with resorbable polymer which are nowadays being used.At 1 year follow up, ZES were not associated with significantly lower or higher definite and probable ST compared to EES. In addition, no significant difference was observed in acute, subacute and late definite or probable ST. However, further trials are recommended to assess the effects of these second-generation DES during the long-term.


M = 12.03, SD = 1.59; 45.4% female) completed assessments at all three points. The cross-lagged model revealed that (a) parental monitoring at T1 predicted lower Internet gaming disorder at T2, and greater Internet gaming disorder at T2 predicted lower parental monitoring at T3; (b) father–child relationship had a reciprocal, indirect effect on the relationship between parental monitoring and Internet gaming disorder, while mother–child relationship did not. These findings suggest that the parental effects  might play a vital role in preventing Internet gaming disorder in adolescents.Although empirical studies have indicated that parents have an important role in preventing Internet gaming disorder in adolescents, longitudinal research on the parental predictors of Internet gaming disorder is lacking. We used a three-wave cross-lagged panel model to explore the reciprocal association between parental monitoring and Internet gaming disorder, and examined the different impacts of mother– and father–child relationships on this association. A sample of 1490 adolescents aged 10–15 years ( Internet gaming disorder in adolescents is becoming a widespread problem, particularly in China. According to the Fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), Internet gaming disorder can be defined as “Persistent and recurrent use of the Internet to engage in games, often with other players, leading to clinically significant impairment or distress” , 2013. OParental monitoring is defined as parents’ practices and knowledge concerning their children’s activities, whereabouts, and peers . In keepSome empirical studies have suggested that parental monitoring can reduce the risk of problematic behaviors such as problematic Internet use, substance use, risky sexual behavior, and delinquency . In receMoreover, parents might practice less monitoring when they feel that their adolescents can control their online activities , which iThe quality of the parent–child relationship might underlie the bidirectional association between parental monitoring and Internet gaming disorder. In other words, parental monitoring could exert an effect on Internet gaming disorder via the parent–child relationship. According to the theoretical framework of self-determination theory, autonomy, competence, and relatedness are the three basic psychological needs that underlie intrinsic motivation in both children and adolescents . AdolescIn addition, The present study examined the longitudinal reciprocal relationships among parental monitoring, parent–child relationship, and Internet gaming disorder in a Chinese sample using an autoregressive cross-lagged model. Although parental monitoring and parent–child relationship have been consistently found as correlates of Internet gaming disorder, we lack prospective evidence and an understanding of the mediating role of parent–child relationship. In this study, we specifically examine: (1) parental monitoring negatively predicts Internet gaming disorder; we also tested whether increased parental monitoring is a reaction to adolescents’ Internet gaming disorder. (2) Higher parental monitoring predicts significantly better father–child and mother–child relationships, which in turn predicts significantly lower Internet gaming disorder. (3) We tested the reverse direction of this previous set of relationships: that is, higher Internet gaming disorder predicts worse father–child and mother–child relationships, which in turn predicts lower parental monitoring.Participants were recruited from three primary schools and two junior middle schools in China and were collected semiannually at three different points. At the baseline assessment , a total of 1830 adolescents were recruited. A total of 1680 of the adolescents recruited at T1  participated in the second assessment  and 1490  went on to complete the third assessment . Attrition was mainly due to the following reasons: (a) students were absent from school on the day of the assessment; (b) students had transferred to another school; (c) some students failed to answer most of the items; and (d) students refused to continue participating in the study. There were no significant differences in any of the studies variables between participants who completed all three assessments and participants with missing data.SD = 1.59, range: 9–15 years) at T1; 692 were from primary schools (5th grade) and 798 were from junior middle schools . About 41.3% were an only child, 92.5% came from intact families, and 7.5% came from single-parent families. Moreover, 22.3% of participants came from rural areas, 27.4% came from county seats, 41.9% came from small cities, and 8.3% came from large cities. Furthermore, 73.4% of participants’ mothers’ and 70.4% of their fathers’ education level was high school or less, and 52.2% of families had a per capita average monthly income of more than ¥ 3000, which is higher than the average in China in 2015  > 0.95, root mean square error of approximation (RMSEA) ≤ 0.06  ≤ 0.06 . Based o) ≤ 0.06 .Table 1 reports the means, standard deviations, and correlations for the major study variables at each of the three waves of data. The results indicated that the bivariate correlations of the major variables in our hypothesized cross-lagged models were significant. Both parental monitoring and parent–child relationship at T1–T3 were negatively correlated with Internet gaming disorder at T1–T3. Parental monitoring at T1 was also positively correlated with father–child and mother–child relationship at T1–T3.2(6) = 2.872; CFI = 0.996, and RMSEA = 0.035. Then, Model 2 added the lagged paths from parental monitoring at T1 to Internet gaming disorder at T2 and parental monitoring at T2 to Internet gaming disorder at T3, as well as the path from Internet gaming disorder at T1 to parental monitoring at T2 and Internet gaming disorder at T2 to parental monitoring at T3. Model 2 proved a better fit than the baseline model (Model 1), χ2(2) = 0.255; CFI = 0.997, and RMSEA = 0.000, Δχ2(4) = 2.617, p > 0.05. Then, the path from parental monitoring at T1 to Internet gaming disorder at T3 and the path from Internet gaming disorder at T1 to parental monitoring at T3 were added, producing Model 3; however, Model 3 was a saturated model and the paths added to it were not significant. Thus, Model 2 was identified as the final model .To examine the reciprocal effects between parental monitoring and Internet gaming disorder, we conducted and compared three parallel autoregressive models. Model 1 included the auto-regressive paths and concurrent correlations, and indicated a good fit to the data, χB = -0.043, β = -0.069, p < 0.01, 95%CI . Furthermore, Internet gaming disorder at T2 negatively predicted parental monitoring at T3, B = -0.073, β = -0.048, p < 0.01, 95%CI . However, the path from parental monitoring at T2 to Internet gaming disorder at T3, as well as the path from Internet gaming disorder at T1 to parental monitoring at T2, were not significant.The analyses of Model 2 indicated a reciprocal relationship between parental monitoring and Internet gaming disorder, which showed that greater parental monitoring at T1 significantly predicted less Internet gaming disorder at T2, 2(18) = 4.994, CFI = 0.981, RMSEA = 0.051. At step 2, lagged paths between parental monitoring and Internet gaming disorder over time were added in Model 5-1, which showed a good fit to the data, χ2(14) = 5.135, CFI = 0.984, RMSEA = 0.053, Δχ2(4) = 0.141, p > 0.05. At step 3, the lagged paths between parental monitoring and father–child relationship, as well as the lagged paths between father–child relationship and Internet gaming disorder, were added in Model 6-1, which showed a good fit to the data, χ2(6) = 0.944, CFI = 1.000, RMSEA = 0.000, Δχ2(8) = 4.194, p > 0.05. At step 4, the path from parental monitoring at T1 to Internet gaming disorder at T3 and the path from Internet gaming disorder at T1 to parental monitoring at T3 were added in Model 7-1. Model 7-1, however, was a saturated model and the paths added in it were not significant. Thus, Model 6-1 was identified as the final model . We followed similar steps to examine the mediating role of mother–child relationship; the final model  provided a good fit to the data, χ2(6) = 1.198, CFI = 1.000, RMSEA = 0.012, Δχ2(8) = 7.922, p > 0.05.Based on the suggestion that a significant relationship between a predictor and a dependent variable is not a necessary condition to examine a mediating effect , we testFigure 2, the path from parental monitoring at T1 to father–child relationship at T2 was significant, B = -0.031, β = 0.211, p < 0.01, 95% CI . In turn, father–child relationship at T2 negatively predicted Internet gaming disorder at T3, B = -0.008, β = -0.119, p < 0.01, 95%CI  . Bootstrapping analyses indicated that the indirect effect of parental monitoring on Internet gaming disorder via father–child relationship was significant and positive, B = -0.002, SE = 0.002, β = -0.003, p < 0.05, 95% CI . Meanwhile, Internet gaming disorder at T1 significantly predicted poorer father–child relationship at T2, B = -0.101, β = -0.102, p < 0.01, 95% CI . In turn, poorer father–child relationship at T2 might decrease parental monitoring at T3, B = -0.067, β = -0.057, p < 0.05, 95% CI  . Bootstrapping analyses indicated that the indirect effect of Internet gaming disorder on parental monitoring via father–child relationship was significant and positive, B = -0.008, SE = 0.003, β = -0.005, p < 0.05 and 95% CI .As shown in Figure 3, parental monitoring at T1 predicted lower mother–child relationship at T2, B = 0.082, β = 0.140, p < 0.01, 95% CI . However, mother–child relationship at T2 did not predict Internet gaming disorder at T3, B = 0.058, β = 0.036, p > 0.05, 95% CI . Meanwhile, adolescents’ Internet gaming disorder at T1 significantly predicted poorer mother–child relationship at T2, B = -0.097, β = -0.096, p < 0.01, 95% CI , but mother–child relationship at T2 did not predict parental monitoring at T3, B = -0.042, β = -0.037, p < 0.05, 95% CI  . A significant indirect effect of parental monitoring on Internet gaming disorder via mother–child relationship was not found.As showed in Several important findings emerged from this longitudinal study that may enhance our understanding of the associations between parental monitoring, Internet gaming disorder, and parent–child relationship. First, we found a reciprocal relationship between parental monitoring and Internet gaming disorder. Second, father–child relationship has a mediating effect on both directions of the path between parental monitoring and Internet gaming disorder; however, significant indirect effects of parental monitoring on Internet gaming disorder (and vice versa) via mother–child relationship were not found, indicating that there were different impacts of father–child and mother–child relationship on the reciprocal relationship between parental monitoring and Internet gaming disorder. Altogether, the findings suggest that parental factors  likely contribute to decreased risk of Internet gaming disorder in early adolescence.The results indicate that parental monitoring negatively predicts adolescents’ Internet gaming disorder. However, the relationship between parental monitoring and Internet gaming disorder in this study was not stable over time. In fact, parental monitoring at T1 predicted lower levels of Internet gaming disorder at T2, but parental monitoring at T2 did not significantly predict Internet gaming disorder at T3. This finding suggests an inconsistency in the effects of parental monitoring on Internet gaming disorder. Consistent with our findings, some earlier studies have shown that a higher level of parental monitoring was effective for reducing problematic Internet use and other behavioral problem e.g., . HoweverThe results of the path from Internet gaming disorder to parental monitoring indicated that Internet gaming disorder at T2 was significantly associated with parental monitoring at T3. The present study indicate that when parents are aware that their adolescents are at risk of Internet gaming disorder, they appear to engage in less monitoring of their children’s behaviors after half a year. Contrary to this finding, previous studies found that parental monitoring was associated with recognition of adolescents’ self-regulation ability: that is, when parents perceive that adolescents cannot control their online activities, they tend to practice more monitoring . One posWe believe that the most important finding in this study is that the father–child and mother–child relationships have different influences on the reciprocal relationship between parental monitoring and adolescent Internet gaming disorder. Specifically, only father–child relationship had a reciprocal indirect effect on the relationship between parental monitoring and Internet gaming disorder; no associations were found for mother–child relationship. Although many studies have been reported the importance of mother–child relationship, the present study revealed that fathers might be more important in preventing adolescents’ Internet gaming disorder. These findings accord with those of another longitudinal study showing that parental monitoring and father–child connectedness are associated with reductions in problem behavior in a sample of 6th to 8th grade students over time . FurtherAnalyses of the reverse direction in our cross-lagged model also suggests that high levels of Internet gaming disorder might initially lead to poorer father– and mother–child relationships, but then only poorer father–child relationship led to higher parental monitoring. Adolescents who are addicted to video games might cause parents anxiety in relation to the consequences of Internet gaming disorder, such as academic and behavioral problems . ConsequAlthough the role of parent–child relationship was the primary focus of the present study, the findings revealed the further possibility that Internet gaming disorder can act as a mediator of the relationships between parental monitoring and parent–child relationship. Specifically, the cross-lagged model indicated that high parental monitoring of Internet use might lead to a decrease in Internet gaming disorder, which might then lead to higher quality parent–child relationships.To our knowledge, the present study is the first to test the reciprocal relationships between parental monitoring, parent–child relationship, and Internet gaming disorder. The findings of this study might have implications for targeted interventions. Particularly, parental monitoring was found to be a direct predictor of Internet gaming disorder in this study. Therefore, parents’ behaviors in managing adolescents’ Internet gaming disorder might be important. This study provides evidence to support the suggestion that general parental monitoring, including obtaining more knowledge about daily adolescent activities and setting rules about Internet use time, place, and content, might decrease adolescents’ Internet gaming disorder. For effective parental monitoring of adolescent Internet use, the best strategy might be to pursue open communication with adolescents, as well as build up connections between parents and their adolescents while considering their Internet use . Second,There are several limitations in the current study. First, because all measures in this study were collected from adolescents’ self-report, not from their parents, the findings might be somewhat limited because of common method variance and social desirability bias. Studies have indicated there might be considerable differences between adolescents’ self-report and parental reports about adolescents’ Internet use and parental monitoring: namely, 62% of parents reported that they have supervised their adolescents’ online activities, but only 33% of adolescents reported believing that parents have correct knowledge of their Internet use . Second,Our findings expand on previous studies suggesting that parents play a vital role in preventing Internet gaming disorder among adolescents. A reciprocal direct relationship between parental monitoring and Internet gaming disorder was found—that is, parental monitoring at T1 predicted lower Internet gaming disorder at T2 and a high level of Internet gaming disorder at T2 predicted less parental monitoring at T3. A reciprocal, indirect effect of father–child relationship on the link between parental monitoring and Internet gaming disorder was also found, but no such indirect effect was found for mother–child relationship. Our findings call for further longitudinal studies using more consistent methods of measuring Internet gaming disorder and parental monitoring, as well as alternative methods of data collection . This would aid in our understanding of how parental factors influence Internet gaming disorder in different cultural and social environments.Conceived and designed the research: BS and WZ. Performed the research: BS, CY, and JZ. Analyzed the data: BS, QS, CY, and YJ. Contributed to the writing of the manuscript: BS, CY, WZ, QS, JZ, and YJ.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.