Objective:To investigate the risk factors for mortality in neonates with necrotizing enterocolitis (NEC).Methods:This retrospective cohort study included patients diagnosed with NEC at stage Ⅱ-Ⅲ (Bell's criteria) and admitted to the Neonatology Department of Guangzhou Women and Children's Medical Center Affiliated to Guangzhou Medical University from January 2017 to December 2022. According to the outcomes, these patients were divided into the mortality and survival groups. Perinatal conditions, clinical manifestations, disease status during hospitalization, and blood routine parameters at different time points after birth were compared between the two groups to analyze the risk factors for mortality of NEC. Statistical analysis was performed using independent-sample t-test, Mann-Whitney U test, Chi-square test (or Fisher's exact test), and multivariate logistic regression analysis. Results:(1) A total of 118 NEC cases were included, with 100 in the survival group and 18 in the mortality group. (2) The gestational age and birth weight were significantly lower in the mortality group than in the survival group [(28.9±4.2) weeks vs. (33.7±3.9) weeks, t=4.78; 940 g (685-1 275 g) vs. 1 830 g (1 352-2 368 g), Z=4.18; both P<0.05]. The incidence of neonatal asphyxia was higher in the mortality group [9/18 vs. 18% (18/100), χ2=7.13, P<0.05]. (3) Compared with the survival group, the mortality group had higher proportions of patients who were at NEC stage Ⅲ, accepted surgery, were treated with vasoactive drugs, had undergone invasive mechanical ventilation, or had metabolic acidosis, hyperlactatemia, shock, or hematochezia [17/18 vs. 24% (24/100), χ2=33.39; 17/18 vs. 31% (31/100), χ2=22.88; 16/18 vs. 22% (22/100), χ2=31.26; 16/18 vs. 39% (39/100), χ2=15.26; 18/18 vs. 28% (28/100), χ2=30.29; 16/18 vs. 20% (20/100), χ2=34.15; 17/18 vs. 21% (21/100), χ2=37.69; 9/18 vs. 82% (82/100), χ2=7.13; all P<0.05]. (4) The proportions of patients who developed late-onset sepsis, hemodynamically- significant patent ductus arteriosus, respiratory distress syndrome, or pulmonary hemorrhage were significantly higher in the mortality group than in the survival group [15/18 vs. 33% (33/100), χ2=16.01; 9/18 vs. 21% (21/100), χ2=5.32; 15/18 vs. 39% (39/100), χ2=12.08; 7/18 vs. 7% (7/100), χ2=11.94; all P<0.05]. (5) Within 24 h of birth, the levels of red blood cells and platelets were lower in the mortality group than in the survival group [4.1×1012/L (3.8×10 12/L-4.6×10 12/L) vs. 4.6×10 12/L (4.0×10 12/L-4.9×10 12/L), Z=2.04; (199.9±68.6)×10 9/L vs. (239.8±72.6)×10 9/L, t=2.16; both P<0.05]; at 6-8 d after birth, the levels of red blood cells, hemoglobin (Hb) and hematocrit (HCT) were lower in the mortality group than in the survival group [(3.2±0.5)×10 12/L vs. (3.9±0.8)×10 12/L, t=3.30; (111.2±19.2) vs. (138.1±28.3) g/L, t=3.51; (33.0±5.4)% vs. (40.9±8.1)%, t=3.61; all P<0.05]; at the diagnosis of NEC, red blood cell count, Hb level, HCT, and platelet count were lower in the mortality group than in the survival group [(3.3±1.0)×10 12/L vs. (3.8±0.8)×10 12/L, t=2.47; (102.8±28.8) vs. (124.4±26.3) g/L, t=3.59; 31.0% (25.9%-38.4%) vs. 37.2% (31.5%-43.7%), Z=2.62; 87.0×10 9/L (50.2×10 9/L-157.0×10 9/L) vs. 228.0×10 9/L (130.0×10 9/L-414.7×10 9/L), Z=3.78; all P<0.05], while mean platelet volume (MPV), platelet distribution width, and the differences in hemoglobin (ΔHb) and hematocrit (ΔHCT) between the first 24 h after birth and 6-8 d after birth were significantly higher in the mortality group than in the survival group [13.1 fl (11.4-13.6 fl) vs. 11.6 fl (10.7-12.4 fl), Z=3.26; 19.6% (13.9%-25.2%) vs. 14.8% (12.0%-18.6%), Z=2.76; 35.5 g/L (28.3-57.3 g/L) vs. 27.0 g/L (8.0-42.5 g/L), Z=2.20;11.5% (9.4%-16.3%) vs. 6.3% (2.2%-11.2%), Z=2.85; all P<0.05]. (6) Late-onset sepsis ( OR=5.568, 95% CI: 1.201-25.816), hyperlactatemia ( OR=6.702, 95% CI: 1.193-37.651), shock ( OR=10.616, 95% CI: 1.157-97.406) and MPV elevation at the diagnosis of NEC ( OR=2.769, 95% CI: 1.468-5.223) were independent risk factors, while gestational age ( OR=0.836, 95% CI: 0.708-0.986), and HCT at 6-8 d after birth ( OR=0.848, 95% CI: 0.759-0.947) were protecting fctors for death in NEC. Conclusions:Preterm infants with smaller gestational age are more prone to mortality of NEC. Early identification and management of late-onset sepsis, shock, and hyperlactatemia may reduce the risk of mortality in NEC.

Objective:To investigate the risk factors for mortality in neonates with necrotizing enterocolitis (NEC).Methods:This retrospective cohort study included patients diagnosed with NEC at stage Ⅱ-Ⅲ (Bell's criteria) and admitted to the Neonatology Department of Guangzhou Women and Children's Medical Center Affiliated to Guangzhou Medical University from January 2017 to December 2022. According to the outcomes, these patients were divided into the mortality and survival groups. Perinatal conditions, clinical manifestations, disease status during hospitalization, and blood routine parameters at different time points after birth were compared between the two groups to analyze the risk factors for mortality of NEC. Statistical analysis was performed using independent-sample t-test, Mann-Whitney U test, Chi-square test (or Fisher's exact test), and multivariate logistic regression analysis. Results:(1) A total of 118 NEC cases were included, with 100 in the survival group and 18 in the mortality group. (2) The gestational age and birth weight were significantly lower in the mortality group than in the survival group [(28.9±4.2) weeks vs. (33.7±3.9) weeks, t=4.78; 940 g (685-1 275 g) vs. 1 830 g (1 352-2 368 g), Z=4.18; both P<0.05]. The incidence of neonatal asphyxia was higher in the mortality group [9/18 vs. 18% (18/100), χ2=7.13, P<0.05]. (3) Compared with the survival group, the mortality group had higher proportions of patients who were at NEC stage Ⅲ, accepted surgery, were treated with vasoactive drugs, had undergone invasive mechanical ventilation, or had metabolic acidosis, hyperlactatemia, shock, or hematochezia [17/18 vs. 24% (24/100), χ2=33.39; 17/18 vs. 31% (31/100), χ2=22.88; 16/18 vs. 22% (22/100), χ2=31.26; 16/18 vs. 39% (39/100), χ2=15.26; 18/18 vs. 28% (28/100), χ2=30.29; 16/18 vs. 20% (20/100), χ2=34.15; 17/18 vs. 21% (21/100), χ2=37.69; 9/18 vs. 82% (82/100), χ2=7.13; all P<0.05]. (4) The proportions of patients who developed late-onset sepsis, hemodynamically- significant patent ductus arteriosus, respiratory distress syndrome, or pulmonary hemorrhage were significantly higher in the mortality group than in the survival group [15/18 vs. 33% (33/100), χ2=16.01; 9/18 vs. 21% (21/100), χ2=5.32; 15/18 vs. 39% (39/100), χ2=12.08; 7/18 vs. 7% (7/100), χ2=11.94; all P<0.05]. (5) Within 24 h of birth, the levels of red blood cells and platelets were lower in the mortality group than in the survival group [4.1×1012/L (3.8×10 12/L-4.6×10 12/L) vs. 4.6×10 12/L (4.0×10 12/L-4.9×10 12/L), Z=2.04; (199.9±68.6)×10 9/L vs. (239.8±72.6)×10 9/L, t=2.16; both P<0.05]; at 6-8 d after birth, the levels of red blood cells, hemoglobin (Hb) and hematocrit (HCT) were lower in the mortality group than in the survival group [(3.2±0.5)×10 12/L vs. (3.9±0.8)×10 12/L, t=3.30; (111.2±19.2) vs. (138.1±28.3) g/L, t=3.51; (33.0±5.4)% vs. (40.9±8.1)%, t=3.61; all P<0.05]; at the diagnosis of NEC, red blood cell count, Hb level, HCT, and platelet count were lower in the mortality group than in the survival group [(3.3±1.0)×10 12/L vs. (3.8±0.8)×10 12/L, t=2.47; (102.8±28.8) vs. (124.4±26.3) g/L, t=3.59; 31.0% (25.9%-38.4%) vs. 37.2% (31.5%-43.7%), Z=2.62; 87.0×10 9/L (50.2×10 9/L-157.0×10 9/L) vs. 228.0×10 9/L (130.0×10 9/L-414.7×10 9/L), Z=3.78; all P<0.05], while mean platelet volume (MPV), platelet distribution width, and the differences in hemoglobin (ΔHb) and hematocrit (ΔHCT) between the first 24 h after birth and 6-8 d after birth were significantly higher in the mortality group than in the survival group [13.1 fl (11.4-13.6 fl) vs. 11.6 fl (10.7-12.4 fl), Z=3.26; 19.6% (13.9%-25.2%) vs. 14.8% (12.0%-18.6%), Z=2.76; 35.5 g/L (28.3-57.3 g/L) vs. 27.0 g/L (8.0-42.5 g/L), Z=2.20;11.5% (9.4%-16.3%) vs. 6.3% (2.2%-11.2%), Z=2.85; all P<0.05]. (6) Late-onset sepsis ( OR=5.568, 95% CI: 1.201-25.816), hyperlactatemia ( OR=6.702, 95% CI: 1.193-37.651), shock ( OR=10.616, 95% CI: 1.157-97.406) and MPV elevation at the diagnosis of NEC ( OR=2.769, 95% CI: 1.468-5.223) were independent risk factors, while gestational age ( OR=0.836, 95% CI: 0.708-0.986), and HCT at 6-8 d after birth ( OR=0.848, 95% CI: 0.759-0.947) were protecting fctors for death in NEC. Conclusions:Preterm infants with smaller gestational age are more prone to mortality of NEC. Early identification and management of late-onset sepsis, shock, and hyperlactatemia may reduce the risk of mortality in NEC.

AIM:To investigate the mechanism of Piezo1 channel activation promoting the increase in intra-cellular Ca2+ concentration([Ca2+]i)of rat coronary artery smooth muscle cells(CASMCs).METHODS:The primary CASMCs of SD rats were cultured,and the expression and subcellular localization of Piezo1 in the cells were observed by immunofluorescence staining.The Piezo1 and stromal interaction molecule 1(STIM1)in CASMCs were knocked down by siRNA transfection,and the expression levels of the proteins were detected by Western blot.Utilizing laser confocal mi-croscopy,the change of[Ca2+]i in CASMCs was detected by Fluo-4 AM fluorescent probes.RESULTS:It was confirmed by immunofluorescence staining that the expression of Piezo1 existed in primary rat CASMCs.Immunofluorescence staining also showed that Piezo1 was co-located with sarco-/endoplasmic reticulum Ca2+-ATPase 2(SERCA2),mitochondrial outer membrane protein TOM20 and nuclear membrane protein lamin B1.Western blot results showed that the protein expres-sion levels of STIM1 and Piezo1 were significantly down-regulated by siRNA transfection(P<0.05).Compared with con-trol group,Yoda1,the agonist of Piezo1,could increase the extracellular Ca2+ influx of CASMCs(P<0.01).However,the Ca2+ influx mediated by Yoda1 was not affected by the inhibition of L-type calcium channels.Treatment with Yoda1 in-creased the intracellular Ca2+ release of CASMCs(P<0.01).However,inhibition of calcium channels on endoplasmic re-ticulum,ryanodine receptor and inositol 1,4,5-triphosphate receptor,did not affect intracellular Ca2+ release mediated by Yoda1.After the Ca2+ in endoplasmic reticulum was emptied using thapsigargin(TG),Yoda1 also mediated the Ca2+ re-lease of other organelles in CASMCs(P<0.01).After inhibition of L-type calcium channels,treatment with store-operated calcium channel(SOCC)inhibitor BTP2 or knockdown of STIM1 led to the decrease in extracellular Ca2+ influx of CASMCs mediated by Yoda1(P<0.01).Treatment with TG increased the release of Ca2+ from the endoplasmic reticulum of CASMCs after knockdown of Piezo1(P<0.05),but the extracellular Ca2+ influx mediated by TG was not affected.After inhibition of L-type calcium channels and SOCC,knockdown of Piezo1 led to the decreases in intracellular Ca2+ release and extracellular Ca2+ influx induced by Yoda1(P<0.01).CONCLUSION:The Piezo1 agonist orchestrates the influx of extracellular Ca2+ by activating Piezo1 channels on the cell membrane and inducing the indirect activation of SOCC.More-over,it facilitates the release of Ca2+ from organelles.Consequently,these pathways synergistically elevate the[Ca2+]i of rat CASMCs.

AIM:To investigate the influence of fat mass and obesity-associated(Fto)gene on the aberrant contraction of aortic smooth muscle in diabetes mellitus(DM)mice,and to explore the mechanism of Fto gene underlying the calcium regulation.METHODS:Smooth muscle-specific Fto gene knockout(FtoSMKO)mice were generated using Cre-loxP technology.The experiment involved 3 groups of mice:wild-type(WT)group,DM model group and FtoSMKO-DM group,with 15 mice in each group.In DM group and FtoSMKO-DM group,type 1 DM was induced by intraperitoneal injec-tion of streptozotocin.The mice in WT group were injected with equal volume of citric acid-sodium citrate buffer solution.The influences of different drugs on the contraction responses of aortic smooth muscle in mice were analyzed using a multi-myograph system.The expression level of FTO protein in the aortic tissues was detected by Western blot.RESULTS:(1)Compared with WT mice,the expression levels of FTO protein in the aortic tissues of DM mice were significantly in-creased(P＜0.01).(2)The expression level of FTO protein in smooth muscle was significantly decreased after knockout of Fto gene(P＜0.01).Compared with WT group,the mice in DM group exhibited a significant decrease in body weight and a marked increase in fasting blood glucose level(P＜0.05).There were no noticeable differences in body weight or fasting blood glucose level between FtoSMKO-DM group and DM group(P＞0.05).(3)The contraction responses of aortic smooth muscle in DM group were substantially increased by phenylephrine compared with WT group.Specifically,vaso-constriction responses mediated by non-L-type calcium channels and store-operated calcium channels(SOCC)were signifi-cantly enhanced in DM group.In addition,the responses mediated by inositol 1,4,5-trisphosphate receptors(IP3R),which facilitate calcium release from the sarcoplasmic reticulum,were significantly enhanced.However,the responses mediated by caffeine-activated ryanodine receptors(RyR),which also facilitate calcium release from the sarcoplasmic re-ticulum,were significantly inhibited(P＜0.05).(4)Compared with DM group,the phenylephrine-induced contraction re-sponses of aortic smooth muscle in FtoSMKO-DM group were greatly weakened(P＜0.05).In particular,the vasoconstriction responses mediated by non-L-type calcium channels and SOCC in FtoSMKO-DM group were greatly suppressed(P＜0.05),while those mediated by caffeine-activated RyR were dramatically boosted(P＜0.05).However,IP3R-mediated responses were not affected(P＞0.05).CONCLUSION:Smooth muscle-specific Fto gene knockout suppresses contractile hyperre-sponsiveness in the aortic smooth muscle of DM mice,which may be attributed to involvement of FTO protein in calcium regulation in the vascular smooth muscle.

OBJECTIVE: To detect variants of IVD gene among 4 neonates with suspected isovalerate acidemia in order to provide a guidance for clinical treatment. METHODS: 111 986 newborns and 7461 hospitalized children with suspected metabolic disorders were screened for acyl carnitine by tandem mass spectrometry. Those showing a significant increase in serum isovaleryl carnitine (C5) were analyzed for urinary organic acid and variants of the IVD gene. RESULTS: Four cases of isovalerate acidemia were detected, which included 2 asymptomatic newborns (0.018‰, 2/111 986) and 2 children suspected for metabolic genetic diseases (0.268‰, 2/7461). The formers had no obvious clinical symptoms. Analysis of acyl carnitine has suggested a significant increase in C5, and urinary organic acid analysis has shown an increase in isovaleryl glycine and 3-hydroxyisovalerate. Laboratory tests of the two hospitalized children revealed high blood ammonia, hyperglycemia, decreased red blood cells, white blood cells, platelets and metabolic acidosis. The main clinical manifestations have included sweaty foot-like odor, feeding difficulty, confusion, drowsiness, and coma. Eight variants (5 types) were detected, which included c.158G>A (p.Arg53His), c.214G>A (p.Asp72Asn), c.548C>T (p.Ala183Val), c.757A>G (p.Thr253Ala) and 1208A>G (p.Tyr403Cys). Among these, c.548C>T and c.757A>G were unreported previously. None of the variants was detected by next generation sequencing of 2095 healthy newborns, and all variants were predicted to be likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics. CONCLUSION The incidence of isovalerate acidemia in Liuzhou area is quite high. Screening of metabolic genetic diseases is therefore recommended for newborns with abnormal metabolism. The discovery of novel variants has enriched the mutational spectrum of the IVD gene.
Infant, Newborn ; Child ; Humans ; Acidosis ; Carnitine ; Erythrocytes ; High-Throughput Nucleotide Sequencing

Objective To investigate the high sensitive C reactive protein(hs-CRP),homocysteine(Hcy), interleukin 2(IL-2)and lipoprotein associated phospholipase A 2(Lp-PLA2)analysis and correlation test for coronary heart disease.Methods 147 cases were selected from our hospital in December 2015 to December 2016 in patients with coronary heart disease(observation group).At the same time,we selected 51 healthy persons in our hospital during December 2015 to December 2016 as control group.The content of hs-CRP was determined by immune transmission turbidimetry.The content of Hcy was determined by cyclic enzymatic method.The content of IL-2 was determined by enzyme linked immunosorbent assay(ELISA).The content of Lp-PLA2 was determined by immune transmission turbidimetry.Results The observation group serum levels of hs-CRP,Hcy,IL-2 and Lp-PLA2 were significantly higher than those in the control group,and the differ-ence were statistically significant(P<0.05).The coronary heart disease each group serum levels of hs-CRP, Hcy,IL-2 and Lp-PLA2 were significantly higher than those in the control group,and the differences were sta-tistically significant(P<0.05).The serum levels of hs-CRP,Hcy,IL-2 and Lp-PLA2 of AMI group were sig-nificantly higher than those in UAP group and SAP group,and the differences were statistically significant (P<0.05).The UAP group serum levels of hs-CRP,Hcy,IL-2 and Lp-PLA2 were significantly higher than those in SAP group,and the difference was statistically significant(P<0.05).Conclusion Hs-CRP,Hcy,IL-2,Lp-PLA2 have significant correlation with coronary heart disease.

Objective To investigate the impact of an infant formula supplemented with 1.34 g/L oligosaccharides mixture (galactooligosaccharides and fructooligosaccharides,GOS+FOS) on growth,stool characteristics and fecal beneficial bacteria content in healthy infants.Methods 25 formula-fed (FF) infants and 29 breast-fed (BF) infants within 3 weeks after birth were included and followed up.FF infants were exclusively fed with GOS+FOS supplemented formula at the median age of 13d (1-27d).Body weight and head circumference at 6 and 12 weeks were measured.The frequency,consistency,and color of the infant's stool,and occurrences of spit-up or vomiting during the study period were recorded daily by the parents.Stool samples were collected at 6 and 12 weeks of age,and then subjected to quantitative real-time polymerase chain reaction assays for the enumeration of lactobacillus,bifidobacteria,Bifidobacterium longum,Bifidobacterium breve and Bifidobacterium adolescentis.Results No difference in daily weight gain was detected between the two groups (33.56 ± 7.03g vs.31.18 + 6.91g,P=0.22)during the observation phase from 6 to 12 weeks.FF infants had less frequent and firmer stools at 6 weeks than BF infants,and they both had similar stool frequency,color and consistency at the end of study.FF infants had less fecal Bifidobacterium,B.longum and B.breve at 6 weeks,but those differences became less significant at 12 weeks with more increase in fecal B.longum and B.breve levels in FF infants during the follow up.Conclusions The GOS+FOS supplemented formula provides adequate nutrition for growth,and promotes soft stools and intestinal bifidogenic effects in healthy infants.

Objective To investigate the association between the RoSCo scoring system and the postopera tive complications of laparoscopic partial nephrectomy.Methods The clinical data of 127 patients with laparoscopic partial nephrectomy in our hospital from 2010 to 2015 were retrospectively analyzed.By studying all patients' preoperative clinical data,we give all the patients the RoSCo score,then divided all the patients into the RoSCo low group (3-4),RoSCo moderate group (5-6),RoSCo high group (7-8).Respectively study the association between the RoSCo score and Clavein score,operative time,intraoperative blood loss,warm ischemia time and length of hospital stay after laparoscopic partial nephrectomy.Results The RoSCo score was associated with postoperative complications of laparoscopic partial nephrectomy (P ＜ 0.05) and was more accurate than the RENAL score alone.There were significant differences in bleeding,warm ischemia time,hospital stay and operation time between the low,middle and high groups of RoSCo (P ＜ 0.05).Conclusion The RoSCo scoring system can be used to assess the complications of laparoscopic partial nephrectomy.

Objective To study the optimum combination of orientation parameter of total hip prosthesis and acetabular safe zone on condition that the range of motion for activities of daily living (ADL) is fulfilled.Methods A three-dimensional generic parametric and visually kinematic simulation module of THA was developed.Range of motion ( ROM ) of hip flexion ≥ 110°,internal-rotation ≥30° at 90° flexion,extension ≥30° and external rotation ≥40° were defined as the normal criteria for ADL.ROM of hip flexion ≥ 120°,internal-rotation ≥45° at 90° flexion,extension ≥30° and external rotation ≥40° were as the severe criteria.The ranges of changes in general ratios (GRs) of head-neck,femoral neck antevemion ( FNA ),operative inclination (OI) and anteversion (OA) of acetabulur components were 2.0-2.92,0°-30°,10°-60°,and 0°-70° respectively.Within the limits of the upper two activity criteria,the synchronous OA of acetabulur components was calculated with every 5°change in OI of the cup,and the collodiaphyseal angle ( CDD ) was set as 135°.The safe-zone of combination of acetabulur operative anteversion (OA) and inclination (OI) was defined as the area that fulfilled the two mentioned criteria of ROM without cup-neck impingement.All parameters were analyzed by using SAS 6.12 software.Results The safe zone of acetabdar angle rose with the increase of GRs of head-neck and the safe zone of severe criteria was smaller than that of normal criteria.When the CDD angle was 135°,the sum of average aeetabular OA and acetahular OI plus 0.816 times of the FNA equaled to 84.76° innormal criteria; and the sum of average acetabular OA and acetabular OI plus 0.873 times of the FNA equaled to 92.04° in severe criteria.Conclusions A high GR of head-neck greatly increases the size of safe-zone of acetabular angle.The higher demand of ROM of hip joint requires the smaller safe zone of acetabular angle,as can be corrected by increasing the GR of head-neck.The optimum combination between the sum of average acetabular OA and acetabular OI ( Y) plus FA (X)in the normal criteria and severe criteria can be estimated by using the following formulae:Y1 =-0.816X1 + 84.76 (R2 =0.993 ),Y2 =-0.873X2 + 92.04( R2 =0.999) respectively.

Objective To discuss biomechanical mechanism of reconstructed joint capsule ligament stabilizing hip joint by means of three-dimensional finite element analysis.Methods A finite element model of total hip arthroplasty(THA)including ischiofemoral ligament reconstruction was construtted by using finite element analysis software Unigraphics NX 2.0 and SolidWorks 2006 to simulate seated leg crossing and obtain peak resisting moment and range of motion prior to impingement.Results The current form of finite element model was characterized by large deformation multi-body contact,large interfacial sliding and high elasticity and could clearly reflect real anatomy and biomechanical behavior of ischiofemoral ligaments.Compared with model with only metal,model of ischiofemoral ligament reconstruction could reduce the peak polyethylene stress at the impingement site and at the head egress site by typically 17%and 31%respectively,increase peak resisting moment by nearly 57%and provide 2.29-fold stability.Conclusions As a discrete structure within the posterior capsule of the hip joint,the ischiofemoral ligament may be the most important contributor to the mechanical integrity of the posterior stability structure.The joint capsule ligament must be reconstructed in hip arthroplasty.