There are many documented sex differences in the clinical course,symptom expression profile,and treatment response of Parkinson’s disease,creating additional challenges for patient management.Although subthalamic nuc...There are many documented sex differences in the clinical course,symptom expression profile,and treatment response of Parkinson’s disease,creating additional challenges for patient management.Although subthalamic nucleus deep brain stimulation is an established therapy for Parkinson’s disease,the effects of sex on treatment outcome are still unclear.The aim of this retrospective observational study,was to examine sex differences in motor symptoms,nonmotor symptoms,and quality of life after subthalamic nucleus deep brain stimulation.Outcome measures were evaluated at 1 and 12 months post-operation in 90 patients with Parkinson’s disease undergoing subthalamic nucleus deep brain stimulation aged 63.00±8.01 years(55 men and 35 women).Outcomes of clinical evaluations were compared between sexes via a Student’s t-test and within sex via a paired-sample t-test,and generalized linear models were established to identify factors associated with treatment efficacy and intensity for each sex.We found that subthalamic nucleus deep brain stimulation could improve motor symptoms in men but not women in the on-medication condition at 1 and 12 months post-operation.Restless legs syndrome was alleviated to a greater extent in men than in women.Women demonstrated poorer quality of life at baseline and achieved less improvement of quality of life than men after subthalamic nucleus deep brain stimulation.Furthermore,Hoehn-Yahr stage was positively correlated with the treatment response in men,while levodopa equivalent dose at 12 months post-operation was negatively correlated with motor improvement in women.In conclusion,women received less benefit from subthalamic nucleus deep brain stimulation than men in terms of motor symptoms,non-motor symptoms,and quality of life.We found sex-specific factors,i.e.,Hoehn-Yahr stage and levodopa equivalent dose,that were related to motor improvements.These findings may help to guide subthalamic nucleus deep brain stimulation patient selection,prognosis,and stimulation programming for optimal therapeutic efficacy in Parkinson’s disease.展开更多
The role of intercalatus nucleus of Staderini(INS),the most caudal of the perihypoglossal nuclei,is much debated.Last research seems to suggest that this nucleus plays a role as a vertical eyes movements integrator.Th...The role of intercalatus nucleus of Staderini(INS),the most caudal of the perihypoglossal nuclei,is much debated.Last research seems to suggest that this nucleus plays a role as a vertical eyes movements integrator.The few clinical reports present in the literature that describe isolated lesions of the INS have described patients presenting in acute with up-beating vertical spontaneous nystagmus.Isolated acute lesion of INS is,in fact,much rare,and,without other neurological signs,is exceptional.We present a case of acute isolated vertigo with no other neurological signs or symptoms,due to INS ischemia provoked by vertebral artery stenosis.The patient presented with spontaneous vertical up-beating nystagmus that showed at videonystagmographic recording,a clear exponential decay of angular slow-phase velocity,that is considered a typical sign of neural integrator impairment.This case seems to represent a further confirm that INS is part,as a vertical-to-position neural integrator,of the neural circuit controlling the vertical eyes movements.展开更多
AIM:To evaluate the lateral geniculate nucleus(LGN)volume and height using magnetic resonance imaging(MRI)in glaucoma patients.METHODS:Literatures retrieval was carried out through Pub Med,Web of Science,Embase,and Co...AIM:To evaluate the lateral geniculate nucleus(LGN)volume and height using magnetic resonance imaging(MRI)in glaucoma patients.METHODS:Literatures retrieval was carried out through Pub Med,Web of Science,Embase,and Cochrane Library.Studies that compared the volume and height of LGN in glaucoma patients with that in control subjects were included.The volume and height of LGN were extracted from the included studies.The Review Manager 5.4.1 software was used for the Meta-analysis.RESULTS:This Meta-analysis included 10 crosssectional studies,including the eyes of 223 glaucoma patients and 185 healthy controls.Compared with the control subjects,the volume and height of LGN in glaucoma patients measured by MRI were significantly reduced{-29.13 mm3,95%[confidence interval(CI):-44.82 to-13.43,P=0.0003;-0.61 mm,95%CI:-0.78 to-0.44,P<0.00001,respectively]}.Subgroup analysis demonstrated that the differences of LGN volume and height between glaucoma patients and control subjects in the older group were smaller than that in the younger group,and LGN volume decreased with the increase of glaucoma severity.CONCLUSION:The results demonstrate that the volume and height of LGN are decreased in glaucoma patients,and LGN volume can be considered a parameter of glaucoma severity.展开更多
In this proceeding,some highlight results on the constraints of the nuclear matter equation of state(EOS)from the data of nucleus resonance and neutron-skin thickness using the Bayesian approach based on the Skyrme-Ha...In this proceeding,some highlight results on the constraints of the nuclear matter equation of state(EOS)from the data of nucleus resonance and neutron-skin thickness using the Bayesian approach based on the Skyrme-Hartree-Fock model and its extension have been presented.Typically,the anti-correlation and positive correlations between the slope parameter and the value of the symmetry energy at the saturation density under the constraint of the neutron-skin thickness and the isovector giant dipole resonance have been discussed respectively.It’s shown that the Bayesian analysis can help to find a compromise for the“PREXII puzzle”and the“soft Tin puzzle”.The possible modifications on the constraints of lower-order EOS parameters as well as the relevant correlation when higher-order EOS parameters are incorporated as independent variables have been further illustrated.For a given model and parameter space,the Bayesian approach serves as a good analysis tool suitable for multi-messengers versus multi-variables,and is helpful for constraining quantitatively the model parameters as well as their correlations.展开更多
Objective Disc calcification is strongly associated with disc degeneration;however,the underlying mechanisms driving its pathogenesis are poorly understood.This study aimed to provide a gene expression profile of nucl...Objective Disc calcification is strongly associated with disc degeneration;however,the underlying mechanisms driving its pathogenesis are poorly understood.This study aimed to provide a gene expression profile of nucleus pulposus cells(NPCs)from calcified discs,and clarify the potential mechanism in disc degeneration.Methods Primary NPCs were isolated from calcified and control discs(CAL-NPC and CON-NPC),respectively.The proliferation and extracellular matrix(ECM)metabolism capacities of the cells were evaluated using MTT and Western blotting,respectively.RNA sequencing was used to identify differentially expressed genes(DEGs)in the CAL-NPCs.The biological functions of the DEGs were analyzed using the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases.The transcription factor database and Cytoscape software were used to construct the transcription factor-DEGs regulatory network.The role of the verified transcription factor in NPC proliferation and ECM metabolism was also investigated.Results The CAL-NPCs exhibited a lower proliferation rate and higher ECM degradation capacity than the CON-NPCs.In total,375 DEGs were identified in the CAL-NPCs.The GO and KEGG analyses showed that the DEGs were primarily involved in the regulation of ribonuclease activity and NF-kappa B and p53 signaling pathways.GATA-binding protein 3(GATA3)with the highest verified levels was selected for further studies.Overexpression of GATA3 in the CON-NPCs significantly inhibited their proliferation and promoted their ECM degradation function,while the knockdown of GATA3 in the CAL-NPCs resulted in the opposite phenotypes.Conclusion This study provided a comprehensive gene expression profile of the NPCs from the calcified discs and supported that GATA3 could be a potential target for reversing calcification-associated disc degeneration.展开更多
BACKGROUND Hydrogen sulfide(H2S)is a recently discovered gaseous neurotransmitter in the nervous and gastrointestinal systems.It exerts its effects through multiple signaling pathways,impacting various physiological a...BACKGROUND Hydrogen sulfide(H2S)is a recently discovered gaseous neurotransmitter in the nervous and gastrointestinal systems.It exerts its effects through multiple signaling pathways,impacting various physiological activities.The nucleus tractus solitarius(NTS),a vital nucleus involved in visceral sensation,was investigated in this study to understand the role of H2S in regulating gastric function in rats.AIM To examine whether H2S affects the nuclear factor kappa-B(NF-κB)and transient receptor potential vanilloid 1 pathways and the neurokinin 1(NK1)receptor in the NTS.METHODS Immunohistochemical and fluorescent double-labeling techniques were employed to identify cystathionine beta-synthase(CBS)and c-Fos co-expressed positive neurons in the NTS during rat stress.Gastric motility curves were recorded by inserting a pressure-sensing balloon into the pylorus through the stomach fundus.Changes in gastric motility were observed before and after injecting different doses of NaHS(4 nmol and 8 nmol),physiological saline,Capsazepine(4 nmol)+NaHS(4 nmol),pyrrolidine dithiocarbamate(PDTC,4 nmol)+NaHS(4 nmol),and L703606(4 nmol)+NaHS(4 nmol).RESULTS We identified a significant increase in the co-expression of c-Fos and CBS positive neurons in the NTS after 1 h and 3 h of restraint water-immersion stress compared to the expressions observed in the control group.Intra-NTS injection of NaHS at different doses significantly inhibited gastric motility in rats(P<0.01).However,injection of saline,first injection NF-κB inhibitor PDTC or transient receptor potential vanilloid 1(TRPV1)antagonist Capsazepine or NK1 receptor blockers L703606 and then injection NaHS did not produce significant changes(P>0.05).CONCLUSION NTS contains neurons co-expressing CBS and c-Fos,and the injection of NaHS into the NTS can suppress gastric motility in rats.This effect may be mediated by activating TRPV1 and NK1 receptors via the NF-κB channel.展开更多
The measures of steam curing and early-strengthening agents to promote the precast components to reach the target strength quickly can bring different degrees of damage to the concrete.Based on this,the new nanomateri...The measures of steam curing and early-strengthening agents to promote the precast components to reach the target strength quickly can bring different degrees of damage to the concrete.Based on this,the new nanomaterial CSH-the hydration product of cement effectively solves these measures’disadvantages,such as excessive energy consumption,thermal stress damage,and the introduction of external ions.In this paper,the effect of CSH on the early strength of precast fly ash concrete components was investigated in terms of setting time,workability,and mechanical properties and analyzed at the microscopic level using hydration temperature,XRD,and SEM.The results showed that under the same workability,CSH could significantly reduce the amount of admixture,shorten the final setting time,almost not affect the initial setting time,and accelerate the hydration of cement.At the optimum dose of 5%,the mechanical properties of the specimens were improved by more than 98%within 12 h of hydration,resulting in an earlier release time of 12 h and no risk of strength inversion later.The results of this paper give theoretical support to the behavior of precast components under steam-free curing.展开更多
BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchym...BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.展开更多
基金support by the National Nature Science Foundation of China,Nos.81830033,61761166004(both to JGZ)。
文摘There are many documented sex differences in the clinical course,symptom expression profile,and treatment response of Parkinson’s disease,creating additional challenges for patient management.Although subthalamic nucleus deep brain stimulation is an established therapy for Parkinson’s disease,the effects of sex on treatment outcome are still unclear.The aim of this retrospective observational study,was to examine sex differences in motor symptoms,nonmotor symptoms,and quality of life after subthalamic nucleus deep brain stimulation.Outcome measures were evaluated at 1 and 12 months post-operation in 90 patients with Parkinson’s disease undergoing subthalamic nucleus deep brain stimulation aged 63.00±8.01 years(55 men and 35 women).Outcomes of clinical evaluations were compared between sexes via a Student’s t-test and within sex via a paired-sample t-test,and generalized linear models were established to identify factors associated with treatment efficacy and intensity for each sex.We found that subthalamic nucleus deep brain stimulation could improve motor symptoms in men but not women in the on-medication condition at 1 and 12 months post-operation.Restless legs syndrome was alleviated to a greater extent in men than in women.Women demonstrated poorer quality of life at baseline and achieved less improvement of quality of life than men after subthalamic nucleus deep brain stimulation.Furthermore,Hoehn-Yahr stage was positively correlated with the treatment response in men,while levodopa equivalent dose at 12 months post-operation was negatively correlated with motor improvement in women.In conclusion,women received less benefit from subthalamic nucleus deep brain stimulation than men in terms of motor symptoms,non-motor symptoms,and quality of life.We found sex-specific factors,i.e.,Hoehn-Yahr stage and levodopa equivalent dose,that were related to motor improvements.These findings may help to guide subthalamic nucleus deep brain stimulation patient selection,prognosis,and stimulation programming for optimal therapeutic efficacy in Parkinson’s disease.
文摘The role of intercalatus nucleus of Staderini(INS),the most caudal of the perihypoglossal nuclei,is much debated.Last research seems to suggest that this nucleus plays a role as a vertical eyes movements integrator.The few clinical reports present in the literature that describe isolated lesions of the INS have described patients presenting in acute with up-beating vertical spontaneous nystagmus.Isolated acute lesion of INS is,in fact,much rare,and,without other neurological signs,is exceptional.We present a case of acute isolated vertigo with no other neurological signs or symptoms,due to INS ischemia provoked by vertebral artery stenosis.The patient presented with spontaneous vertical up-beating nystagmus that showed at videonystagmographic recording,a clear exponential decay of angular slow-phase velocity,that is considered a typical sign of neural integrator impairment.This case seems to represent a further confirm that INS is part,as a vertical-to-position neural integrator,of the neural circuit controlling the vertical eyes movements.
基金Supported by National Natural Science Foundation of China (No.81860170)。
文摘AIM:To evaluate the lateral geniculate nucleus(LGN)volume and height using magnetic resonance imaging(MRI)in glaucoma patients.METHODS:Literatures retrieval was carried out through Pub Med,Web of Science,Embase,and Cochrane Library.Studies that compared the volume and height of LGN in glaucoma patients with that in control subjects were included.The volume and height of LGN were extracted from the included studies.The Review Manager 5.4.1 software was used for the Meta-analysis.RESULTS:This Meta-analysis included 10 crosssectional studies,including the eyes of 223 glaucoma patients and 185 healthy controls.Compared with the control subjects,the volume and height of LGN in glaucoma patients measured by MRI were significantly reduced{-29.13 mm3,95%[confidence interval(CI):-44.82 to-13.43,P=0.0003;-0.61 mm,95%CI:-0.78 to-0.44,P<0.00001,respectively]}.Subgroup analysis demonstrated that the differences of LGN volume and height between glaucoma patients and control subjects in the older group were smaller than that in the younger group,and LGN volume decreased with the increase of glaucoma severity.CONCLUSION:The results demonstrate that the volume and height of LGN are decreased in glaucoma patients,and LGN volume can be considered a parameter of glaucoma severity.
基金Supported by National Natural Science Foundation of China (11922514)。
文摘In this proceeding,some highlight results on the constraints of the nuclear matter equation of state(EOS)from the data of nucleus resonance and neutron-skin thickness using the Bayesian approach based on the Skyrme-Hartree-Fock model and its extension have been presented.Typically,the anti-correlation and positive correlations between the slope parameter and the value of the symmetry energy at the saturation density under the constraint of the neutron-skin thickness and the isovector giant dipole resonance have been discussed respectively.It’s shown that the Bayesian analysis can help to find a compromise for the“PREXII puzzle”and the“soft Tin puzzle”.The possible modifications on the constraints of lower-order EOS parameters as well as the relevant correlation when higher-order EOS parameters are incorporated as independent variables have been further illustrated.For a given model and parameter space,the Bayesian approach serves as a good analysis tool suitable for multi-messengers versus multi-variables,and is helpful for constraining quantitatively the model parameters as well as their correlations.
基金funded by the Youth Research Fund of the Peking Union Medical College Hospital(No.pumch201911708).
文摘Objective Disc calcification is strongly associated with disc degeneration;however,the underlying mechanisms driving its pathogenesis are poorly understood.This study aimed to provide a gene expression profile of nucleus pulposus cells(NPCs)from calcified discs,and clarify the potential mechanism in disc degeneration.Methods Primary NPCs were isolated from calcified and control discs(CAL-NPC and CON-NPC),respectively.The proliferation and extracellular matrix(ECM)metabolism capacities of the cells were evaluated using MTT and Western blotting,respectively.RNA sequencing was used to identify differentially expressed genes(DEGs)in the CAL-NPCs.The biological functions of the DEGs were analyzed using the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases.The transcription factor database and Cytoscape software were used to construct the transcription factor-DEGs regulatory network.The role of the verified transcription factor in NPC proliferation and ECM metabolism was also investigated.Results The CAL-NPCs exhibited a lower proliferation rate and higher ECM degradation capacity than the CON-NPCs.In total,375 DEGs were identified in the CAL-NPCs.The GO and KEGG analyses showed that the DEGs were primarily involved in the regulation of ribonuclease activity and NF-kappa B and p53 signaling pathways.GATA-binding protein 3(GATA3)with the highest verified levels was selected for further studies.Overexpression of GATA3 in the CON-NPCs significantly inhibited their proliferation and promoted their ECM degradation function,while the knockdown of GATA3 in the CAL-NPCs resulted in the opposite phenotypes.Conclusion This study provided a comprehensive gene expression profile of the NPCs from the calcified discs and supported that GATA3 could be a potential target for reversing calcification-associated disc degeneration.
基金the Natural Science Foundation of Shandong Province,No.ZR2019MC020。
文摘BACKGROUND Hydrogen sulfide(H2S)is a recently discovered gaseous neurotransmitter in the nervous and gastrointestinal systems.It exerts its effects through multiple signaling pathways,impacting various physiological activities.The nucleus tractus solitarius(NTS),a vital nucleus involved in visceral sensation,was investigated in this study to understand the role of H2S in regulating gastric function in rats.AIM To examine whether H2S affects the nuclear factor kappa-B(NF-κB)and transient receptor potential vanilloid 1 pathways and the neurokinin 1(NK1)receptor in the NTS.METHODS Immunohistochemical and fluorescent double-labeling techniques were employed to identify cystathionine beta-synthase(CBS)and c-Fos co-expressed positive neurons in the NTS during rat stress.Gastric motility curves were recorded by inserting a pressure-sensing balloon into the pylorus through the stomach fundus.Changes in gastric motility were observed before and after injecting different doses of NaHS(4 nmol and 8 nmol),physiological saline,Capsazepine(4 nmol)+NaHS(4 nmol),pyrrolidine dithiocarbamate(PDTC,4 nmol)+NaHS(4 nmol),and L703606(4 nmol)+NaHS(4 nmol).RESULTS We identified a significant increase in the co-expression of c-Fos and CBS positive neurons in the NTS after 1 h and 3 h of restraint water-immersion stress compared to the expressions observed in the control group.Intra-NTS injection of NaHS at different doses significantly inhibited gastric motility in rats(P<0.01).However,injection of saline,first injection NF-κB inhibitor PDTC or transient receptor potential vanilloid 1(TRPV1)antagonist Capsazepine or NK1 receptor blockers L703606 and then injection NaHS did not produce significant changes(P>0.05).CONCLUSION NTS contains neurons co-expressing CBS and c-Fos,and the injection of NaHS into the NTS can suppress gastric motility in rats.This effect may be mediated by activating TRPV1 and NK1 receptors via the NF-κB channel.
文摘The measures of steam curing and early-strengthening agents to promote the precast components to reach the target strength quickly can bring different degrees of damage to the concrete.Based on this,the new nanomaterial CSH-the hydration product of cement effectively solves these measures’disadvantages,such as excessive energy consumption,thermal stress damage,and the introduction of external ions.In this paper,the effect of CSH on the early strength of precast fly ash concrete components was investigated in terms of setting time,workability,and mechanical properties and analyzed at the microscopic level using hydration temperature,XRD,and SEM.The results showed that under the same workability,CSH could significantly reduce the amount of admixture,shorten the final setting time,almost not affect the initial setting time,and accelerate the hydration of cement.At the optimum dose of 5%,the mechanical properties of the specimens were improved by more than 98%within 12 h of hydration,resulting in an earlier release time of 12 h and no risk of strength inversion later.The results of this paper give theoretical support to the behavior of precast components under steam-free curing.
基金Supported by the National Natural Science Foundation of China,No.82172462,No.81972136the Traditional Chinese Medicine Science and Technology Development Plan Project of Jiangsu Province,No.YB2020085Cross Cooperation Project of Northern Jiangsu People’s Hospital,No.SBJC21014.
文摘BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.
