Objective The aim of the present study was not only to assess the retrograde degenerative changes in the dopaminergic neurons of the substantia nigra （SN） and ventral tegmental area （VTA） after injection of 6-hydr...Objective The aim of the present study was not only to assess the retrograde degenerative changes in the dopaminergic neurons of the substantia nigra （SN） and ventral tegmental area （VTA） after injection of 6-hydroxydopamine （6-OHDA） into the striatum, but also to use this 6-OHDA model of Parkinson＇s disease to explore the possible neuroprotective effect of R-apomorphine （R-APt）. Methods The partial lesion was obtained by intrastriatal administration of 6-OHDA. R- APt administration （10 mg/kg, s.c.） started 15 min prior to lesioning and continued daily for another 22 days post surgery. Testing was carried out 5 weeks after lesioning. We investigated the histology and associated behavior and neurochemical changes. Structural and functional deficits were quantified by tyrosine hydroxylase （TH） / Nissl-staining cell number counting, striatal dopamine （DA） content determination and amphetamine-induced rotation analysis. Results R-APt- treatment attenuated the amphetamine-induced ipsiversive rotation 5 weeks after the lesion induction. R-APt administra- tion for 22 days significantly reduced the size of the lesion at the level of the SN from 50% （control group） to 69%. Moreover, the cell shape resembled that observed in the intact animals. R-APt treatment significantly increased the number of cells in both the lesion and the intact sides of VTA by 60%, suggesting selective neurotrophic effect of R-APt in this area. Finally, R-APt-treatment significantly attenuated the 6-OHDA-induced striatal DA depletion and normalized dihydroxyphenylacetic acid （DOPAC）/DA ratios. Conclusion We conclude that R-APt has neuroprotective and pos- sible neurotrophic effect on a striatal lesion with 6-OHDA, suggesting that this drug may have rescuing properties in patients with early stage Parkinson＇s disease. These effects are more pronounced in VTA and enhance with duration of treatment.展开更多
Objective Combine olfactory ensheathing glia （OEG） implantation with ex vivo non-viral vector-based neurotrophin- 3 （NT-3） gene therapy in attempting to enhance regeneration after thoracic spinal cord injury （SCI...Objective Combine olfactory ensheathing glia （OEG） implantation with ex vivo non-viral vector-based neurotrophin- 3 （NT-3） gene therapy in attempting to enhance regeneration after thoracic spinal cord injury （SCI）. Methods Primary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1 （＋）-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord （T9） contusion by the New York University impactor. The animals in 3 different groups received 4x 1050EG transfected with pcDNA3.1 （＋）-NT3 or pcDNA3.1 （＋） plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted. Results NT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1 （＋）-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting. Conclusion Non-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT- 3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.展开更多
This article reviewed the beneficial effects of moderate voluntary physical exercise on brain health according to the studies on humans and animals, which includes improving psychological status and cognitive function...This article reviewed the beneficial effects of moderate voluntary physical exercise on brain health according to the studies on humans and animals, which includes improving psychological status and cognitive function, enhancing psychological well-being, decreasing the risks of Alzheimer＇s disease （AD） and dementia, and promoting the effects of antidepressant and anxiolytic. The possible underlying neurobiological mechanisms are involved up-active and down-active pathways. The up-active pathway is associated with enhancements of several neurotransmitters systems afferent to hippocampus, including norepinephrine （NE）, serotonin （5-Hydroxytryptamine, 5-HT）, acetylcholine （ACh） and γ-aminobutyric acid （GABA）. The down-active pathway is mainly concerned with up-regulation of the brain-derived neurotrophic factor （BDNF） and neurogenesis. It is suggested that NE activation via β-adrenergic receptors may be essential for exercise-induced BDNF up-regulation. The possible intracellular signaling pathways of NE-mediated BDNF up-expression may be involved in GPCR-MAPK-PI-3K crosstalk and positive feedback.展开更多
A dissolved oxygen fuzzy system predicting model based on neural network was put forward in this study. 106 groups of data were used to confirm the fitness of the predicting model. The first 80 groups of data were act...A dissolved oxygen fuzzy system predicting model based on neural network was put forward in this study. 106 groups of data were used to confirm the fitness of the predicting model. The first 80 groups of data were acted as training input and the other 26 groups of data were acted as the confirmed data in the system. The result showed that the testing data was approximately the same as the predicted data. So it gave a new way to solve the problem that the status of the water quality couldn＇t be predicted in time and it＇s hard to watching and measuring the factors dynamic.展开更多
Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by...Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells （BMSCs）. Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration （800 pg/mL） than BMSCs did （less than 100 pg/mL）. The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin （10 μmol/L）. Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.展开更多
Objective To observe the activating effect of ciliary neurotrophic factor （CNTF） on astrocyte in vitro. Methods Astrocytes cultured purely from newborn rats. Cerebral cortex was raised in normal and serum deprivatio...Objective To observe the activating effect of ciliary neurotrophic factor （CNTF） on astrocyte in vitro. Methods Astrocytes cultured purely from newborn rats. Cerebral cortex was raised in normal and serum deprivation condition with different concentrations （in ng/ml： 0, 2, 20, or 200） of CNTF. After cultured for 24 h, the shape and the cell cycle of astrocytes were examined by immunocytochemistry and flow cytometer, respectively. Results The immunoactivity of glial fibrillary acidic protein （GFAP） and the nuclear size of astrocytes were increased when CNTF was applied, whether cells were cultured in medium with or without serum. CNTF promoted astrocytes to enter the cell cycle in medium with serum, but had no this effect in medium without serum. Conclusion In medium without serum, astrocytes could differentiate into activated state ceils with CNTF application, but could not proliferate; in medium with serum, astrocytes could proliferate with aid of CNTF.展开更多
[Objective] The aim was to explore evaluated precision on quality of soil environment polluted with zinc in agricultural production areas and to provide references for verification of production area.[Method] In Shula...[Objective] The aim was to explore evaluated precision on quality of soil environment polluted with zinc in agricultural production areas and to provide references for verification of production area.[Method] In Shulan City in Jilin Province,soils were sampled and analyzed in a laboratory using single-factor pollution index and GIS based spatial interpolation.The quality of environment polluted with zinc was assessed and related methods were compared according to Environment Quality Standard of Green Food Production Area.[Result] Spatial interpolation of zinc in soils based on GIS proved more precise than traditional methods;cokriging method with co-factors was higher in precision than common cokriging;cokriging method with zinc and organic matter was higher in precision than cokriging with zinc alone.[Conclusion] Quality assessment on environment polluted with zinc based on GIS interpolation is more scientific and reasonable than traditional methods.展开更多
Objective To investigate the expression of nerve growth factor （NGF） and glial cell line-derived neurotrophic factor （GDNF） in monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood ...Objective To investigate the expression of nerve growth factor （NGF） and glial cell line-derived neurotrophic factor （GDNF） in monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood flow occlusion. Methods The monkeys were immediately removed brain after death in operation of group A （identical temperature perfusion group） and group B （ultraprofound hypothermia perfusion group）. Immunohistochemical technique was used to determine frontal cellular expression of NGF and GDNF. Statistics were analyzed by ANOVA analyses with significance level at P 〈 0.05. Results The expressions of NGF and GDNF in the group B were significantly higher than those in the group A （P 〈 0.05）. Conclusion NGF and GDNF increased significantly in the monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood flow occlusion. It may be a protective mechanism for neuron survival and neural function recovery.展开更多
Microglial activation plays an important role in a panel of neurological disorders such as multiple sclerosis(MS) and Parkinson's disease(PD),and is a key target for developing therapeutic strategies for these dis...Microglial activation plays an important role in a panel of neurological disorders such as multiple sclerosis(MS) and Parkinson's disease(PD),and is a key target for developing therapeutic strategies for these diseases.Ketogenic diet (KD),which is able to inhibit microglial activation in substantia nigra pars compacta of mice,has been shown effective in a mouse model of PD,possibly through increasing D-β-hydroxybutyrate(D-β-HB),a major component of ketone bodies.To verify this,we developed an in vitro model of microglia activation with a microglia line,BV-2,and investigated how D-β-HB have an effect on the LPS-stimulated BV-2 cells.We found D-β-HB is able to recover the cell viability,and inhibit the production of inflammatory mediators and cytokines such as ROS,nitrite,IL-1β,TNF-α,and IL-6,which otherwise were increased in LPS-stimulated BV-2 cells.We conclude that the LPS induced BV-2 cells activation is a valid in vitro model of microglia activation.D-β-HB is able to suppress the activation of BV-2 cells, which might account for one of the possible reasons of KD therapy on the PD model.展开更多
Objective The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein （GFAP） production and glial cell line-derived neurotrophic factor （GDNF） secretion of astrocytes under isch...Objective The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein （GFAP） production and glial cell line-derived neurotrophic factor （GDNF） secretion of astrocytes under ischemic insult and the related signaling pathways. Methods Using transient right middle cerebral artery occlusion （tMCAO） and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR）, Western blotting, enzyme linked immunosorbent assay （ELISA） were used to investigate location of P2Y1 receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules. Results Blockage of P2Y1 receptor with the selective antagonist N^6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate diammonium （MRS2179） reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y1 receptor caused elevation of phosphorylated Akt and cAMP response element binding protein （CREB）, and reduction of phosphorylated Janus kinase2 （JAK2） and signal transducer and activator of transcription3 （STAT3, Ser727）. After blockage of P2Y1 receptor and deprivation of oxygen-glucose-serum, AG490 （inhibitor of JAK2） reduced phosphorylation of STAT3 （Ser727） as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase （PI3-K）, decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase 1/2 （MEK 1/2） U0126, an important molecule of Ras/extracellular signal- regulated kinase （ERK） signaling pathway, decreased the phosphorylation of JAK2, STAT3 （Ser727）, Akt and CREB. Conclusion These results suggest that P2Y1 receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.展开更多
文摘Objective The aim of the present study was not only to assess the retrograde degenerative changes in the dopaminergic neurons of the substantia nigra （SN） and ventral tegmental area （VTA） after injection of 6-hydroxydopamine （6-OHDA） into the striatum, but also to use this 6-OHDA model of Parkinson＇s disease to explore the possible neuroprotective effect of R-apomorphine （R-APt）. Methods The partial lesion was obtained by intrastriatal administration of 6-OHDA. R- APt administration （10 mg/kg, s.c.） started 15 min prior to lesioning and continued daily for another 22 days post surgery. Testing was carried out 5 weeks after lesioning. We investigated the histology and associated behavior and neurochemical changes. Structural and functional deficits were quantified by tyrosine hydroxylase （TH） / Nissl-staining cell number counting, striatal dopamine （DA） content determination and amphetamine-induced rotation analysis. Results R-APt- treatment attenuated the amphetamine-induced ipsiversive rotation 5 weeks after the lesion induction. R-APt administra- tion for 22 days significantly reduced the size of the lesion at the level of the SN from 50% （control group） to 69%. Moreover, the cell shape resembled that observed in the intact animals. R-APt treatment significantly increased the number of cells in both the lesion and the intact sides of VTA by 60%, suggesting selective neurotrophic effect of R-APt in this area. Finally, R-APt-treatment significantly attenuated the 6-OHDA-induced striatal DA depletion and normalized dihydroxyphenylacetic acid （DOPAC）/DA ratios. Conclusion We conclude that R-APt has neuroprotective and pos- sible neurotrophic effect on a striatal lesion with 6-OHDA, suggesting that this drug may have rescuing properties in patients with early stage Parkinson＇s disease. These effects are more pronounced in VTA and enhance with duration of treatment.
文摘Objective Combine olfactory ensheathing glia （OEG） implantation with ex vivo non-viral vector-based neurotrophin- 3 （NT-3） gene therapy in attempting to enhance regeneration after thoracic spinal cord injury （SCI）. Methods Primary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1 （＋）-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord （T9） contusion by the New York University impactor. The animals in 3 different groups received 4x 1050EG transfected with pcDNA3.1 （＋）-NT3 or pcDNA3.1 （＋） plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted. Results NT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1 （＋）-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting. Conclusion Non-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT- 3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.
基金the National Natural Science Fundation of China (No. 30570895,No. 30700389)
文摘This article reviewed the beneficial effects of moderate voluntary physical exercise on brain health according to the studies on humans and animals, which includes improving psychological status and cognitive function, enhancing psychological well-being, decreasing the risks of Alzheimer＇s disease （AD） and dementia, and promoting the effects of antidepressant and anxiolytic. The possible underlying neurobiological mechanisms are involved up-active and down-active pathways. The up-active pathway is associated with enhancements of several neurotransmitters systems afferent to hippocampus, including norepinephrine （NE）, serotonin （5-Hydroxytryptamine, 5-HT）, acetylcholine （ACh） and γ-aminobutyric acid （GABA）. The down-active pathway is mainly concerned with up-regulation of the brain-derived neurotrophic factor （BDNF） and neurogenesis. It is suggested that NE activation via β-adrenergic receptors may be essential for exercise-induced BDNF up-regulation. The possible intracellular signaling pathways of NE-mediated BDNF up-expression may be involved in GPCR-MAPK-PI-3K crosstalk and positive feedback.
基金Supported by National Natural Science Foundation of China (40801227)Open Foundation of Marine and Estuarine Fisheries Resources of Ministry of Agriculture and the Key Laboratory of Ecology (Open-2-04-09)~~
文摘A dissolved oxygen fuzzy system predicting model based on neural network was put forward in this study. 106 groups of data were used to confirm the fitness of the predicting model. The first 80 groups of data were acted as training input and the other 26 groups of data were acted as the confirmed data in the system. The result showed that the testing data was approximately the same as the predicted data. So it gave a new way to solve the problem that the status of the water quality couldn＇t be predicted in time and it＇s hard to watching and measuring the factors dynamic.
基金This work was supported by the Natural Science Foundation of Shanghai Municipality(No.03ZR14016).
文摘Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells （BMSCs）. Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration （800 pg/mL） than BMSCs did （less than 100 pg/mL）. The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin （10 μmol/L）. Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.
文摘Objective To observe the activating effect of ciliary neurotrophic factor （CNTF） on astrocyte in vitro. Methods Astrocytes cultured purely from newborn rats. Cerebral cortex was raised in normal and serum deprivation condition with different concentrations （in ng/ml： 0, 2, 20, or 200） of CNTF. After cultured for 24 h, the shape and the cell cycle of astrocytes were examined by immunocytochemistry and flow cytometer, respectively. Results The immunoactivity of glial fibrillary acidic protein （GFAP） and the nuclear size of astrocytes were increased when CNTF was applied, whether cells were cultured in medium with or without serum. CNTF promoted astrocytes to enter the cell cycle in medium with serum, but had no this effect in medium without serum. Conclusion In medium without serum, astrocytes could differentiate into activated state ceils with CNTF application, but could not proliferate; in medium with serum, astrocytes could proliferate with aid of CNTF.
基金Supported by National 973 Program(2010CB951500)National 863 Program(2006AA-120103)~~
文摘[Objective] The aim was to explore evaluated precision on quality of soil environment polluted with zinc in agricultural production areas and to provide references for verification of production area.[Method] In Shulan City in Jilin Province,soils were sampled and analyzed in a laboratory using single-factor pollution index and GIS based spatial interpolation.The quality of environment polluted with zinc was assessed and related methods were compared according to Environment Quality Standard of Green Food Production Area.[Result] Spatial interpolation of zinc in soils based on GIS proved more precise than traditional methods;cokriging method with co-factors was higher in precision than common cokriging;cokriging method with zinc and organic matter was higher in precision than cokriging with zinc alone.[Conclusion] Quality assessment on environment polluted with zinc based on GIS interpolation is more scientific and reasonable than traditional methods.
基金This work was supported by the Key Program of Natural Science Foundation of Yunnan Province, China （No. 2003C0010Z）.
文摘Objective To investigate the expression of nerve growth factor （NGF） and glial cell line-derived neurotrophic factor （GDNF） in monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood flow occlusion. Methods The monkeys were immediately removed brain after death in operation of group A （identical temperature perfusion group） and group B （ultraprofound hypothermia perfusion group）. Immunohistochemical technique was used to determine frontal cellular expression of NGF and GDNF. Statistics were analyzed by ANOVA analyses with significance level at P 〈 0.05. Results The expressions of NGF and GDNF in the group B were significantly higher than those in the group A （P 〈 0.05）. Conclusion NGF and GDNF increased significantly in the monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood flow occlusion. It may be a protective mechanism for neuron survival and neural function recovery.
文摘Microglial activation plays an important role in a panel of neurological disorders such as multiple sclerosis(MS) and Parkinson's disease(PD),and is a key target for developing therapeutic strategies for these diseases.Ketogenic diet (KD),which is able to inhibit microglial activation in substantia nigra pars compacta of mice,has been shown effective in a mouse model of PD,possibly through increasing D-β-hydroxybutyrate(D-β-HB),a major component of ketone bodies.To verify this,we developed an in vitro model of microglia activation with a microglia line,BV-2,and investigated how D-β-HB have an effect on the LPS-stimulated BV-2 cells.We found D-β-HB is able to recover the cell viability,and inhibit the production of inflammatory mediators and cytokines such as ROS,nitrite,IL-1β,TNF-α,and IL-6,which otherwise were increased in LPS-stimulated BV-2 cells.We conclude that the LPS induced BV-2 cells activation is a valid in vitro model of microglia activation.D-β-HB is able to suppress the activation of BV-2 cells, which might account for one of the possible reasons of KD therapy on the PD model.
基金the National Natural Science Foundation of China (No. 30500189)
文摘Objective The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein （GFAP） production and glial cell line-derived neurotrophic factor （GDNF） secretion of astrocytes under ischemic insult and the related signaling pathways. Methods Using transient right middle cerebral artery occlusion （tMCAO） and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR）, Western blotting, enzyme linked immunosorbent assay （ELISA） were used to investigate location of P2Y1 receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules. Results Blockage of P2Y1 receptor with the selective antagonist N^6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate diammonium （MRS2179） reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y1 receptor caused elevation of phosphorylated Akt and cAMP response element binding protein （CREB）, and reduction of phosphorylated Janus kinase2 （JAK2） and signal transducer and activator of transcription3 （STAT3, Ser727）. After blockage of P2Y1 receptor and deprivation of oxygen-glucose-serum, AG490 （inhibitor of JAK2） reduced phosphorylation of STAT3 （Ser727） as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase （PI3-K）, decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase 1/2 （MEK 1/2） U0126, an important molecule of Ras/extracellular signal- regulated kinase （ERK） signaling pathway, decreased the phosphorylation of JAK2, STAT3 （Ser727）, Akt and CREB. Conclusion These results suggest that P2Y1 receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.
