Hypobaric hypoxia (HH) exposure can cause serious brain injury as well as life-threatening cerebral edema in severe cases. Previous studies on the mechanisms of HH-induced brain injury have been conducted primarily us...Hypobaric hypoxia (HH) exposure can cause serious brain injury as well as life-threatening cerebral edema in severe cases. Previous studies on the mechanisms of HH-induced brain injury have been conducted primarily using non-primate animal models that are genetically distant to humans, thus hindering the development of disease treatment. Here, we report that cynomolgus monkeys (Macaca fascicularis) exposed to acute HH developed human-like HH syndrome involving severe brain injury and abnormal behavior. Transcriptome profiling of white blood cells and brain tissue from monkeys exposed to increasing altitude revealed the central role of the HIF-1 and other novel signaling pathways, such as the vitamin D receptor (VDR) signaling pathway, in co-regulating HH-induced inflammation processes. We also observed profound transcriptomic alterations in brains after exposure to acute HH, including the activation of angiogenesis and impairment of aerobic respiration and protein folding processes, which likely underlie the pathological effects of HH-induced brain injury. Administration of progesterone (PROG) and steroid neuroprotectant 5α-androst-3β,5,6β-triol (TRIOL) significantly attenuated brain injuries and rescued the transcriptomic changes induced by acute HH. Functional investigation of the affected genes suggested that these two neuroprotectants protect the brain by targeting different pathways, with PROG enhancing erythropoiesis and TRIOL suppressing glutamate-induced excitotoxicity. Thus, this study advances our understanding of the pathology induced by acute HH and provides potential compounds for the development of neuroprotectant drugs for therapeutic treatment.展开更多
Postpartum depression （PPD） is a modified form of major depressive disorders （MDD） that can exert profound negative effects on both mothers and infants than MDD. Within the postpartum period, both mothers and infa...Postpartum depression （PPD） is a modified form of major depressive disorders （MDD） that can exert profound negative effects on both mothers and infants than MDD. Within the postpartum period, both mothers and infants are susceptible; but because PPD typically occurs for short durations and has moderate symptoms, there exists challenges in exploring and addressing the underlying cause of the depression. This fact highlights the need for relevant animal models. In the present study, postpartum adult female cynomolgus monkeys （Macaca fascicularis） living in breeding groups were observed for typical depressive behavior. The huddle posture behavior was utilized as an indicator of behavioral depression postpartum （BDP） as it has been established as the core depressive-like behavior in primates. Monkeys were divided into two groups： A t3DP group （n=6）, which were found to spend more time huddling over the first two weeks postpartum than other individuals that formed a non-depression control group （n=4）. The two groups were then further analyzed for locomotive activity, stressful events, hair cortisol levels and for maternal interactive behaviors. No differences were found between the BDP and control groups in locomotive activity, in the frequencies of stressful events experienced and in hair cortisol levels. These findings suggested that the postpartum depression witnessed in the monkeys was not related to external factors other than puerperium period. Interestingly, the BDP monkeys displayed an abnormal maternal relationship consisting of increased infant grooming. Taken together, these findings suggest that the adult female cynomolgus monkeys provide a natural model of behavioral postpartum depression that holds a number of advantages over commonly used rodent systems in PPD modeling. The cynomolgus monkeys have a highly-organized social hierarchy and reproductive characteristics without seasonal restriction--similar to humans--as well as much greater homology to humans than rodents. As such,展开更多
Blastocyst complementation by pluripotent stem cell(PSC)injection is believed to be the most promising method to generate xenogeneic organs.However,ethical issues prevent the study of human chimeras in the late embryo...Blastocyst complementation by pluripotent stem cell(PSC)injection is believed to be the most promising method to generate xenogeneic organs.However,ethical issues prevent the study of human chimeras in the late embryonic stage of development.Primate embryonic stem cells(ESCs),which have similar pluripotency to human ESCs,are a good model for studying interspecies chimerism and organ generation.However,whether primate ESCs can be used in xenogenous grafts remains unclear.In this study,we evaluated the chimeric ability of cynomolgus monkey(Macaca fascicularis)ESCs(cmESCs)in pigs,which are excellent hosts because of their many similarities to humans.We report an optimized culture medium that enhanced the anti-apoptotic ability of cmESCs and improved the development of chimeric embryos,in which domesticated cmESCs(D-ESCs)injected into pig blastocysts differentiated into cells of all three germ layers.In addition,we obtained two neonatal interspecies chimeras,in which we observed tissue-specific D-ESC differentiation.Taken together,the results demonstrate the capability of D-ESCs to integrate and differentiate into functional cells in a porcine model,with a chimeric ratio of 0.001-0.0001 in different neonate tissues.We believe this work will facilitate future developments in xenogeneic organogenesis,bringing us one step closer to producing tissue-specific functional cells and organs in a large animal model through interspecies blastocyst complementation.展开更多
Neural tube defects(NTDs)are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure.Although folate supplementation has been shown to mitigate the incidence of NTDs,some cases,often...Neural tube defects(NTDs)are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure.Although folate supplementation has been shown to mitigate the incidence of NTDs,some cases,often attributable to genetic factors,remain unpreventable.The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation;at present,however,the underlying mechanism remains unclear.Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate.To determine the role of SHROOM3 in early developmental morphogenesis,we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase.Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei.These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins,namely fibrous actin(F-actin),myosin II,and phospho-myosin light chain(PMLC),to the apical side of the neuroepithelial cells.Notably,these defects were not rescued by folate supplementation.RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis.In summary,we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.展开更多
AIM To establish a simplified, reproducible D-galactosamineinduced cynomolgus monkey model of acute liver failure having an appropriate treatment window. METHODS Sixteen cynomolgus monkeys were randomly dividedinto fo...AIM To establish a simplified, reproducible D-galactosamineinduced cynomolgus monkey model of acute liver failure having an appropriate treatment window. METHODS Sixteen cynomolgus monkeys were randomly dividedinto four groups(A, B, C and D) after intracranial pressure(ICP) sensor implantation. D-galactosamine at 0.3, 0.25, 0.20 + 0.05(24 h interval), and 0.20 g/kg body weight, respectively, was injected via the small saphenous vein. Vital signs, ICP, biochemical indices, and inflammatory factors were recorded at 0, 12, 24, 36, 48, 72, 96, and 120 h after D-galactosamine administration. Progression of clinical manifestations, survival times, and results of H&E staining, TUNEL, and Masson staining were recorded. RESULTS Cynomolgus monkeys developed different degrees of debilitation, loss of appetite, and jaundice after D-galactosamine administration. Survival times of groups A, B, and C were 56 ± 8.7 h, 95 ± 5.5 h, and 99 ± 2.2 h, respectively, and in group D all monkeys survived the 144-h observation period except for one, which died at 136 h. Blood levels of ALT, AST, CK, LDH, TBi L, Cr, BUN, and ammonia, prothrombin time, ICP, endotoxin, and inflammatory markers [(tumor necrosis factor(TNF)-α, interleukin(IL)-1β, and IL-6)] significantly increased compared with baseline values in different groups(P < 0.05). Pathological results showed obvious liver cell necrosis that was positively correlated with the dose of D-galactosamine.CONCLUSION We successfully established a simplified, reproducible D-galactosamine-induced cynomolgus monkey model of acute liver failure, and the single or divided dosage of 0.25 g/kg is optimal for creating this model.展开更多
Mutations of PTEN-induced kinase I(PINK1)cause early-onset Parkinson’s disease(PD)with selective neurodegeneration in humans.However,current PINK1 knockout mouse and pig models are unable to recapitulate the typical ...Mutations of PTEN-induced kinase I(PINK1)cause early-onset Parkinson’s disease(PD)with selective neurodegeneration in humans.However,current PINK1 knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients.This suggests that generating PINK1 disease models in non-human primates(NHPs)that are close to humans is essential to investigate the unique function of PINK1 in primate brains.Paired single guide RNA(sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9,both of which can reduce off-target effects without compromising on-target editing,are two optimized strategies in the CRISPR/Cas9 system for establishing disease animal models.Here,we combined the two strategies and injected Cas9-D10A mRNA and two truncated sgRNAs into one-cell-stage cynomolgus zygotes to target the PINK1 gene.We achieved precise and efficient gene editing of the target site in three newborn cynomolgus monkeys.The frame shift mutations of PINK1 in mutant fibroblasts led to a reduction in mRNA.However,western blotting and immunofluorescence staining confirmed the PINK1 protein levels were comparable to that in wild-type fibroblasts.We further reprogramed mutant fibroblasts into induced pluripotent stem cells(iPSCs),which showed similar ability to differentiate into dopamine(DA)neurons.Taken together,our results showed that co-injection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enabled the generation of human disease models in NHPs and target editing by pair truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact protein expression.展开更多
In this study, we developed a systemic PD model in middle-aged cynomolgus monkeys using individualized low-dose MPTP, to explore effective indicators for the early prediction of clinical outcomes. MPTP was not stopped...In this study, we developed a systemic PD model in middle-aged cynomolgus monkeys using individualized low-dose MPTP, to explore effective indicators for the early prediction of clinical outcomes. MPTP was not stopped until the animals showed typical PD motor symptoms on days 10 to 13 after MPTP administration when the Kurlan score reached 10; this abrogated the dif- ferences in individual susceptibility to MPTP. The clinical symptoms persisted, peaking on days 3 to 12 after MPTP withdrawal （rapid progress stage）, and then the Kurlan score plateaued. A Kurlan score at the end of the rapid progress stage 〉15 reflected stable or slowly-progressive PD, while a score 〈15 indicated spontaneous recovery. The entire clinical evolution and outcome of the systemic PD model was characterized in this study, thus providing options for therapeutic and translational research.展开更多
Bone marrow mesenchymal stem cells(BM-MSCs) are a potential tool for cell therapy and tissue engineering.In this study,we carried on a comparative study of the characteristics of MSCs from different age cynomolgus mon...Bone marrow mesenchymal stem cells(BM-MSCs) are a potential tool for cell therapy and tissue engineering.In this study,we carried on a comparative study of the characteristics of MSCs from different age cynomolgus monkeys.A variety of factors,including donor age,must be considered before further applications,and various tests should be used to properly assess MSCs before the clinical application,especially when a prolonged culture and ex vivo expansion is necessary.展开更多
To characterize recombinant AAV2 （rAAV2）-mediated expression of L 132C/T 159C ChR2 mutant in retinal ganglion cells （RGCs） of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 m...To characterize recombinant AAV2 （rAAV2）-mediated expression of L 132C/T 159C ChR2 mutant in retinal ganglion cells （RGCs） of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP （ChR2-GFP） were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing （RBPMS） was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS＋ and GFP＋ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 p.m from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 ~tm from the edge. On average, the ratio of the double-labeled RGCs versus RBPMS＋ RGCs approached 0.324-0.15 （n=14 fields） at the central foveal region （0.1 to 0.53 mm）. We observed that the ratio reached 0.784-0.16 （n=21 fields） at peripheral retinal locations （eccentricity 〉7 mm）. This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.展开更多
Rhesus macaques(Macaca mulatta) and cynomolgus macaques(Macaca fascicularis) are frequently used in establishing animal models for human diseases. To determine the differences in gut microbiota between these species, ...Rhesus macaques(Macaca mulatta) and cynomolgus macaques(Macaca fascicularis) are frequently used in establishing animal models for human diseases. To determine the differences in gut microbiota between these species, rectal swabs from 20 rhesus macaques and 21 cynomolgus macaques were collected, and the microbial composition was examined by deep sequencing of the 16 S rR NA gene. We found that the rectal microbiota of cynomolgus macaques exhibited significantly higher alpha diversity than that of rhesus macaques, although the observed number of operational taxonomic units(OTUs) was almost the same. The dominant taxa at both the phylum and genus levels were similar between the two species, although the relative abundances of these dominant taxa were significantly different between them. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States(PICRUSt) showed significant differences in the functional components between the microbiota of the two species, in particular the lipopolysaccharide(LPS) synthesis proteins. The above data indicated significant differences in microbial composition and function between these two closely related macaque species, which should be taken into consideration in the future selection of these animals for disease models.展开更多
Background : Cynomolgus disease models that are similar to the preclinical stage of human type 2 diabetes mellitus(T2 DM) were established by feeding middle-aged cynomolgus monkeys different high energy diets to study...Background : Cynomolgus disease models that are similar to the preclinical stage of human type 2 diabetes mellitus(T2 DM) were established by feeding middle-aged cynomolgus monkeys different high energy diets to study the differential expression of diabetes-related genes. Methods : A total of 36 male monkeys were randomly divided into four groups and fed human diets with high sugar, high fat, double high sugar and fat, and a normal diet. The preclinical diabetes phase was determined by monitoring the metabolic characteristic indices and the results of oral glucose tolerance tests( OGTT). The mRNA expression of 45 diabetes-related genes in peripheral blood leukocytes was analyzed using real-time PCR. Results : A total of 22, 25, and 21 genes were significantly up-regulated( P < 0.05) and 5, 7, and 5 genes were significantly down-regulated( P < 0.05) in the above three induced groups, respectively, compared with the control group. Of the 45 tested genes, the expression profiles of 21 genes were consistent. Most of the expression levels in the double high sugar-and-fat individuals were slightly lower than those in the high glucose and high fat groups, although the expression patterns of the three groups were essentially similar. Conclusion : The different high energy diets all induced diabetes and shared some phenotypic properties with human T2 DM. Most of the expression patterns of the related genes were identical. The gene expression profiles could be used as references for the study of early diagnostic indicators and T2 DM pathogenesis.展开更多
Juvenile （2-3 years old） cynomolgus monkeys are frequently used as recipients in non-human primate islet transplantation studies. The aim of this study was to examine the effects of different doses of streptozotocin...Juvenile （2-3 years old） cynomolgus monkeys are frequently used as recipients in non-human primate islet transplantation studies. The aim of this study was to examine the effects of different doses of streptozotocin （STZ）, and find the optimal dose for inducing diabetes in these monkeys. Fifteen juvenile （2-3 years old） cynomolgus monkeys were separated into three groups and administered with different doses of STZ （100, 68 or 60 mg kg-l）. Basal and glucose-stimulated blood glucose, in- sulin, and C-peptide levels, as well as body weights were monitored. Hepatic and renal function tests and pancreatic immuno- histochemistry were performed before and after STZ treatment. Monkeys treated with both 100 and 68 mg kg-1 of STZ exhib- ited continuous hyperglycemia, which coincided with a nearly complete loss of islet 13-cells. Two monkeys received 60 mg kg-1 of STZ, but only one became completely diabetic. During the first week following STZ treatment, hepatic and renal func- tion slightly increased in these three groups. However. 24 hours post-STZ, serum total bile acid levels were significantly in- creased in monkeys treated with 100 mg kg-1 than those treated with 68 mg kg-I of STZ （P〈0.05）. These data suggest that 100 mg kg-1 and 68 mg kg-1 of STZ can safely induce diabetes in cynomolgus monkeys aged 2-3 years, but 68 mg kg-1 of STZ, rather than 100 mg kg-1 of STZ, may be more appropriate for inducing diabetes in these monkeys. Furthermore, body surface area, rather than body weight, was a more reliable determinant of dosage, where 700 mg m-2 of STZ should be the lower limit for inducing diabetes in juvenile monkeys.展开更多
Stress from dominance ranks in human societies, or that of other social animals, especially nonhuman primates, can have negative influences on health. Individuals holding different social status may be burdened with v...Stress from dominance ranks in human societies, or that of other social animals, especially nonhuman primates, can have negative influences on health. Individuals holding different social status may be burdened with various stress levels. The middle class experiences a special stress situation within the dominance hierarchy due to its position between the higher and lower classes. Behaviorally, questions about where middle-class stress comes from and how individuals adapt to middle-class stress remain poorly understood in nonhuman primates. In the present study, social interactions, including aggression, avoidance, grooming and mounting behaviors, between beta males, as well as among group members holding higher or lower social status, were analyzed in captive male-only cynomolgus monkey groups. We found that aggressive tension from the higher hierarchy members was the main origin of stress for middle- class individuals. However, behaviors such as attacking lower hierarchy members immediately after being the recipient of aggression, as well as increased avoidance, grooming and mounting toward both higher and lower hierarchy members helped alleviate middle-class stress and were particular adaptations to middle-class social status.展开更多
基金Supported by National High Technology Research and Development Program of China 863 Programs No.2006AA02A141 and No.2012AA020505the Medical Research Fund of Guangdong Province No.2009164
文摘AIM: To evaluate a hybrid bioartificial liver support system (HBALSS) in cynomolgus monkeys with acute liver failure.
基金supported by the National Natural Science Foundation of China(81773711)to W.Y.Strategic Priority Research Program of the Chinese Academy of Sciences(XDB13000000)+6 种基金Lundbeck Foundation Grant(R190-2014-2827)Carlsberg Foundation Grant(CF16-0663)to G.J.Z.Science and Technology Program of Guangzhou,China(201704020103)to W.Y.Introduction of Innovative R&D Team Program of Guangdong Province(2013Y104)Leading Talent Project in Science and Technology of Guangzhou Development District(2019-L002)National Major Scientific and Technological Special Project for “Significant New Drugs Development”(2016ZX09101026)to S.Z.L.Key Projects of the Military Science and Technology PLA(AWS14C007 and AWS16J023)to Y.Q.G
文摘Hypobaric hypoxia (HH) exposure can cause serious brain injury as well as life-threatening cerebral edema in severe cases. Previous studies on the mechanisms of HH-induced brain injury have been conducted primarily using non-primate animal models that are genetically distant to humans, thus hindering the development of disease treatment. Here, we report that cynomolgus monkeys (Macaca fascicularis) exposed to acute HH developed human-like HH syndrome involving severe brain injury and abnormal behavior. Transcriptome profiling of white blood cells and brain tissue from monkeys exposed to increasing altitude revealed the central role of the HIF-1 and other novel signaling pathways, such as the vitamin D receptor (VDR) signaling pathway, in co-regulating HH-induced inflammation processes. We also observed profound transcriptomic alterations in brains after exposure to acute HH, including the activation of angiogenesis and impairment of aerobic respiration and protein folding processes, which likely underlie the pathological effects of HH-induced brain injury. Administration of progesterone (PROG) and steroid neuroprotectant 5α-androst-3β,5,6β-triol (TRIOL) significantly attenuated brain injuries and rescued the transcriptomic changes induced by acute HH. Functional investigation of the affected genes suggested that these two neuroprotectants protect the brain by targeting different pathways, with PROG enhancing erythropoiesis and TRIOL suppressing glutamate-induced excitotoxicity. Thus, this study advances our understanding of the pathology induced by acute HH and provides potential compounds for the development of neuroprotectant drugs for therapeutic treatment.
基金supported by National Natural Science Foundation of China(31271167,81271495,31070963,30921064)the Yunnan Provincial Project to attract ore-hundred exceptional talents from Overseas
文摘Postpartum depression （PPD） is a modified form of major depressive disorders （MDD） that can exert profound negative effects on both mothers and infants than MDD. Within the postpartum period, both mothers and infants are susceptible; but because PPD typically occurs for short durations and has moderate symptoms, there exists challenges in exploring and addressing the underlying cause of the depression. This fact highlights the need for relevant animal models. In the present study, postpartum adult female cynomolgus monkeys （Macaca fascicularis） living in breeding groups were observed for typical depressive behavior. The huddle posture behavior was utilized as an indicator of behavioral depression postpartum （BDP） as it has been established as the core depressive-like behavior in primates. Monkeys were divided into two groups： A t3DP group （n=6）, which were found to spend more time huddling over the first two weeks postpartum than other individuals that formed a non-depression control group （n=4）. The two groups were then further analyzed for locomotive activity, stressful events, hair cortisol levels and for maternal interactive behaviors. No differences were found between the BDP and control groups in locomotive activity, in the frequencies of stressful events experienced and in hair cortisol levels. These findings suggested that the postpartum depression witnessed in the monkeys was not related to external factors other than puerperium period. Interestingly, the BDP monkeys displayed an abnormal maternal relationship consisting of increased infant grooming. Taken together, these findings suggest that the adult female cynomolgus monkeys provide a natural model of behavioral postpartum depression that holds a number of advantages over commonly used rodent systems in PPD modeling. The cynomolgus monkeys have a highly-organized social hierarchy and reproductive characteristics without seasonal restriction--similar to humans--as well as much greater homology to humans than rodents. As such,
基金This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA16030400 to W.L.)the National Key Research and Development Program(2016YFA0100202 and 2017YFA0104401 to T.H.,2018YFA0109701 to R.F.)+1 种基金the National Natural Science Foundation of China(31621004 to Q.Z.and W.L.,31571533 to H.W.,and 31701286 to G.F.)the Key Research Projects of the Frontier Science of the Chinese Academy of Sciences(QYZDY-SSWSMC002 to Q.Z.).
文摘Blastocyst complementation by pluripotent stem cell(PSC)injection is believed to be the most promising method to generate xenogeneic organs.However,ethical issues prevent the study of human chimeras in the late embryonic stage of development.Primate embryonic stem cells(ESCs),which have similar pluripotency to human ESCs,are a good model for studying interspecies chimerism and organ generation.However,whether primate ESCs can be used in xenogenous grafts remains unclear.In this study,we evaluated the chimeric ability of cynomolgus monkey(Macaca fascicularis)ESCs(cmESCs)in pigs,which are excellent hosts because of their many similarities to humans.We report an optimized culture medium that enhanced the anti-apoptotic ability of cmESCs and improved the development of chimeric embryos,in which domesticated cmESCs(D-ESCs)injected into pig blastocysts differentiated into cells of all three germ layers.In addition,we obtained two neonatal interspecies chimeras,in which we observed tissue-specific D-ESC differentiation.Taken together,the results demonstrate the capability of D-ESCs to integrate and differentiate into functional cells in a porcine model,with a chimeric ratio of 0.001-0.0001 in different neonate tissues.We believe this work will facilitate future developments in xenogeneic organogenesis,bringing us one step closer to producing tissue-specific functional cells and organs in a large animal model through interspecies blastocyst complementation.
基金supported by the National Natural Science Foundation of China (81930121,82125008 to Y.C.C.)National Key Research and Development Program of China (2018YFA0107902 to Y.C.C.and 2018YFA0801403 to Z.B.W.)+1 种基金Major Basic Research Project of Science and Technology of Yunnan (202001BC070001 to Y.C.C.)Natural Science Foundation of Yunnan Province (202102AA100053 to Y.C.C.)。
文摘Neural tube defects(NTDs)are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure.Although folate supplementation has been shown to mitigate the incidence of NTDs,some cases,often attributable to genetic factors,remain unpreventable.The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation;at present,however,the underlying mechanism remains unclear.Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate.To determine the role of SHROOM3 in early developmental morphogenesis,we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase.Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei.These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins,namely fibrous actin(F-actin),myosin II,and phospho-myosin light chain(PMLC),to the apical side of the neuroepithelial cells.Notably,these defects were not rescued by folate supplementation.RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis.In summary,we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.
基金Supported by The National Natural Science Foundation of China,No.81470875The Natural Science Foundation of Guangdong Province,China,No.2014A030312013+1 种基金The Science and Technology Planning Project of Guangdong Province,China,No.2014B020227002,No.2015B090903069,and No.2015B020229002The Science and Technology Program of Guangzhou,China,No.201604020002
文摘AIM To establish a simplified, reproducible D-galactosamineinduced cynomolgus monkey model of acute liver failure having an appropriate treatment window. METHODS Sixteen cynomolgus monkeys were randomly dividedinto four groups(A, B, C and D) after intracranial pressure(ICP) sensor implantation. D-galactosamine at 0.3, 0.25, 0.20 + 0.05(24 h interval), and 0.20 g/kg body weight, respectively, was injected via the small saphenous vein. Vital signs, ICP, biochemical indices, and inflammatory factors were recorded at 0, 12, 24, 36, 48, 72, 96, and 120 h after D-galactosamine administration. Progression of clinical manifestations, survival times, and results of H&E staining, TUNEL, and Masson staining were recorded. RESULTS Cynomolgus monkeys developed different degrees of debilitation, loss of appetite, and jaundice after D-galactosamine administration. Survival times of groups A, B, and C were 56 ± 8.7 h, 95 ± 5.5 h, and 99 ± 2.2 h, respectively, and in group D all monkeys survived the 144-h observation period except for one, which died at 136 h. Blood levels of ALT, AST, CK, LDH, TBi L, Cr, BUN, and ammonia, prothrombin time, ICP, endotoxin, and inflammatory markers [(tumor necrosis factor(TNF)-α, interleukin(IL)-1β, and IL-6)] significantly increased compared with baseline values in different groups(P < 0.05). Pathological results showed obvious liver cell necrosis that was positively correlated with the dose of D-galactosamine.CONCLUSION We successfully established a simplified, reproducible D-galactosamine-induced cynomolgus monkey model of acute liver failure, and the single or divided dosage of 0.25 g/kg is optimal for creating this model.
基金supported by the National Key Research and Development Program(2016YFA0101401 and 2018YFA0801400)Major Basic Research Project of Science and Technology of Yunnan(2019FY002 and 202001BC070001)。
文摘Mutations of PTEN-induced kinase I(PINK1)cause early-onset Parkinson’s disease(PD)with selective neurodegeneration in humans.However,current PINK1 knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients.This suggests that generating PINK1 disease models in non-human primates(NHPs)that are close to humans is essential to investigate the unique function of PINK1 in primate brains.Paired single guide RNA(sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9,both of which can reduce off-target effects without compromising on-target editing,are two optimized strategies in the CRISPR/Cas9 system for establishing disease animal models.Here,we combined the two strategies and injected Cas9-D10A mRNA and two truncated sgRNAs into one-cell-stage cynomolgus zygotes to target the PINK1 gene.We achieved precise and efficient gene editing of the target site in three newborn cynomolgus monkeys.The frame shift mutations of PINK1 in mutant fibroblasts led to a reduction in mRNA.However,western blotting and immunofluorescence staining confirmed the PINK1 protein levels were comparable to that in wild-type fibroblasts.We further reprogramed mutant fibroblasts into induced pluripotent stem cells(iPSCs),which showed similar ability to differentiate into dopamine(DA)neurons.Taken together,our results showed that co-injection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enabled the generation of human disease models in NHPs and target editing by pair truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact protein expression.
基金supported by grants from the National High-Tech Development Project of Ministry of Sciences and Technology of China(2012AA020703)the National Natural Science Foundation of China(31472056)+1 种基金Scientific Project of the Science and Technology Department of Guangxi Zhuang Autonomous Region,China(1598025-31)Scientific Project of the Science and Technology Bureau of Nanning Municipality,Guangxi Zhuang Autonomous Region,China(20145194,20155192)
文摘In this study, we developed a systemic PD model in middle-aged cynomolgus monkeys using individualized low-dose MPTP, to explore effective indicators for the early prediction of clinical outcomes. MPTP was not stopped until the animals showed typical PD motor symptoms on days 10 to 13 after MPTP administration when the Kurlan score reached 10; this abrogated the dif- ferences in individual susceptibility to MPTP. The clinical symptoms persisted, peaking on days 3 to 12 after MPTP withdrawal （rapid progress stage）, and then the Kurlan score plateaued. A Kurlan score at the end of the rapid progress stage 〉15 reflected stable or slowly-progressive PD, while a score 〈15 indicated spontaneous recovery. The entire clinical evolution and outcome of the systemic PD model was characterized in this study, thus providing options for therapeutic and translational research.
基金supported in part by the National High Technology Research and Development Program of China (Grant No 2006AA02A114)the Science Project of Beijing Municipal Science Technology Commission (Grant No D07050701350703)
文摘Bone marrow mesenchymal stem cells(BM-MSCs) are a potential tool for cell therapy and tissue engineering.In this study,we carried on a comparative study of the characteristics of MSCs from different age cynomolgus monkeys.A variety of factors,including donor age,must be considered before further applications,and various tests should be used to properly assess MSCs before the clinical application,especially when a prolonged culture and ex vivo expansion is necessary.
基金supported by National Science Foundation of China(31571091 to Mingliang Pu),National Basic Research Program of China (2015CB351806 to Mingliang Pu)National Institutes of Health Grant (NIH) (EY17130 to Zhuo-Hua Pan)Dryer Foundation, the Ligon Research Center of Vision, and Research to Prevent Blindness to Department of Ophthalmology at Wayne State University
文摘To characterize recombinant AAV2 （rAAV2）-mediated expression of L 132C/T 159C ChR2 mutant in retinal ganglion cells （RGCs） of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP （ChR2-GFP） were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing （RBPMS） was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS＋ and GFP＋ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 p.m from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 ~tm from the edge. On average, the ratio of the double-labeled RGCs versus RBPMS＋ RGCs approached 0.324-0.15 （n=14 fields） at the central foveal region （0.1 to 0.53 mm）. We observed that the ratio reached 0.784-0.16 （n=21 fields） at peripheral retinal locations （eccentricity 〉7 mm）. This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.
基金supported by the National Natural Science Foundation of China(81571607)Beijing Natural Science Foundation(7162136)Ministry of Science and Technology(2017ZX10202102003005,2015BAI08B03)of China
文摘Rhesus macaques(Macaca mulatta) and cynomolgus macaques(Macaca fascicularis) are frequently used in establishing animal models for human diseases. To determine the differences in gut microbiota between these species, rectal swabs from 20 rhesus macaques and 21 cynomolgus macaques were collected, and the microbial composition was examined by deep sequencing of the 16 S rR NA gene. We found that the rectal microbiota of cynomolgus macaques exhibited significantly higher alpha diversity than that of rhesus macaques, although the observed number of operational taxonomic units(OTUs) was almost the same. The dominant taxa at both the phylum and genus levels were similar between the two species, although the relative abundances of these dominant taxa were significantly different between them. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States(PICRUSt) showed significant differences in the functional components between the microbiota of the two species, in particular the lipopolysaccharide(LPS) synthesis proteins. The above data indicated significant differences in microbial composition and function between these two closely related macaque species, which should be taken into consideration in the future selection of these animals for disease models.
基金Science and Technology Planning Project of Guangdong Province,Grant/Award Number:2016A030303037 and 2017A070702014Natural Science Foundation of Guangdong Province,(2018A030313307)+1 种基金National Natural Science Foundation of China,Grant/Award Number:81102358GDAS Special Project of Science and Technology Development,Grant/Award Number:2017GDASCX-0107
文摘Background : Cynomolgus disease models that are similar to the preclinical stage of human type 2 diabetes mellitus(T2 DM) were established by feeding middle-aged cynomolgus monkeys different high energy diets to study the differential expression of diabetes-related genes. Methods : A total of 36 male monkeys were randomly divided into four groups and fed human diets with high sugar, high fat, double high sugar and fat, and a normal diet. The preclinical diabetes phase was determined by monitoring the metabolic characteristic indices and the results of oral glucose tolerance tests( OGTT). The mRNA expression of 45 diabetes-related genes in peripheral blood leukocytes was analyzed using real-time PCR. Results : A total of 22, 25, and 21 genes were significantly up-regulated( P < 0.05) and 5, 7, and 5 genes were significantly down-regulated( P < 0.05) in the above three induced groups, respectively, compared with the control group. Of the 45 tested genes, the expression profiles of 21 genes were consistent. Most of the expression levels in the double high sugar-and-fat individuals were slightly lower than those in the high glucose and high fat groups, although the expression patterns of the three groups were essentially similar. Conclusion : The different high energy diets all induced diabetes and shared some phenotypic properties with human T2 DM. Most of the expression patterns of the related genes were identical. The gene expression profiles could be used as references for the study of early diagnostic indicators and T2 DM pathogenesis.
基金supported by the National Basic Research Program of China(Grant No.2007CB947704)the National High Technology Research and Development Program of China(Grant Nos. 2006AA02A112 and 2006AA02A116)High Level Talent Fund of the Beijing Healthcare System(Grant No.2009-2-14)
文摘Juvenile （2-3 years old） cynomolgus monkeys are frequently used as recipients in non-human primate islet transplantation studies. The aim of this study was to examine the effects of different doses of streptozotocin （STZ）, and find the optimal dose for inducing diabetes in these monkeys. Fifteen juvenile （2-3 years old） cynomolgus monkeys were separated into three groups and administered with different doses of STZ （100, 68 or 60 mg kg-l）. Basal and glucose-stimulated blood glucose, in- sulin, and C-peptide levels, as well as body weights were monitored. Hepatic and renal function tests and pancreatic immuno- histochemistry were performed before and after STZ treatment. Monkeys treated with both 100 and 68 mg kg-1 of STZ exhib- ited continuous hyperglycemia, which coincided with a nearly complete loss of islet 13-cells. Two monkeys received 60 mg kg-1 of STZ, but only one became completely diabetic. During the first week following STZ treatment, hepatic and renal func- tion slightly increased in these three groups. However. 24 hours post-STZ, serum total bile acid levels were significantly in- creased in monkeys treated with 100 mg kg-1 than those treated with 68 mg kg-I of STZ （P〈0.05）. These data suggest that 100 mg kg-1 and 68 mg kg-1 of STZ can safely induce diabetes in cynomolgus monkeys aged 2-3 years, but 68 mg kg-1 of STZ, rather than 100 mg kg-1 of STZ, may be more appropriate for inducing diabetes in these monkeys. Furthermore, body surface area, rather than body weight, was a more reliable determinant of dosage, where 700 mg m-2 of STZ should be the lower limit for inducing diabetes in juvenile monkeys.
基金Foundation items： This study was supported by the Ministry of Science and Technology of China Grant （2012CB825500） and the Youth Innovation Promotion Association of Chinese Academy of Sciences （2012075）
文摘Stress from dominance ranks in human societies, or that of other social animals, especially nonhuman primates, can have negative influences on health. Individuals holding different social status may be burdened with various stress levels. The middle class experiences a special stress situation within the dominance hierarchy due to its position between the higher and lower classes. Behaviorally, questions about where middle-class stress comes from and how individuals adapt to middle-class stress remain poorly understood in nonhuman primates. In the present study, social interactions, including aggression, avoidance, grooming and mounting behaviors, between beta males, as well as among group members holding higher or lower social status, were analyzed in captive male-only cynomolgus monkey groups. We found that aggressive tension from the higher hierarchy members was the main origin of stress for middle- class individuals. However, behaviors such as attacking lower hierarchy members immediately after being the recipient of aggression, as well as increased avoidance, grooming and mounting toward both higher and lower hierarchy members helped alleviate middle-class stress and were particular adaptations to middle-class social status.
