Gamma-glutamyl transferase 5 overexpression in cerebrovascular endothelial cells improves brain pathology,cognition,and behavior in APP/PS1 mice

In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of Alzheimer’s disease remains unclear.This study aimed to explore the effect of GGT5 on cognitive function and brain pathology in an APP/PS1 mouse model of Alzheimer’s disease,as well as the underlying mechanism.We observed a significant reduction in GGT5 expression in two in vitro models of Alzheimer’s disease(Aβ_(1-42)-treated hCMEC/D3 and bEnd.3 cells),as well as in the APP/PS1 mouse model.Additionally,injection of APP/PS1 mice with an adeno-associated virus encoding GGT5 enhanced hippocampal synaptic plasticity and mitigated cognitive deficits.Interestingly,increasing GGT5 expression in cerebrovascular endothelial cells reduced levels of both soluble and insoluble amyloid-βin the brains of APP/PS1 mice.This effect may be attributable to inhibition of the expression ofβ-site APP cleaving enzyme 1,which is mediated by nuclear factor-kappa B.Our findings demonstrate that GGT5 expression in cerebrovascular endothelial cells is inversely associated with Alzheimer’s disease pathogenesis,and that GGT5 upregulation mitigates cognitive deficits in APP/PS1 mice.These findings suggest that GGT5 expression in cerebrovascular endothelial cells is a potential therapeutic target and biomarker for Alzheimer’s disease.

Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice

BACKGROUND Atherosclerosis(AS),a chronic inflammatory disease of blood vessels,is a major contributor to cardiovascular disease.Dental pulp stem cells(DPSCs)are capable of exerting immunomodulatory and anti-inflammatory effects by secreting cytokines and exosomes and are widely used to treat autoimmune and inflam-mation-related diseases.Hepatocyte growth factor(HGF)is a pleiotropic cytokine that plays a key role in many inflammatory and autoimmune diseases.AIM To modify DPSCs with HGF(DPSC-HGF)and evaluate the therapeutic effect of DPSC-HGF on AS using an apolipoprotein E-knockout(ApoE-/-)mouse model and an in vitro cellular model.METHODS ApoE-/-mice were fed with a high-fat diet(HFD)for 12 wk and injected with DPSC-HGF or Ad-Null modified DPSCs(DPSC-Null)through tail vein at weeks 4,7,and 11,respectively,and the therapeutic efficacy and mechanisms were analyzed by histopathology,flow cytometry,lipid and glucose measurements,real-time reverse transcription polymerase chain reaction(RT-PCR),and enzyme-linked immunosorbent assay at the different time points of the experiment.An in vitro inflammatory cell model was established by using RAW264.7 cells and human aortic endothelial cells(HAOECs),and indirect co-cultured with supernatant of DPSC-Null(DPSC-Null-CM)or DPSC-HGF-CM,and the effect and mechanisms were analyzed by flow cytometry,RT-PCR and western blot.Nuclear factor-κB(NF-κB)activators and inhibitors were also used to validate the related signaling pathways.RESULTS DPSC-Null and DPSC-HGF treatments decreased the area of atherosclerotic plaques and reduced the expression of inflammatory factors,and the percentage of macrophages in the aorta,and DPSC-HGF treatment had more pronounced effects.DPSCs treatment had no effect on serum lipoprotein levels.The FACS results showed that DPSCs treatment reduced the percentages of monocytes,neutrophils,and M1 macrophages in the peripheral blood and spleen.DPSC-Null-CM and DPSC-HGF-CM reduced adhesion molecule expression in tumor necrosis factor-αstimulated HAOECs and regulated M1 polarization and inflammatory factor expression in lipopolysaccharide-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway.CONCLUSION This study suggested that DPSC-HGF could more effectively ameliorate AS in ApoE-/-mice on a HFD,and could be of greater value in stem cell-based treatments for AS.

Optimal dose of busulfan for depleting testicular germ cells of recipient mice before spermatogonial transplantation

Successful spermatogonial transplantation requires depletion of the host germ cells to allow efficient colonization of the donor spermatogonial stem cells. Although a sterilizing drug,busulfan （Myleran）,is commonly used for preparing a recipient mouse before transplantation,the optimal dose of this drug has not yet been defined.The present study investigated the effects of different doses of busulfan （10-50 mg per kg body weight） on survival rate,testicular mass and histomorphology,and on the haploid spermatids and spermatozoa of male BALB/c mice.The results suggest that a dosage of 30 mg kg^-1 is optimal for the ablative treatment withbusulfan used to prepare the recipient mice. This dose results in an adequate depletion of the host germ cells for colonization of donorderived spermatogonial stem cells and causes the lowest death rate of the animals.

Repair of glutamate-induced excitotoxic neuronal damage mediated by intracerebroventricular transplantation of neural stem cells in adult mice

Objective To investigate a possibility of repairing damaged brain by intracerebroventricular transplantation of neural stem cells （NSCs） in the adult mice subjected to glutamate-induced excitotoxic injury. Methods Mouse NSCs were isolated from the brains of embryos at 15-day postcoitum （dpc）. The expression of nestin, a special antigen for NSC, was detected by immunocytochemistry. Immunofluorescence staining was carried out to observe the survival and location of transplanted NSCs. The animals in the MSG＋NSCs group received intracerebroventricular transplantation of NSCs （approximately 1.0×10^5 cells） separately on day 1 and day 10 after 10-d MSG exposure （4.0 g/kg per day）. The mice in control and MSG groups received intracerebroventricular injection of Dulbecco＇s minimum essential medium （DMEM） instead of NSCs. On day 11 after the last NSC transplantation, the test of Y-maze discrimination learning was performed, and then the histopathology of the animal brains was studied to analyze the MSG-induced functional and morphological changes of brain and the effects of intracerebroventricular transplantation of NSCs on the brain repair. Results The isolated cells were Nestin-positive. The grafted NSCs in the host brain were region-specifically survived at 10-d post-transplantation. Intracerebroventricular transplantation of NSCs obviously facilitated the brain recovery from glutamate-induced behavioral disturbances and histopathological impairs in adult mice. Conclusion Intracerebroventricular transplantation of NSCs may be feasible in repairing diseased or damaged brain tissue.

Antitumor effects of artesunate on human breast carcinoma MCF-7 cells and IGF-IR expression in nude mice xenografts

Purpose： The objective of this study was to investigate the anti-tumor effects and analyze the mechanism of artesunate （ART） action on breast cancer in vivo using tumor transplanted nude mice. Methods： The human breast tumor cell line MCF-7 was transplanted into nude mice, and the animals were treated with various doses of ART alone or in combination with cyclophosphamide （CTX） or normal saline （NS）. The tumor inhibitory effects were observed and compared, and the ultrastructural morphology of the transplanted tumor cells was observed by electron microscopy. The apoptosis rates and cell cycle status were detected by flow cytometry （FCM）. The expression of apoptosis-related proteins p53, Bcl-2, Bax and Caspase-3 were detected by immunohistochemistry and IGF-IR was detected by western blot. The expression correlation for these proteins was also analyzed. Results： The tumor inhibition rates in the low dose ART group, high dose ART group, CTX group and combined drug therapy group were （24.39±10.20）%, （40.24±7.02）%, （57.01±5.84）% and （68.29±5.1）%, respectively. The cell cycle was arrested in phase G0/Gt after treatment with ART. The expression of Bcl-2 was significantly reduced, and the expression levels of Bax and Caspase-3 were significantly increased in the ART group compared to the negative control saline group. There was no significant difference detected in p53 expression. The Bcl-2 level was negatively related to Bax and Caspase-3. The western blotting results showed IGF-IR downregulation. Conclusions： ART inhibits the growth of MCF-7 breast tumor cell xenografts in nude mice. The anti-tumor mechanism of ART for human breast carcinoma in nude mice might be correlated with the alteration of apoptosis related protein expression, which may further induce apoptosis and inhibit cell proliferation.

Functional Mechanism of Resveratrol in Inhabiting Growth of Cells ls174t and Its Mechanism in Subcutaneously Transplanted Tumor of Nude Mice

To explore the functional mechanism of Resveratrol against colon cancer cells ls174t and the growth of colon cancer tissue of tumor-bearing mice, MTT method was used to observe the functions of resveratrol for inhibition against cells ls174t in vitro. Transmission electron microscope was used to observe the cell apoptosis. FCM assay was performed to measure the change of the cell apoptosis rate and of cell cycle. RT-PCR method was used to detect the expressions of bcl-2 and bax mRNA. Western blot method was used to detect the expressions of bcl-2 and bax protein. Ceils ls174t were transplanted subcutaneously to nude mice to observe the effect of resveratrol on the growth of subcutaneously transplanted tumor, RT-PCR method was used to detect the expressions of bcl-2 and bax mRNA in the tumor tissue. Western blot method was used to detect the expressions of bcl-2 and bax protein in the tumor tissue. Resveratrol has an effect of inhibiting proliferation of cells ls174t in vitro（P〈0.01）. It is able to induce the apoptosis of cells ls174t, causing the decrease in the expression of bcl-2 and the increase in the expression of bax. Resveratrol could inhibit the growth of subcutaneously transplanted tumor of nude mice（P〈0.05）, causing the decrease in the expression of bcl-2 and the increase in the expression of bax. Resveratrol can inhibit the growth of cells 174t and the growth of subcutaneously transplanted tumor. The mechanism is possibly related to the induction of the cell apoptosis and the regulation of bcl-2/bax expression.

Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice

AIM: To investigate the correlation between lymphogenous metastasis and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymph nodes. METHODS: Fifty-six inbred 615-mice were equally divided into 2 groups and inoculated with Hca-F and Hca-P cells. Their lymph node metastatic rates were examined. Growth fraction of lymphocytes in host lymph nodes was detected by flow cytometry. The Hca-F and Hca-P cells were cultured with extract of lymph node, liver or spleen. The quantity of MMPs in these supernatants was examined by zymographic analysis. The expression of Fas ligand, PCNA, Bcl-2 protein of Hca-F and Hca-P cells in the mice were examined by immunohistochemistry. The apoptosis signals of macro-phages in lymph nodes were observed with in situ DNA fragmentation. RESULTS: On the 28th day post-inoculation, the lymph node metastatic rate of HcaF was 80%(16/20), whereas that of Hca-P was 25%(5/20). The growth fraction of lymphocytes was as follows: in the Hca-F cells, the proliferating peak of lymphocytes appeared on the 14th day post inoculation and then decreased rapidly, while in HcaP cells, the peak appeared on the 7th day post inoculation and then kept at a high level. With the extract of lymph node, the quantity of the MMP-9 activity increased (P【0.01) and active MMP-9 and MMP-2 were produced by both Hca-F and Hca-P tumor cells, which did not produce MMPs without the extract of lymph node or with the extracts of the liver and spleen. The expression of Fas Ligand of Hca-F cells was stronger than that of Hca-P cells (P 【0.01). The expressions of PCNA and Bcl-2 protein of Hca-F cells in the tumors of inoculated area were the same as that of Hca-P cells. In situ DNA fragmentation showed that the positive signals of macrophages were around Hca-F cells. CONCLUSION: Secretion of MMPs which was associated with metastatic ability of Hca-F and Hca-P tumor cells depends on the environment of lymph nodes. The increased expression of Fas ligand protein of Hca-F tumor cells with high lymphogenous metastatic potential in lymph nodes may help tumor cells escape from being killed by host lymphocytes.

Antitumor Activities and Apoptosis-regulated Mechanisms of Fermented Wheat Germ Extract in the Transplantation Tumor Model of Human HT-29 Cells in Nude Mice

Objective A subcutaneous transplantation tumor model of human HT-29 cells in nude mice was established to evaluate anticarcinogenic activities, and the apoptosis-regulated mechanism effect of aqueous extract of fermented wheat germ with Lactobacillus plantarum dy-1 （LFWGE）. Methods The HT-29 cells were transplanted via subcutaneous injection of 1×10^7cells into the right flank of each nude mouse. Then, nude mice were treated for 30 d with LFWGE （high-dose 2 g/kg/d; low-dose 1 g/kg/d） and for 7 d with 5-fluorouracil （5-FU, 25 mg/kg/d） by gavage and intraperitoneal injection, respectively. An inhibition of tumor growth was observed. Results Tumor volume and weights decreased significantly in both groups of nude mice treated with LFWGE. In addition, the cell apoptosis rate of the LFWGE group （2 g/kg/d, 60.2%＋4.4%; 1 g/kg/d, 58.6%＋6.9%） was significantly higher than that of the control group （11.5%＋1.6%） and 5-FU group （32.1%＋3.5%） as measured by the TUNEL assay. Moreover, the real-time fluorescent quantitative PCR and Western blot method further confirmed these enhancing apoptosis and growth inhibition effects. The involvement of LFWGE in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, Caspase-3, and CyclinD1. Conclusion The results showed that LFWGE could induce subcutaneous transplantation tumor apoptosis in nude mice and could be as a natural nutrient supplements or chemopreventive agent in the treatment of human colon cancer.

Antitumor Activities and Apoptosis-regulated Mechanisms of Fermented Barley Extract in the Transplantation Tumor Model of Human HT-29 Cells in Nude Mice

Objective A subcutaneous transplantation tumor model of human HT-29 cells was established in nude mice to study the anticarcinogenic activities and apoptosis-regulatory mechanistic effect of aqueous extract of fermented barley with Lactobacillus plantarum dy-1 （LFBE）. Methods HT-29 cells were transplanted via subcutaneous injection of 1 × 107cells into the right flank of each nude mouse. Then, nude mice were treated for 30 days with LFBE （high-dose 2 g·kg-1·d-1; low-dose 1 g·kg-1·d-1） and for 7 days with 5-fluorouracil （5-FU, 25 g·kg-1·d-1） by gavage and intraperitoneal injection, respectively. Results Tumor volume and weight decreased significantly in both groups of nude mice treated with LFBE. In addition, the cell apoptosis rate of the LFBE group was significantly higher than that of the control group and 5-FU groups as measured by the TUNEL assay. Moreover, the real-time fluorescent quantitative PCR and Western blot methods further confirmed these apoptosis-enhancing and growth-inhibiting effects. The involvement of LFBE in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, caspase-3, and cyclin D1. Conclusion The results showed that LFBE could induce subcutaneous transplantation tumor apoptosis in nude mice and could be used as a natural nutrient supplement or chemopreventive agent in the treatment of human colon cancer.

Bioassay of Eucalyptus extracts for anticancer activity against Ehrlich ascites carcinoma(eac) cells in Swiss albino mice

Objective:To evaluate the antineoplastic activity of Eucalyptus extract(EUE) against Ehrlich ascites carcinoma(EAC)in Swiss albino mice.Methods:Preliminary examination of four plant extracts(namely Eucalyptus,Costus,Azadirachla.Feroniai has been done by observing the reduction ability of number of EAC cells in previously inoculated Swiss alliino mice.Among them as EuE showed maximum capability,the whole study has been conducted with EuE only. Important parameters viz.enhancement of life span,reduction of average tumor weight etc.have been studied.In addition the effects of EuE on hematological parameters in both normal and EAC inoculated mice have been measured.Effect of EuE on normal peritoneal cells has also been studied.Results:EuE reduced tumor burden remarkably.It reduced the tumor growth rate and enhanced the life span of EAC bearing mice noticeably.It reversed back the hematological parameters towards normal,reduced the Irasplanlability of EAC cells and enhanced the immunomodulatory effects in mice.The host toxic effect of EuE in mice is minimum and mostly reversible with time.All such data have been compared with those obtained by running parallel experiments with bleomycin at dose 0.3 mg/kg(i.p.).Conclusions:The Eucalyptus extract may be considered as a potent anticancer agent for advanced researches.

Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells

BACKGROUND Liver fibrosis is the common pathological process associated with the occurrence and development of various chronic liver diseases.At present,there is still a lack of effective prevention and treatment methods in clinical practice.Hepatic stellate cell(HSC)plays a key role in liver fibrogenesis.In recent years,the study of liver fibrosis targeting HSC autophagy has become a hot spot in this research field.Angiotensin-converting enzyme 2(ACE2)is a key negative regulator of reninangiotensin system,and its specific molecular mechanism on autophagy and liver fibrosis needs to be further explored.AIM To investigate the effect of ACE2 on hepatic fibrosis in mice by regulating HSC autophagy through the Adenosine monophosphate activates protein kinases(AMPK)/mammalian target of rapamycin(mTOR)pathway.METHODS Overexpression of ACE2 in a mouse liver fibrosis model was induced by injection of liver-specific recombinant adeno-associated virus ACE2 vector(rAAV2/8-ACE2).The degree of liver fibrosis was assessed by histopathological staining and the biomarkers in mouse serum were measured by Luminex multifactor analysis.The number of apoptotic HSCs was assessed by terminal deoxynucleoitidyl transferase-mediated dUTP nick-end labeling(TUNEL)and immunofluorescence staining.Transmission electron microscopy was used to identify the changes in the number of HSC autophagosomes.The effect of ACE2 overexpression on Wu Y et al.ACE2 improves liver fibrosis through autophagy WJG https://www.wjgnet.com 4976 September 7,2023 Volume 29 Issue 33 autophagy-related proteins was evaluated by multicolor immunofluorescence staining.The expression of autophagy-related indicators and AMPK pathway-related proteins was measured by western blotting.RESULTS A mouse model of liver fibrosis was successfully established after 8 wk of intraperitoneal injection of carbon tetrachloride(CCl4).rAAV2/8-ACE2 administration reduced collagen deposition and alleviated the degree of liver fibrosis in mice.The serum levels of platelet-derived growth factor,angiopoietin-2,vascular endothelial growth factor and angiotensin II were decreased,while the levels of interleukin(IL)-10 and angiotensin-(1-7)were increased in the rAAV2/8-ACE2 group.In addition,the expression of alpha-smooth muscle actin,fibronectin,and CD31 was down-regulated in the rAAV2/8-ACE2 group.TUNEL and immunofluorescence staining showed that rAAV2/8-ACE2 injection increased HSC apoptosis.Moreover,rAAV2/8-ACE2 injection notably decreased the number of autophagosomes and the expression of autophagy-related proteins(LC3I,LC3II,Beclin-1),and affected the expression of AMPK pathway-related proteins(AMPK,p-AMPK,p-mTOR).CONCLUSION ACE2 overexpression can inhibit HSC activation and promote cell apoptosis by regulating HSC autophagy through the AMPK/mTOR pathway,thereby alleviating liver fibrosis and hepatic sinusoidal remodeling.

Mesenchymal stromal cells modulate unfolded protein response and preserve β-cell mass in type 1 diabetes

Introduction:Transplantation of mesenchymal stromal cells(MSCs)is a promising therapy for type 1 diabetes(T1D).However,whether the infused MSCs affect the endoplasmic reticulum stress or subsequent unfolded protein response inβcells remains unclear.Methods:To investigate this,we induced early-onset T1D in non-obese diabetic mice using streptozotocin.Subsequently,T1D mice were randomly assigned to receive either MSCs or phosphate-buffered saline.We observed the in vivo homing of MSCs and assessed their effectiveness by analyzing blood glucose levels,body weight,histopathology,pancreatic protein expression,and serum levels of cytokines,proinsulin,and C-peptide.Results:Infused MSCs were found in the lungs,liver,spleen,and pancreas of T1D mice.They exhibited various effects,including reducing blood glucose levels,regulating immunity,inhibiting inflammation,increasingβ-cell areas,and reducing the expression of key proteins in the unfolded protein response pathway.Fasting serum proinsulin and C-peptide levels were significantly higher in the MSCs treatment group than in the T1D model group.However,there was no significant difference in the biomarker ofβ-cell endoplasmic reticulum stress,the ratio of fasting serum proinsulin to C-peptide,between the two groups.Conclusion:Ourfindings reveal that MSCs infusion does not alleviate endoplasmic reticulum stress inβcells directly but modulates the unfolded protein response pathway to preserveβ-cell mass and function in T1D mice.

No Evidence of Effect on Male Mice Germ Cells After Acute Treatment With Thiram

Thiram is a dithiocarbamate compound widely used for industrial processes and agriculture. Animal studies reveal that this compound may afftct the male reproductive system. Aim of this study was to test, using sensitive testicular parameters, whether thiram directly affects germinal cells. For this purpose, B6C3F1 mice were intraperitoneally injected with thiram in oil (single dose:75 mg/kg; repeated five daily doses: 25 mg/kg).Although both treatments were toxic, none of the parameters examined, i.e., testis weighi, spermatid head number,specific enzyme levels at different times after treatment (14, 28, 35, 56 days) showed significant variations from the controls, On the contrary, in the positive controls (treated with chlorambucil), a marked reduction of sperm head number as well as a deerease of lactate dehydrogenasex and sorbitol dehydrogenase activity letels were evidenced at day 28, with a tendency to recover at day 35. Under these conditions thiram did not cause cytotoxicity on differentiating spermatogonia and on late spermatocyte stages of mice gonads

The recruitment of exogenous endothelial progenitor cells in lung tumor model of nude mice

Background and Objective: Endothelial progenitor cells (EPCs) play an important role in hypoxia-triggered tumor vasculogenesis. However, the homing of exogenous EPCs in tumors is still unclear. In this study, we investigated the recruitment of exogenous EPCs in human lung adenocarcinoma model of nude mice. Methods: EPCs labeled with green fluorescence protein (GFP) were transplanted into nude mice bearing human lung adenocarcinoma. The growth of tumor was observed. After the mice were killed, GFP-EPCs in different tissues were examined by fluorescence. The tumor tissues were stained for CD133, hypoxia-inducible factor-1alpha (HIF-1α), stromal cell-derived factor-1α (SDF-1α), and vascular endothelial growth factor receptor (KDR). Real-time polymerase chain reaction of CD133, HIF-1α, SDF-1α, and VEGF-1 were also performed. Results: The growth of tumor in EPC group was significantly faster than that in saline solution group (P < 0.05). Under fluorescence microscope, GFP-EPCs were strongly expressed in both tumor and bone marrow. EPCs were recruited to the tumor periphery to participate in tumor vasculogenesis. The expression of CD133, HIF-1α, and SDF-1 mRNA in tumor and bone marrow were significantly higher than that in the liver, spleen, and skin (P < 0.05). Conclusions: Exogenous EPCs can be recruited to tumor and accelerate tumor growth. Except tumor, bone marrow can also recruit EPCs.

Differentiation of Embryonic Stem Cells into Neurons and Retina-like Structure in Nude Mice

Purpose:To investigate the intraocular growth and biological characteristics of mice embryonic stem cells in nude mice.Methods:Murine embryonic stem cells(D3 cell line)were cultured and maintained in an undifferentiated state in vitro,then transplanted into the anterior chamber of nude mice.Mophological and immunohistochemical examinations were implemented.Results:Two to three days after transplantation,yellow-white floating granules,sheets and masses were seen inside the anterior chamber and vitreous cavity,and enlarged gradually,14-20days later,the mice were executed.Morphological examination showed that there were undifferentiated cells and some round or polygonal differentiated cells in anterior chamber and vitreous cavity.The morphology of these differentiated cells were similar to that of the retina.The cells were highly positive in NSE staining.Conclusion:The tranplanted embryonic stem cells cold grow in the eyes of nude mice with tendency to differentiate into neurons and retina-like structure.

Effects of P301L-TAU on post-translational modifications of microtubules in human iPSC-derived cortical neurons and TAU transgenic mice

TAU is a microtubule-associated protein that promotes microtubule assembly and stability in the axon.TAU is missorted and aggregated in an array of diseases known as tauopathies.Microtubules are essential for neuronal function and regulated via a complex set of post-translational modifications,changes of which affect microtubule stability and dynamics,microtubule interaction with other proteins and cellular structures,and mediate recruitment of microtubule-severing enzymes.As impairment of microtubule dynamics causes neuronal dysfunction,we hypothesize cognitive impairment in human disease to be impacted by impairment of microtubule dynamics.We therefore aimed to study the effects of a disease-causing mutation of TAU(P301L)on the levels and localization of microtubule post-translational modifications indicative of microtubule stability and dynamics,to assess whether P301L-TAU causes stability-changing modifications to microtubules.To investigate TAU localization,phosphorylation,and effects on tubulin post-translational modifications,we expressed wild-type or P301L-TAU in human MAPT-KO induced pluripotent stem cell-derived neurons(i Neurons)and studied TAU in neurons in the hippocampus of mice transgenic for human P301L-TAU(p R5 mice).Human neurons expressing the longest TAU isoform(2N4R)with the P301L mutation showed increased TAU phosphorylation at the AT8,but not the p-Ser-262 epitope,and increased polyglutamylation and acetylation of microtubules compared with endogenous TAU-expressing neurons.P301L-TAU showed pronounced somatodendritic presence,but also successful axonal enrichment and a similar axodendritic distribution comparable to exogenously expressed 2N4R-wildtype-TAU.P301L-TAU-expressing hippocampal neurons in transgenic mice showed prominent missorting and tauopathy-typical AT8-phosphorylation of TAU and increased polyglutamylation,but reduced acetylation,of microtubules compared with non-transgenic littermates.In sum,P301L-TAU results in changes in microtubule PTMs,suggestive of impairment of microtubule stability.This is accompanied by missorting and aggregation of TAU in mice but not in i Neurons.Microtubule PTMs/impairment may be of key importance in tauopathies.

Investigating Müller glia reprogramming in mice: a retrospective of the last decade, and a look to the future

Müller glia,as prominent glial cells within the retina,plays a significant role in maintaining retinal homeostasis in both healthy and diseased states.In lower vertebrates like zebrafish,these cells assume responsibility for spontaneous retinal regeneration,wherein endogenous Müller glia undergo proliferation,transform into Müller glia-derived progenitor cells,and subsequently regenerate the entire retina with restored functionality.Conversely,Müller glia in the mouse and human retina exhibit limited neural reprogramming.Müller glia reprogramming is thus a promising strategy for treating neurodegenerative ocular disorders.Müller glia reprogramming in mice has been accomplished with remarkable success,through various technologies.Advancements in molecular,genetic,epigenetic,morphological,and physiological evaluations have made it easier to document and investigate the Müller glia programming process in mice.Nevertheless,there remain issues that hinder improving reprogramming efficiency and maturity.Thus,understanding the reprogramming mechanism is crucial toward exploring factors that will improve Müller glia reprogramming efficiency,and for developing novel Müller glia reprogramming strategies.This review describes recent progress in relatively successful Müller glia reprogramming strategies.It also provides a basis for developing new Müller glia reprogramming strategies in mice,including epigenetic remodeling,metabolic modulation,immune regulation,chemical small-molecules regulation,extracellular matrix remodeling,and cell-cell fusion,to achieve Müller glia reprogramming in mice.

Efficient production of chimeric mice from embryonic stem cells injected into 4-to 8-cell and blastocyst embryos

Background： Production of chimeric mice is a useful tool for the elucidation of gene function. After successful isolation of embryonic stem （ES） cell lines, there are many methods for producing chimeras, including co-culture with the embryos, microinjection of the ES ceils into pre-implantation embryos, and use of tetraploid embryos to generate the full ES-derived transgenic mice. Here, we aimed to generate the transgenic ES cell line, compare the production efficiency of chimeric mice and its proportion to yield the male chimeric mice by microinjected ES cells into 4- to 8-cell and blastocysts embryos with the application of Piezo-Micromanipulator （PMM）, and trace the fate of the injected ES cells. Results： We successfully generated a transgenic ES cell line and proved that this cell line still maintained pluripotency. Although we achieved a satisfactory chimeric mice rate, there was no significant difference in the production of chimeric mice using the two different methods, but the proportion of the male chimeric mice in the 4- to 8-cell group was higher than in the blastocyst group. We also found that there was no tendency for ES cells to aggregate into the inner cell mass using in vitro culture of the chimeric embryos, indicating that they aggregated randomly. Conclusions： These results showed that the PMM method is a convenient way to generate chimeric mice and microinjection of ES cells into 4- to 8-cell embryos can increase the chance of yielding male chimeras compared to the blastocyst injection. These results provide useful data in transgenic research mediated by ES cells.

RESEARCHES ON THE ANTI-AUTOLOGOUS TUMOR EFFECTS OF HUMAN CANCER-KILLING CELLS IN NUDE MICE

An investigation of human splenic LAK cells killing autologous tumor in vivo has been performed in this study. Briefly, an ovary embryo carcinoma (OEC) removed surgically from patient was transplanted Into nude mice (s. c. ) and cultured in vitro, that would be used as targets. Lymphokine-activated killer (LAK) cells were generated from splenic lymphocytes of the OEC patient, who died two and half months after operation, by co- culture with recombinant human interleukin- 2 ( rIL- 2 ) in vitro. The results from Winn' s test in nude mice suggested that these LAK cells could effectively inhibit the tumorlgenicity of autologous tumor m vitro.

The Origin of Neointimal Smooth Muscle Cells in Transplant Arteriosclerosis from Recipient Bone-marrow Cells in Rat Aortic Allograft

In order to investigate the origin of neointimal smooth muscle cells in transplant arterio- sclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD-female Wistar aortic allografts, male SD-male Wis- tar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohisto- chemistry. The results indicated that excessive accumulation of α-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts. Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were sig- nificantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P<0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a dis- tinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic al- lograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.