No apparent transmission of transgenic α–synuclein into nigrostriatal dopaminergic neurons in multiple mouse models

Background:α–synuclein(α–syn)is the main component of intracytoplasmic inclusions deposited in the brains of patients with Parkinson’s disease(PD)and certain other neurodegenerative disorders.Recent studies have explored the ability ofα–syn to propagate between or across neighboring neurons and supposedly“infect”them with a prion–like mechanism.However,much of this research has used stereotaxic injections of heterologousα–syn fibrils to induce the spreading of inclusions in the rodent brains.Whetherα–syn is able to transmit from the host cells to their neighboring cells in vivo is unclear.Methods:Using immunestaining,we examined the potential propagation ofα–syn into nigrostriatal dopaminergic(DA)neurons in three lines of transgenic mice that overexpress human wild–typeα–syn(hα–syn)in different neuron populations.Results:After testing for three different routes by which hα–syn propagation might occur,we were unable to find any evidence that hα–syn behaved like a prion and could be transmitted overtime into the DA neurons initially lack of hα–syn expression.Conclusions:In transgenic mice hα–syn does not have the ability to propagate at pathologically significant levels between or across neurons.It must be noted that these observations do not disprove the studies that show its prion–like qualities,but rather that propagation is not detectable in transgenic models that do not use any injections of heterologous proteins or viral vectors to induce a spreading state.

Protective effect of capsaicin against methyl methanesulphonate induced toxicity in the third instar larvae of transgenic Drosophila melanogaster(hsp70-lacZ)Bg9

Capsaicin(trans-8-methyl-N-vanillyl-6-nonenamide) is the main component in hot peppers, including red chili peppers, alapenos, and habanero, belonging to the genus Capsicum. Capsaicin is a potent antioxidant that interferes with free radical activities. In the present study, the possible protective effect of capsaicin was studied against methyl methanesulphonate(MMS) induced toxicity in third instar larvae of transgenic Drosophila melanogaster(hsp70-lacZ)Bg^9. The third instar was allowed to feed on the diet having different doses of capsaicin and MMS separately and in combination. The results suggested that the exposure of third instar larvae to the diet having MMS alone showed significant hsp70 expression as well as tissue DNA and oxidative damage, whereas the larvae feed on the diet having MMS and capsaicin showed a decrease in the toxic effects for 48-h of exposure. In conclusion, capsaicin showed a dose-dependent decrease in the toxic effects induced by MMS in the third instar larvae of transgenic Drosophila melanogaster.

Role of Ocimum sanctum leaf extract on dietary supplementation in the transgenic Drosophila model of Parkinson's disease

AIM: To evaluate the effect of Ocimum sanctum leaf extract on the dietary supplementation in the transgenic Drosophila model of Parkinson's disease. METHOD: The effect of Ocimum sanctum leaf extract was studied on the transgenic Drosophila model of flies expressing normal human alpha synuclein(h-αs) in the neurons. O. sanctum extract at final concentrations of 0.042 8 × 10-4, 0.87 × 10-4, and 1.85 × 10-4 g·mL-1 of diet were established and the flies were allowed to feed for 21 days. The climbing assay and lipid peroxidation were taken as parameters for the study. RESULTS: The supplementation of O. sanctum extract showed a dose-dependent significant delay in the loss of climbing ability and reduction in oxidative stress in the brain of PD model flies. CONCLUSION: The results of the present study showed that the O. sanctum extract is potent in reducing the PD symptoms in transgenic Drosophila model.

See the color,see the seed:GmW1 as a visual reporter for transgene and genome editing in soybean

A fast and efficient recognition method of transgenic lines will greatly improve detection efficiency and reduce cost.In this study,we successfully identified the transgenic soybean plants by the color.We isolated a GmW1 gene encoding a flavonoid 3'5'-hydroxylase from a soybean cultivar ZH42(purple flower).We found that purple flowers occurred in the overexpression lines in the Jack and Williams 82 backgrounds(white flower).All plants with purple flowers were positive,and this trait seems stably inherited in the offspring.We have also obtained the editing plants,which were classified into three types according to the different flower colors appeared.We analyzed the phenotype and the homozygous types of the T_1mutants.We also found that a correspondence between flower color and stem color.This study provides a visible color reporter on soybean transformation.It can be quickly and early to identify the transgenic soybean plants by stem color of seedlings,which substantially reduces the amount of labor and cost.

The sound of movement,a noninvasive surgical procedure for treating Parkinson’s disease

Parkinson’s disease(PD)is the second most common degenerative neurological disorder after Alzheimer’s disease.As one of fastest growing neurological conditions,PD affects millions of elderly people worldwide.PD patients display progressive motor symptoms,including resting tremor,slowed movement,impaired posture and balance,and rigid muscles[1].Additionally,they also often suffer from chronic pain,depression,dementia,and other non-motor symptoms[2].Medications and surgery can improve patient’s motor performance to some degree,while the treatment for non-motor conditions is limited.Moreover,long-term medication can cause severe side effects,such as dyskinesia and impulse control disorders[3,4].Therefore,new mechanistic insights and therapeutic agents/procedures are still needed to improve the treatment of increasing number of PD patients.

DNA重组酶Cre介导载体间基因的重组转移

DNA重组酶Cre可以识别LoxP位点,使含有LoxP位点的DNA分子发生重组:2个同向LoxP之间的DNA片段被删除,2个环状DNA分子被整合为一个大分子.基于Cre酶的这些作用特性,构建了一套载体间基因的重组转移体系,在Cre酶的作用下,gfp基因被从基因供体pTLG上切除下来,然后转移到基因受体pET-LoxP上,从而快速、简便地完成了gfp基因高效表达载体pET-gfp的构建.gfp基因在大肠杆菌BL21(DE3)中被诱导表达,使菌落产生了可视的绿色荧光.通过对荧光菌落的计数分析,比较了环状基因供体pTLG和线性基因供体pTLG对有效重组率的影响.使繁琐的传统载体构建变为简单的酶促反应,极大地简化了载体构建步骤,为Cre酶在基因克隆和亚克隆中的应用提供了很好的研究基础.

Development and application of plant transformation techniques

Genetic transformation is a powerful biotechnology for introducing novel genes into economically important plants from distantly-related plants or even unrelated species such as microbes and animals.This feat is impossible to be achieved by conventional breeding techniques.Development of transgenic plants has been a controversial subject since 1971 when the first genetically modified organism（GMO）was developed（James and Krattiger1996）.

RNAi-Mediated Silencing of ITPK Gene Reduces Phytic Acid Content,Alters Transcripts of Phytic Acid Biosynthetic Genes,and Modulates Mineral Distribution in Rice Seeds

Phytic acid is the principal storage form of phosphorus in plant seeds and an essential signalling molecule in several regulatory processes of plant development.However,it is known as an anti-nutrient compound owing to its potent chelating property.Thus,reducing the phytic acid content in crops is desirable.Studies involving regulation of MIPS and IPK1 genes to generate low phytate rice have been reported earlier.However,the functional significance of OsITPK and the effect of its down-regulation on phytic acid content and the associated pleiotropic effects on rice have not yet been investigated.In this study,tissue specific RNA interference(RNAi)-mediated down-regulation of a major ITPK homolog(OsITP5/6K-1)resulted in 46.2%decrease in phytic acid content of T2 transgenic seeds with a subsequent 3-fold enhancement in the inorganic phosphorus content.Silencing of OsITP5/6K-1 altered the transcript levels of essential phytic acid pathway genes,without significantly affecting the transcript levels of other OsITPK homologs.Furthermore,the mapping of elements through X-ray microfluorescence analysis revealed significant changes in the spatial distribution pattern and translocation of elements in low phytate seeds.Additionally,low phytate polished seeds exhibited 1.3-fold and 1.6-fold enhancement in iron and zinc content in the grain endosperm,respectively.Silencing of OsITP5/6K-1 also altered the amino acid and myo-inositol content of the transgenic seeds.Our results successfully established that RNAi-mediated silencing of OsITP5/6K-1 gene significantly reduced the phytate levels in seeds without hampering the germination potential of seeds and plant growth.The present study provided an insight into the mechanism of phytic acid biosynthesis pathway.

Soybean hairy roots produced in vitro by Agrobacterium rhizogenes-mediated transformation

Soybean is one of the world's most important oil and protein crops. Efficient transformation is a key factor for the improvement of soybean by genetic modification. We describe an optimized protocol for the Agrobacterium rhizogenes-mediated transformation of soybean and the induction of hairy root development in vitro. Cotyledons with 0.5-cm hypocotyls were cut from 5-day-old seedlings and used as explants. After infection and co-cultivation,hairy roots were produced in induction culture medium after 10–12 days. Using this method, 90%–99% of the infected explants of five different cultivars produced hairy roots within one month. Observations using reporter constructs showed that 30%–60% of the hairy roots induced were transformed. Based on high transformation efficiency and short transformation period, this method represents an efficient and rapid platform for study of soybean gene function.

调节基于老鼠样品改进人的间充质的干细胞的扩大

AIM: To improve the isolation and expansion of human marrow-derived mesenchymal stem cells (MSCs) based on rat samples. METHODS: Based on the fact that rat MSCs are relatively easy to obtain from a small aspirate, bone marrow-derived MSCs from rat were cultured and characterized to set up the different protocols used in this study. Then, accordingly, almost the same protocols were performed on human healthy bone marrow samples, after obtaining approval of the ethics committee and gaining informed consent. We used different protocols and culture conditions, including the type of basal media and the culture composition. The MSCs were characterized by immunophenotyping and differentiation. RESULTS: There was no difference in morphology and proliferation capacity between different culture media at the first passage. During the 5-7th passages, the cells gradually lost their morphology and proliferation potential on Dulbecco’s modified Eagle’s medium (DMEM) high glucose and α modified Eagle’s medium. Although the cells expanded rapidly for up to 10 passages on DMEM low glucose containing 10% to 15% fetal calf serum (FCS), their proliferation was arrested without change in morphology and differentiation capacity at the third passage on 5% FCS. Flow cytometric analysis and functional tests confirmed that more than 90% of marrow cells which were isolated and expanded by our selective protocols were MSCs. CONCLUSION: We improved the isolation and expansion of human bone marrow derived MSCs, based on rat sample experiments, for further experimental and clinical use.

Therapeutic effect of intravesical or intratumoral injection of oncolysate transfected with recombinant vaccinia virus encoding human IL-2 gene on murine melanoma model

Oncolysate, a debris of tumor cells, has been provento be effective in tumor active immunotherapy, it wasreported that the vaccinia virus, especially recombinantvaccinia viruses encoding human IL-2 (rVV-IL-2 ),enhanced the immunogenicity of transfected tumor cells.In this experiment, the murine melanoma cell B16-F10oncolysates trans fected by rVV-IL-2 (IL-2VBO) wereused as vaccine. The IL-2VBO or TK-VBO was preparedby incubating B16-F10 cells with rVV-IL-2 or rVV-TK

Effects of 5-aza-2’-deoxyctidine on the development of porcine parthenogenetic and nuclear transfer embryos

The current study was to investigate whether embryo or fetal fibroblast cells treated with 5-aza-2’-deoxyctidine (5-aza-dC) have a positive effect on the in vitro development of porcine parthenogenetic and cloned embryos. To this end, porcine fetal fibroblast cells were treated with different concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours), the results showed that DNA methylation in PRE-1 SINE region was gradually reduced over time in cells treated with 5-aza-dC. To determine the effect of 5-aza-dC on in vitro development of porcine activated oocytes, the parthenogenetic embryo was treated with 5-aza- dC. Notably, treatment with 5 nM 5-aza-dC for 1 hour led to a significant improvement in blastocyst development, compared with the control group. The effects of donor cell treatment with 5-aza-dC on porcine cloned embryos development were further examined by treating fetal fibroblast cells with various concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours). Exposure of cells in 5 nM 5-aza-dC for 1 - 20 hours led to a significant improvement in the percentage of developed blastocysts, while treatment with 500 nM 5-aza-dC did not affect blastocyst development, compared to untreated controls. These findings indicate that treatment of fetal fibro-blast cells with relatively low concentrations of 5-aza-dC for short exposure times improves subsequent blastocyst development of porcine cloned embryos.

Enhanced bio-catalytic and tolerance properties of an indigenous cellulase through xerogel immobilization

Today, demand exists for cost-effective production of industrially important enzymes from entire scientific sectors. By keeping in mind the extensive industrial applications of cellulase, this study was performed to immobilize the indigenous enzyme produced from Trichoderma viride under pre-optimized SSF of an agricultural waste material, wheat straw. To enhance the bio-catalytic and tolerance properties of the present enzyme gel matrix immobilization engineering was applied. Previously, 2.33~fold purified novel cellulase was immobilized in to a xerogel matrix of TMOS and PTMS. FTIR spectroscopy confirmed the successful immobilization of cellulase. The free and immobilized cellulase was characterized and stability profile showed that after 24 h incubation, immobilization enhanced the thermo-stability up to 75% against 80℃ as compare to the free enzyme. Xerogel matrix immobilization enhanced the catalytic efficiency of entrapped enzyme than that of the free cellulase. Among activators/inhibitors SDS, EDTA, and Hg2+ showed inhibitory effect while, gel matrix immobilization enhanced 80% tolerance capacity of the cellulase against inactivating agents.

In vivo gene therapy of murine melanoma mediated by recombinant vaccinia virus encoding human IL-2 gene

Direct gene transfer into somatic tissue in vivo is adeveloping technology with potential application forcancer gene therapy. Retrovirus vector, which was aneffective vehicle, still has some disadvantages ingenerating high titer recombinant vectors andmanipulating to mediate in viro gene transfer. In thispaper, recombinant vaccinia virus vector encoding

Impacts of prenatal nutrition on animal production and performance： a focus on growth and metabolic and endocrine function in sheep

The concept of foetal programming（FP） originated from human epidemiological studies, where foetal life nutrition was linked to health and disease status later in life. Since the proposal of this phenomenon, it has been evaluated in various animal models to gain further insights into the mechanisms underlying the foetal origins of health and disease in humans. In FP research, the sheep has been quite extensively used as a model for humans. In this paper we will review findings mainly from our Copenhagen sheep model, on the implications of late gestation malnutrition for growth, development, and metabolic and endocrine functions later in life, and discuss how these implications may depend on the diet fed to the animal in early postnatal life. Our results have indicated that negative implications of foetal malnutrition, both as a result of overnutrition and, particularly, late gestation undernutrition, can impair a wide range of endocrine functions regulating growth and presumably also reproductive traits. These implications are not readily observable early in postnatal life, but are increasingly manifested as the animal approaches adulthood. No intervention or cure is known that can reverse this programming in postnatal life. Our findings suggest that close to normal growth and slaughter results can be obtained at least until puberty in animals which have undergone adverse programming in foetal life, but manifestation of programming effects becomes increasingly evident in adult animals.Due to the risk of transfer of the adverse programming effects to future generations, it is therefore recommended that animals that are suspected to have undergone adverse FP are not used for reproduction. Unfortunately, no reliable biomarkers have as yet been identified that allow accurate identification of adversely programmed offspring at birth,except for very low or high birth weights, and, in pigs, characteristic changes in head shape（dolphin head）. Future efforts should be therefore dedicated to identify reliable biomarkers and evaluate their effectiveness for alleviation/reversal of the adverse programming in postnatal life. Our sheep studies have shown that the adverse impacts of an extreme, high-fat diet in early postnatal life, but not prenatal undernutrition, can be largely reversed by dietary correction later in life. Thus, birth（at term） appears to be a critical set point for permanent programming in animals born precocial,such as sheep. Appropriate attention to the nutrition of the late pregnant dam should therefore be a priority in animal production systems.

Dissecting pathophysiology of a human dominantly inherited disease, familial amyloidotic polyneuropathy, by using genetically engineered mice

Familial amyloidotic polyneuropathy(FAP)is a type of systemic amyloidosis characterized by peripheral and autonomic neuropathy.Although FAP is a typical autosomal dominant disorder caused by a point mutation in the TTR gene,the average age at onset varies significantly among different countries.This discrepancy clearly suggests that a combination of intrinsic factors as well as extrinsic(environmental)factors shapes the development of FAP.However,these factors are difficult to analyze in humans,because detailed pathologic tissue analysis is only possible at autopsy.Thus,mouse models have been produced and used to disentangle these factors.This review covers the mouse models produced thus far and how these models are applied to analyze intrinsic and extrinsic factors involved in disease development and to test drug efficacy.

Elevated methionine content in soybean seed by overexpressing maizeβ-zein protein

Soybean provides superior and readily available protein for human and livestock.However,nutritional value of soybean is limited due to the deficiency of an essential amino acid,methionine.To improve total methionine content of soybean,a methionine-rich seed storage protein,β-zein,was introduced into soybean cultivar’Jack’under the control of legumin B4 promoter or Ca MV 35S promoter.Totally 4 T3transgenic lines exhibited higher expression levels of foreign genes,and legumin B4 promoter directed a stronger accumulation ofβ-zein protein than Ca MV 35S promoter.Compared to wild type plant,total methionine content in transgenic soybean seeds significantly increased by up to approximately 15%.Although the introduction ofβ-zein gene improved total methionine content,the level was negligible compared to native soybean storage proteins,implying that the inadequate soluble methionine is the limiting factor.Based on these observations,a new strategy for simultaneously increasing the"source"and"sink"of methionine metabolism is proposed to further improvement of total methionine content in soybean seed.

Fertility Reduction in Male Persian Fallow Deer (Dama dama mesopotamica): Inbreeding Detection and Morphometric Parameters Evaluation of Semen

Persian fallow deer (Dama dama mesopotamica) is only found in a few protected and refuges areas in the northwest, north, and southwest of Iran. The aims of this study were analysis of inbreeding and morphometric parameters of semen in male Persian fallow deer to investigate the cause of reduced fertility of this endangered species in Dasht-e-Naz National Refuge, Sari, Iran. The Persian fallow deer semen was collected by an electroejaculator from four adult bucks randomly during the breeding season and from five dehorned and horned deer’s in non-breeding season. Twelve blood samples were taken and mitochondrial DNA was extracted, a non-coding region called d-loop was amplified, sequenced and then were considered for genetic analysis. The Persian fallow deer’s normal and abnormal spermatozoa were similar to that of domestic ruminants but very smaller and difficult to observe at the primary observation. The post-mating season collected ejaculates contained abnormal spermatozoa, debris and secretion of accessory glands in horned bucks and accessory glands secretion free of any spermatozoa in dehorned or early velvet budding bucks. Microscopic evaluation in all four bucks during the mating season showed the mean concentration of 9 × 106 spermatozoa/ml. The mean ± standard deviation of age, testes length and testes width was 4.60 ± 1.52 years, 3.58 ± 0.32 and 1.86 ± 0.09 cm, respectively. The results identified 1120 loci in which 377 were polymorphic. In conclusion, reduced fertility of male Persian fallow deer may be caused by inbreeding of the protected herd in a limited area of Dasht-e-Naz National Refuge.

Progress on early diagnosing Alzheimer’s disease

Alzheimer’s disease(AD)is a progressive neurodegenerative disorder that affects both cognition and non-cognition functions.The disease follows a continuum,starting with preclinical stages,progressing to mild cognitive and behavioral impairment,ultimately leading to dementia.Early detection of AD is crucial for better diagnosis and more effective treatment.However,the current AD diagnostic tests of biomarkers using cerebrospinal fluid and/or brain imaging are invasive or expensive,and mostly are still not able to detect early disease state.Consequently,there is an urgent need to develop new diagnostic techniques with higher sensitivity and specificity during the preclinical stages of AD.Various non-cognitive manifestations,including behavioral abnormalities,sleep disturbances,sensory dysfunctions,and physical changes,have been observed in the preclinical AD stage before occurrence of notable cognitive decline.Recent research advances have identified several biofluid biomarkers as early indicators of AD.This review focuses on these non-cognitive changes and newly discovered biomarkers in AD,specifically addressing the preclinical stages of the disease.Furthermore,it is of importance to explore the potential for developing a predictive system or network to forecast disease onset and progression at the early stage of AD.

LSD1调控颗粒细胞自噬并抑制Wt1参与FSH调节的有腔卵泡形成

In a growing follicle,the survival and maturation of the oocyte largely depend on support from somatic cells to facilitate FSH-induced mutual signaling and chemical communication.Although apoptosis and autophagy in somatic cells are involved in the process of FSH-induced follicular development,the underlying mechanisms require substantial study.According to our study,along with FSH-induced antral follicles(AFs)formation,both lysine-specific demethylase 1(LSD1)protein levels and autophagy increased simultaneously in granulosa cells(GCs)in a time-dependent manner,we therefore evaluated the importance of LSD upon facilitating the formation of AFs correlated to autophagy in GCs.Conditional knockout of Lsdl in GCs resulted in significantly decreased AF number and subfertility in females,accompanied by marked suppression of the autophagy in GCs.On the one hand,depletion of Lsd1 resulted in accumulation of Wilms tumor 1 homolog(WT1),at both the protein and mRNA levels.WT1 prevented the expression of FSH receptor(Fshr)in GCs and thus reduced the responsiveness of the secondary follicles to FSH induction.On the other hand,depletion of LSD1 resulted in suppressed level of autophagy by upregulation of ATG16L2 in GCs.We finally approved that LSD1 contributed to these sequential activities in GCs through its H3K4me2 demethylase activity.Therefore,the importance of LSD1 in GCs is attributable to its roles in both accelerating autophagy and suppressing WT1 expression to ensure the responsiveness of GCs to FSH during AFs formation.