Development of SSR markers and analysis of diversity in Turkish populations of Brachypodium distachyon

Background  

Brachypodium distachyon (Brachypodium) is rapidly emerging as a powerful model system to facilitate research aimed at improving grass crops for grain, forage and energy production. To characterize the natural diversity of Brachypodium and provide a valuable new tool to the growing list of resources available to Brachypodium researchers, we created and characterized a large, diverse collection of inbred lines.     

Evaluation of Blastomere Biopsy Using a Mouse Model Indicates the Potential High Risk of Neurodegenerative Disorders in the Offspring

Preimplantation genetic diagnosis (PGD), used in clinical practice, is offered to couples that may suffer from a monogenetic disorder, chromosome aneuploidy, or X-linked disease. However, blastomere biopsy, as an indispensable manipulation during the PGD procedure has not been assessed for its long term health implications. Using a mouse model, we investigated the effect of blastomere biopsy of in vitro cultured four-cell embryos on preimplantation development efficiency, postnatal growth, and physiological and behavioral activity compared with control, non-biopsied embryos. The mice generated after blastomere biopsy showed weight increase and some memory decline compared with the control group. Further protein expression profiles in adult brains were analyzed by a proteomics approach. A total of 36 proteins were identified with significant differences between the biopsied and control groups, and the alterations in expression of most of these proteins have been associated with neurodegenerative diseases. Furthermore hypomyelination of the nerve fibers was observed in the brains of mice in the biopsied group. This study suggested that the nervous system may be sensitive to blastomere biopsy procedures and indicated an increased relative risk of neurodegenerative disorders in the offspring generated following blastomere biopsy. Thus, more studies should be performed to address the possible adverse effects of blastomere biopsy on the development of offspring, and the overall safety of PGD technology should be more rigorously assessed.Preimplantation genetic diagnosis (PGD)¹ has been one of the main clinical components of assisted reproductive technologies (ARTs) since 1990 (1). At present, infertile couples experiencing recurrent miscarriage or X chromosome-linked diseases are most likely to benefit from PGD.The treatment of human infertility by ARTs has gained widespread application, but it is disconcerting to many researchers that the clinical procedures used in ARTs are rapidly outpacing the underlying science. ART procedures are generally considered to be safe, but recent studies suggest a small increase in birth defects and low birth weights in ART children (2, 3). In addition, several clinical studies have reported an increased frequency of Beckwith-Wiedemann syndrome or Angelman syndrome caused by an imprinting defect among children conceived with ARTs (4, 5). These potential risks cause serious unease and justify more serious assessments of ARTs. However, moral, ethical, and legal issues complicate assessments of the genetic quality of ART-derived human conceptions, and significant genetic and demographic differences exist among couples participating in the ARTs, so a definitive assessment of the risks associated with this technology has been difficult to achieve. Therefore, appropriate animal models provide an important tool for studying potential effects of ARTs on the health and development of mammalian embryos (6).In many ART procedures, embryos are kept for a short time in a synthetic culture medium before transfer into their recipient mothers. Animal data have demonstrated that in vitro embryo culture and related procedures may be associated with epigenetic changes, perturbed genomic imprinting, and alterations in fetal growth (7). Some evidence also suggested that the culture environment may produce specific abnormalities during fetal and postnatal development (8–10). In the studies using mouse models, more marked changes in adult physiology, including onset of hypertension, were observed (11).As with other ARTs, the protocol required for PGD necessitates embryo manipulation and culture in vitro. However, in contrast to other ARTs, PGD involves embryo biopsy of one or two blastomeres at the eight-cell stage. Some studies have shown that blastomere biopsy does not have negative effects on embryo viability (12, 13), and offspring have been produced using embryo splitting technology, which is similar to the biopsy process, in at least six different domesticated species, including mouse (14), rabbit (15), sheep (16), bovine (17), goat (18), horse (19), pig (20), and in a preliminary non-human primate study one rhesus monkey (21). However, there is still a shortage of proof to convince the public that there is no potential risk to such animals conceived by this technology.To address this issue, we developed a mouse model to study the effects of blastomere biopsy on early embryo development and on postnatal physiological phenotype and behavior. A global proteomics method was also performed to study correlative protein expression profiles in adult brains and to indicate the possibility of neural degenerative disorders in adult conceived following biopsy technology.     

Morphological and physiological characteristics of transgenic cherry tomato mutant with HBsAg gene

HBsAg gene was previously introduced into cherry tomato (Lycopersicum esculentum var. cerasiforme) by Agrobacterium-mediated transformation. To investigate the side effect of HBsAg gene in cherry tomato, we analyzed morphological and physiological characteristics of the transgenic mutant N244. The process was performed under field conditions. The results suggested that the mutant N244 exhibited morphological, cytological and physiological variation. First of all, compared with the wild plants NK, N244 had fleshy and dark green leaves, the fewer notches of leaf edge, more adventitious roots and barren seeds. Moreover, the chromosome of N244 were found to be triploid (n = 36) by flow cytometric analysis. Furthermore, N244 has obvious physiological alterations, as compared to NK. It was speculated that transformation of the genes probably led to ploidy variation, and further caused phenotype and physiological changes of plants. Our study will reveal side effects of the mutants, and promote cultivation of transgenic plants in the field.     

Effects of immunization against a DNA vaccine encoding somatostatin gene (pGM-CSF/SS) by attenuated Salmonella typhimurium on growth, reproduction and lactation in female mice

A novel somatostatin (SS) DNA vaccine (pGM-CSF/SS), delivered orally by attenuated Salmonella typhimurium (CSO22), was used to immunize female mice at 5, 7, and 11 wk of age; the objective was to investigate the humoral immune response and effects of this vaccine on growth, reproduction and lactation. The pGM-CSF/SS induced SS-specific antibodies, which peaked (3.69 ± 0.89; mean ± S.D) 4 wk after the first booster immunization. Compared with a saline-treated control group, body weight gain of a pGM-CSF/SS immunized group increased 30.3% (23.88 vs. 18.32 g, P < 0.05) during the growth period (from 2 wk after primary immunization to 4 wk after the first booster immunization). Immunized mice had higher plasma estradiol concentrations (84.10 ± 2.16 vs. 81.45 ± 2.12 pg/ml, P < 0.05) and a shorter estrous cycle (4.06 ± 0.75 vs. 5.33 ± 0.49 d, P < 0.05), but serum progesterone concentrations were not significantly affected. Since offspring produced by immunized mice gained weight faster (P < 0.05) in the first 2 wk of life (4.27 ± 0.62 and 7.81 ± 1.30 g in Weeks 1 and 2, respectively vs. 3.70 ± 0.23 and 7.14 ± 0.48 g), we inferred that pGM-CSF/SS could have a direct or indirect role in regulating lactation in mice. In conclusion, GM-CSF and CSO22 served as adjuvant and attenuated live vector, respectively, with efficient oral delivery of an SS DNA vaccine which successfully induced a humoral immune response and enhanced rate of weight gain. Furthermore, the DNA vaccine pGM-CSF/SS affected plasma estradiol concentrations and the estrous cycle, and seemed to enhance lactation performance of female mice.     

Cloning, expression, and characterization of a new beta-agarase from Vibrio sp. strain CN41

A new agarase, AgaA(CN41), cloned from Vibrio sp. strain CN41, consists of 990 amino acids, with only 49% amino acid sequence identity with known β-agarases. AgaA(CN41) belongs to the GH50 (glycoside hydrolase 50) family but yields neoagarotetraose as the end product. AgaA(CN41) was expressed and characterized.     

Efficacy of Lovastatin on Learning and Memory Deficits Caused by Chronic Intermittent Hypoxia-Hypercapnia: Through Regulation of NR2B-Containing NMDA Receptor-ERK Pathway

Background

Chronic intermittent hypoxia-hypercapnia (CIHH) exposure leads to learnning and memory deficits in rats. Overactivation of N-methyl-D-aspartate receptors(NMDARs) can lead to the death of neurons through a process termed excitotoxicity, which is involved in CIHH-induced cognitive deficits. Excessively activated NR2B (GluN2B)-containing NMDARs was reported as the main cause of excitotoxicity. The ERK1/2 (extracellular signal-regulated kinase 1/2) signaling cascade acts as a key component in NMDARs-dependent neuronal plasticity and survival. Ca2+/calmodulin-dependent protein kinase II (CaMKII), synapse-associated protein 102 (SAP102) and Ras GTPase-activating protein (SynGAP) have been shown to be involved in the regulation of NMDAR-ERK signalling cascade. Recent studies revealed statins (the HMG-CoA reductase inhibitor) have effect on the expression of NMDARs. The present study intends to explore the potential effect of lovastatin on CIHH-induced cognitive deficits and the NR2B-ERK signaling pathway.

Methods and Findings

Eighty male Sprague Dawley rats were randomly divided into five groups. Except for those in the control group, the rats were exposed to chronic intermittent hypoxia-hypercapnia (CIHH) (9∼11%O₂, 5.5∼6.5%CO₂) for 4 weeks. After lovastatin administration, the rats performed better in the Morris water maze test. Electron microscopy showed alleviated hippocampal neuronal synaptic damage. Further observation suggested that either lovastatin or ifenprodil (a selective NR2B antagonist) administration similarly downregulated NR2B subunit expression leading to a suppression of CaMKII/SAP102/SynGAP signaling cascade, which in turn enhanced the phosphorylation of ERK1/2. The phosphorylated ERK1/2 induced signaling cascade involving cAMP-response element-binding protein (CREB) phosphorylation and brain-derived neurotrophic factor (BDNF) activation, which is responsible for neuroprotection.

Conclusions

These findings suggest that the ameliorative cognitive deficits caused by lovastatin are due to the downregulation of excessive NR2B expression accompanied by increased expression of ERK signaling cascade. The effect of NR2B in upregulating pERK1/2 maybe due, at least in part, to inactivation of CaMKII/SAP102/SynGAP signaling cascade.     

Functional Study of One Nucleotide Mutation in Pri-MiR-125a Coding Region which Related to Recurrent Pregnancy Loss

MicroRNAs (miRNAs) are short non-coding RNAs which modulate gene expression by binding to complementary segments present in the 3′UTR of the mRNAs of protein coding genes. MiRNAs play very important roles in maintaining normal human body physiology conditions, meanwhile, abnormal miRNA expressions have been found related to many human diseases spanning from psychiatric disorders to malignant cancers. Recently, emerging reports have indicated that disturbed miRNAs expression contributed to the pathogenesis of recurrent pregnancy loss (RPL). In this study, we identified a new mutation site (+29A>G, position relative to pre-miR-125a) by scanning pri-miR-125a coding region in 389 Chinese Han RPL patients. This site was co-existed with two polymorphisms (rs12976445 and rs41275794) in patients heterogeneously and changed the predicted secondary structures of pri-miR-125a. Subsequent in vitro analysis indicated that the A>G mutation reduced mature miR-125a expression, and further led to less efficient inhibition of verified target genes. Functional analysis showed that mutant pri-mir-125a can enhance endometrial stromal cells (ESCs) invasive capacity and increase the sensitivity of ESCs cells to mifepristone. Moreover, we further analyzed the possible molecular mechanism by RIP-chip assay and found that mutant pri-mir-125a disturbed the expression of miR-125a targetome, the functions of which includes embryonic development, cell proliferation, migration and invasion. These data suggest that A>G mutation in pri-miR-125a coding region contributes to the genetic predisposition to RPL by disordering the production of miR-125a, which consequently meddled in gene regulatory network between mir-125a and mRNA.     

番茄下胚轴离体培养植株再生及其组织学观察

研究了上海地区的主要栽培品种之一“鲜丰”番茄下胚轴离体培养过程中的激素调控 ,结果表明 :“鲜丰”番茄下胚轴进行离体培养过程中 ,MS培养基上附加不同浓度的生长素( IAA)和细胞分裂素 ( BA) ,对愈伤组织的形成影响不大 ,但对不定芽的分化有较大的影响 ,得出最佳培养基为 MS+ BA1 .0～ 2 .0 mg· L- 1+ IAA0 .2 mg· L- 1。用不同浓度的 ZT、BA、KT进行单因子芽器官的诱导实验 ,发现 ZT的作用力强于 BA和 KT,KT最弱 ;用不同浓度NAA、IAA、IBA、2 ,4- D进行发根培养实验 ,发现番茄的内源生长素浓度较高 ,用外植体直接发根外加生长素有一定的作用 ,若用不定芽扦插发根 ,不附加生长素也极易发根 ,故番茄的生根培养基为 1 /2 MS或 MS附加 IAA 0 .0 5～ 0 .1 mg· L- 1。另外 ,对有关细胞启动、分裂、分化以及器官发生的组织学观察表明 :番茄离体培养中不定芽通常发生在愈伤组织的周边区 ,也可起源于维管组织结节周围的形成层状细胞。不定根则由茎中柱鞘处发生。     

Tumor suppressor PALB2 maintains redox and mitochondrial homeostasis in the brain and cooperates with ATG7/autophagy to suppress neurodegeneration

The PALB2 tumor suppressor plays key roles in DNA repair and has been implicated in redox homeostasis. Autophagy maintains mitochondrial quality, mitigates oxidative stress and suppresses neurodegeneration. Here we show that Palb2 deletion in the mouse brain leads to mild motor deficits and that co-deletion of Palb2 with the essential autophagy gene Atg7 accelerates and exacerbates neurodegeneration induced by ATG7 loss. Palb2 deletion leads to elevated DNA damage, oxidative stress and mitochondrial markers, especially in Purkinje cells, and co-deletion of Palb2 and Atg7 results in accelerated Purkinje cell loss. Further analyses suggest that the accelerated Purkinje cell loss and severe neurodegeneration in the double deletion mice are due to excessive oxidative stress and mitochondrial dysfunction, rather than DNA damage, and partially dependent on p53 activity. Our studies uncover a role of PALB2 in mitochondrial homeostasis and a cooperation between PALB2 and ATG7/autophagy in maintaining redox and mitochondrial homeostasis essential for neuronal survival.     

Opioid activity of synthetic deltorphin II analogues and their spin labeled derivatives

Deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH₂, Del II), an endogenous linear heptapeptide, is a highly selective agonist of the -opioid receptor. To study the effect of the position 4 residue (Glu) on the opioid activity of Del II, we designed and synthesized three analogues of Del II by solid-phase peptide synthesis. They were [Val⁴,Glu⁵]Del II, [Val⁴,Glu⁶]Del II and [Gly⁴,Glu⁷]Del II. To study the effect of spin labeling on peptide bioactivities, all the peptides were labeled using a free radical. The labeling material was a stable nitrogen–oxygen free radical which was linked to the N-terminal via an amide bond. We investigated the opioid bioactivities of these analogues both in vivo and in vitro, and concluded that the differences in opioid activity of Del II and its analogues were due to structural differences. When the Glu residue is at position 5 or 6, the internal hydrogen bonds in Del II are affected and there is a change in three-dimensional structure and opioid activity. The antinociceptive activity of all the peptides decreased after spin labeling. This indicates that the stable nitrogen–oxygen free radical is a dual-function spin-labeling molecule.