Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons

Developing neuronal populations undergo significant attrition by natural cell death. Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis during synaptogenesis. Following this time window, destruction of the anatomic target of dopaminergic neurons results in dopaminergic cell death but the morphology is no longer apoptotic. We describe ultrastructural changes that appear unique to dying embryonic dopaminergic neurons. In primary cultures of mesencephalon, death of dopaminergic neurons is triggered by activation of glutamate receptors sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and differs ultrastructurally from both neuronal apoptosis or typical excitotoxicity. AMPA causes morphological changes selectively in dopaminergic neurons, without affecting other neurons in the same culture dishes. Two hours after the onset of treatment swelling of Golgi complexes is apparent. At 3 h, dopaminergic neurons display loss of membrane asymmetry (coinciding with commitment to die), as well as nuclear membrane invagination, irregular aggregation of chromatin, and mitochondrial swelling. Nuclear changes continue to worsen until loss of cytoplasmic structures and cell death begins to occur after 12 h. These changes are different from those described in neurons undergoing either apoptosis or excitotoxic death, but are similar to ultrastructural changes observed in spontaneous death of dopaminergic neurons in the natural mutant weaver mouse.     

A Mouse Surgical Model for Metastatic Ovarian Granulosa Cell Tumor

We recently described a genetically engineered mouse model that develops ovarian granulosa cell tumors (GCTs) that mimic many aspects of the advanced human disease, including distant dissemination. However, because the primary tumors killed their hosts before metastases were able to form, the use of these mice to study metastatic disease required the development of a simple, reliable, and humane surgical protocol for the excision of large GCTs from debilitated mice. Here we describe a protocol involving multimodal anesthesia, tumor removal through ventral midline celiotomy and perioperative fluid therapy, and analgesia that led to the postoperative survival of more than 90% of mice, despite the removal of tumors representing as much as 10% of the animal''s body weight. Intraabdominal recurrence of the GCT did not occur in surviving animals, but most developed pulmonary or adrenal metastases (or both) by 12 wk after surgery. We propose that this mouse model of metastatic GCT will serve as a useful preclinical model for the development of novel treatment modalities and diagnostic techniques. Furthermore, our results delineate anesthetic and surgical principles for the removal of large abdominal tumors from mice that will be applicable to other models of human cancers.Abbreviation: GCT, granulosa cell tumorThe ovarian granulosa cell tumor (GCT) is the most prevalent sex cord tumor in women and is thought to represent up to 5% of all ovarian cancers.⁸ It is generally considered a low-grade malignancy, but a small percentage of GCTs are considered aggressive, and a large proportion of patients develop recurrent disease postoperatively.¹⁰ Few therapeutic options are available for the recurrent disease, and fastidious long-term follow-up is required in most patients, given that recurrent lesions have been diagnosed as long as 40 y after removal of the original tumor.⁴One of the key factors that has limited progress in the development of therapeutic approaches for ovarian cancer is the dearth of relevant animal models.⁹ Our laboratory has recently developed a genetically engineered mouse model (Pten^{tm1Hwu/tm1Hwu}; Ctnnb1^tm1Mmt/+;Amhr2^{tm3(cre)Bhr/+}) that develops an aggressive form of GCT in animals of both sexes.¹ In female mice, the disease is characterized by perinatal onset and rapid tumor development in both ovaries, causing death by approximately 8 wk of age. Histopathologic analyses of 6- to 8-wk-old Pten^{tm1Hwu/tm1Hwu}; Ctnnb1^tm1Mmt/+; Amhr2^{tm3(cre)Bhr/+} tissues revealed the presence of tumor cell embolisms in the lungs, suggesting metastatic potential for GCTs.⁶ However, the mice invariably died or required euthanasia for humane reasons before true metastases were able to form. Surgical excision of the primary tumors allowed for the postoperative survival of the mice for several months, and permitted the development of metastases in several tissues including lung and adrenal gland, thereby confirming the metastatic potential of the tumor cells.⁶Because of its mimicry of advanced human disease from the molecular to tumor biology level, we have proposed that the Pten^{tm1Hwu/tm1Hwu};Ctnnb1^tm1Mmt/+;Amhr2^{tm3(cre)Bhr/+} model could represent a powerful tool for the development of new therapeutic strategies for metastatic GCT. However, because the GCTs kill their hosts before metastases are able to form, their use as a model of the metastatic disease requires the development of a simple, reliable, and humane surgical protocol for the excision of the primary tumors. Such a protocol would need to take into account that pulmonary tumor cell embolisms have not been noted in these mice before the age of 6 wk, necessitating that surgery be performed in debilitated animals with tumor burdens approaching 10% of their body weight. A review of the existing literature on anesthesia, fluid therapy, pain management, and surgical techniques in mice yielded little information applicable to our model, necessitating that we develop a novel protocol that addressed both the medical needs of the mice and the scientific objectives of the research program.     

Targeting to the HIV-1 RRE of the influenza virus NS1 protein effector domain as a potent, specific anti-HIV agent

The Rev axis of HIV autoregulation is one of two critical viral regulatory pathways required for expression of viral genomic and mRNA and for replication. Consequently it is an attractive therapeutic target. Previous studies have investigated the anti-HIV efficacy of targeting to the RRE (the viral RNA target sequence of the Rev axis) a trans-dominant negative inhibitor mutant Rev, M10. In this study we have fused a portion of the influenza virus NS1 protein (which normally inhibits polyA(+) mRNA transport and splicing) to the Rev M10 gene while deleting the NS1 poly(A) binding region. The resulting chimera demonstrates specific and enhanced inhibition of viral-RRE-containing RNA expression.     

Spheroids from adipose‐derived stem cells exhibit an miRNA profile of highly undifferentiated cells

Two‐dimensional (2D) cell cultures have been extensively used to investigate stem cell biology, but new insights show that the 2D model may not properly represent the potential of the tissue of origin. Conversely, three‐dimensional cultures exhibit protein expression patterns and intercellular junctions that are more representative of their in vivo condition. Multiclonal cells that grow in suspension are defined as “spheroids,” and we have previously demonstrated that spheroids from adipose‐derived stem cells (S‐ASCs) displayed enhanced regenerative capability. With the current study, we further characterized S‐ASCs to further understand the molecular mechanisms underlying their stemness properties. Recent studies have shown that microRNAs (miRNAs) are involved in many cellular mechanisms, including stemness maintenance and proliferation, and adipose stem cell differentiation. Most studies have been conducted to identify a specific miRNA profile on adherent adipose stem cells, although little is still known about S‐ASCs. In this study, we investigate for the first time the miRNA expression pattern in S‐ASCs compared to that of ASCs, demonstrating that cell lines cultured in suspension show a typical miRNA expression profile that is closer to the one reported in induced pluripotent stem cells. Moreover, we have analyzed miRNAs that are specifically involved in two distinct moments of each differentiation, namely early and late stages of osteogenic, adipogenic, and chondrogenic lineages during long‐term in vitro culture. The data reported in the current study suggest that S‐ASCs have superior stemness features than the ASCs and they represent the true upstream stem cell fraction present in adipose tissue, relegating their adherent counterparts.     

Evaluation of self-similar features in time series of serum growth hormone and prolactin levels by fractal analysis: Effect of delayed sleep and complexity of diurnal variation

We assayed the diurnal concentrations of growth hormone (GH) and prolactin (PRL) in 6 healthy male volunteers to evaluate the self-similar features in the time series of each hormone on the basis of fractal theory and to determine the fractal dimension as an index of the complexity of the diurnal variation. In addition, we assessed the effects of a 6-hour delay in the sleep period on the complexity of the diurnal variaton of these hormones. There was a statistically significant fractal feature in the serum levels of GH both under the nocturnal-sleep and delayed-sleep conditions in all subjects. The time series of the serum PRL concentrations also showed a statistically significant fractal feature under the nocturnal-sleep and delayed-sleep conditions in all subjects. The fractal dimensions of the patterns of the GH or PRL levels were 1.879 and 1.929 or 1.754 and 1.785 under the nocturnal-sleep and delayed-sleep conditions, respectively. Two-way ANOVA revealed no significant difference in the fractal dimension between the two sleep conditions but did reveal a significant difference between the fractal dimensions of the GH and PRL levels. These results showed (1) that delayed sleep had no significant effect on the complexity of the diurnal pattern of these hormones, and (2) that the diurnal pattern of the GH levels was more complex than that of the PRL levels.     

The effects of type I collagen on bone defects and gene expression changes for osteogenesis: In a rat model

The aim of this study is to investigate the effects of type I collagen on bone defects and on genes specifically for osteogenesis in a rat model. Two millimeter drill hole bone defect was created in the femur of rats. In the experimental group, type I collagen was applied in bone defects whereas in control group defects were left empty. Inflammation, development of connective tissue, osteogenesis, and foreign body reaction parameters evaluated with histologically and genes evaluated by blood samples. In the experimental group, the histopathologically significant change was found in favor of bone healing only at the first week. A significant increase was found in genetic expressions of BMP-1, 2, 3, 4, 5, 6, 7, TGF-βRII, Smad-1, IL-6, BMPR-IA, BMPR-IB, Eng, BMPR-II, c-fos, Cdkn1a, Chrd, Gdf-5, Id-1, PDGF-β, IGF-1, Serpine-1, and TGF-βRI at the first hour. At the first, third, and sixth week, no significant increase was found in any of the gene expressions. Type I collagen is found to be effective in favor of bone healing through increased inflammatory cytokines and expression of BMP genes in the early stages of fracture healing.     

Identification of gene coexpression modules,hub genes,and pathways related to spinal cord injury using integrated bioinformatics methods

Spinal cord injury (SCI) is characterized by dramatic neurons loss and axonal regeneration suppression. The underlying mechanism associated with SCI-induced immune suppression is still unclear. Weighted gene coexpression network analysis (WGCNA) is now widely applied for the identification of the coexpressed modules, hub genes, and pathways associated with clinic traits of diseases. We performed this study to identify hub genes associated with SCI development. Gene Expression Omnibus (GEO) data sets GSE45006 and GSE20907 were downloaded and the significant correlativity and connectivity between them were detected using WGCNA. Three significant consensus modules, including 567 eigengenes, were identified from the master GSE45006 data following the preconditions of approximate scale-free topology for WGCNA. Further bioinformatics analysis showed these eigengenes were involved in inflammatory and immune responses in SCI. Three hub genes Rac2, Itgb2, and Tyrobp and one pathway “natural killer cell-mediated cytotoxicity” were identified following short time-series expression miner, protein-protein interaction network, and functional enrichment analysis. Gradually upregulated expression patterns of Rac2, Itgb2, and Tyrobp genes at 0, 3, 7, and 14 days after SCI were confirmed based on GSE45006 and GSE20907 data set. Finally, we found that Rac2, Itgb2, and Tyrobp genes might take crucial roles in SCI development through the “natural killer cell–mediated cytotoxicity” pathway.     

两株采自惠州的细鞘丝藻亚科(Leptolyngbyaceae)嗜热蓝细菌的分离鉴定及细胞组分分析

【背景】随着CO_2排放增加,全球变暖愈发严峻,嗜热蓝细菌作为能够在45°C及以上环境中生长并实现生物固碳的微生物,具有重要的研究意义。【目的】对从广东惠州地区采集的藻种进行分离鉴定,并筛选出2株嗜热蓝细菌,研究其生长特性,为嗜热蓝细菌的后续应用提供依据。【方法】通过16SrRNA基因、藻蓝蛋白链A基因(PhycoA)序列分析确定从惠州地区采集到的菌株的分类学位置。对PKUAC-GDTS1-24和PKUAC-GDTS1-29两株嗜热蓝细菌进行形态观察和主要细胞成分(灰分、糖类、脂质、蛋白质和色素)分析。【结果】共分离出12株嗜热蓝细菌,其中PKUAC-GDTS1-24和PKUAC-GDTS1-29菌株,形态上呈蓝绿色球形毛状体,是由细胞形成密集的簇,彼此附着形成的。两株嗜热蓝细菌的主要细胞成分是糖类,分别占细胞干重的36.42%和28.46%。PKUAC-GDTS1-24的灰分、脂质和蛋白质含量分别为24.41%、21.40%和26.64%。PKUAC-GDTS1-29的细胞中,灰分、脂质和蛋白质含量分别为24.72%、23.92%和12.93%。藻蓝蛋白(Phycocyanin,PC)在PKUAC-GDTS1-24和PKUAC-GDTS1-29中的含量分别为157.29 mg/g DW和374.86 mg/g DW,类胡萝卜素分别为65.13 mg/g DW和18.87 mg/g DW。【结论】基于系统发育树研究,本实验的2个分离株属于细鞘丝藻亚科(Leptolyngbyaceae),与研究较少的纤发鞘丝蓝细菌属(Leptolyngbya)菌株相近,可能是广东和四川温泉中存在的一种新型丝状轻度嗜热蓝细菌属或Leptolyngbya新种。嗜热菌株PKUAC-GDTS1-24和PKUAC-GDTS1-29的形态和细胞组成相似,通过比较,2个菌株的藻胆蛋白含量远远高于其他研究报道的Leptolyngbya蓝细菌,尤其是PKUAC-GDTS1-29可以作为藻蓝蛋白生产的潜在菌株。