Factors affecting the outcome of human blastocyst vitrification

With single blastocyst transfer practice becoming more common in ART, there is a greater demand for a convenient and reliable cryostorage of surplus blastocysts. Vitrification has emerged in the last decade as an alternative promising substitute for slow freezing. Blastocysts represent a unique challenge in cryostorage due to their size, multicellular structure and presence of blastocoele. The continuous acquisition of experience and introduction of many different technological developments has led to the improvement of vitrification as a technology and improved the results of its application in blastocyst cryostorage. The current information concerning safety and efficacy of the vitrification of blastocysts will be reviewed along with the variables that can impact the outcome of the procedure.     

Cytosolic calcium and pH signaling in plants under salinity stress

Calcium is one of the essential nutrients for growth and development of plants. It is an important component of various structures in cell wall and membranes. Besides some fundamental roles under normal condition, calcium functions as a major secondary-messenger molecule in plants under different developmental cues and various stress conditions including salinity stress. Also changes in cytosolic pH, pH_cyt, either individually, or in coordination with changes in cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, evoke a wide range of cellular functions in plants including signal transduction in plant-defense responses against stresses. It is believed that salinity stress, like other stresses, is perceived at cell membrane, either extra cellular or intracellular, which then triggers an intracellular-signaling cascade including the generation of secondary messenger molecules like Ca²⁺ and protons. The variety and complexity of Ca²⁺ and pH signaling result from the nature of the stresses as well as the tolerance level of the plant species against that specific stress. The nature of changes in [Ca²⁺]_cyt concentration, in terms of amplitude, frequency and duration, is likely very important for decoding the specific downstream responses for salinity stress tolerance in planta. It has been observed that the signatures of [Ca²⁺]_cyt and pH differ in various studies reported so far depending on the techniques used to measure them, and also depending on the plant organs where they are measured, such as root, shoot tissues or cells. This review describes the recent advances about the changes in [Ca²⁺]_cyt and pH_cyt at both cellular and whole-plant levels under salinity stress condition, and in various salinity-tolerant and -sensitive plant species.Key words: cytosolic calcium, ionic toxicity, osmotic stress, pH, salinity stress, salt tolerance, signaling     

A Novel Bicistronic High-Capacity Gutless Adenovirus Vector That Drives Constitutive Expression of Herpes Simplex Virus Type 1 Thymidine Kinase and Tet-Inducible Expression of Flt3L for Glioma Therapeutics

Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, affecting ∼18,000 new patients every year; its prognosis remains poor despite standard treatment with surgery, radiotherapy, and chemotherapy (temozolomide) (36-38, 44; J. C. Buckner, presented at the ASCO Annual Meeting, 2009). Complete resection is mostly impossible due to the highly infiltrative nature of this disease. Residual GBM cells remaining within the nonneoplastic brain parenchyma eventually lead to tumor recurrence that is resistant to conventional chemotherapy and radiotherapy, ultimately leading to the patient''s death (44). Several dendritic cell vaccination strategies aiming to stimulate the patient''s immune system to seek out and destroy residual brain tumor cells are currently under preclinical and clinical development and constitute a promising adjuvant treatment for GBM (18, 24, 42, 43, 45).We have developed a novel immunotherapeutic approach for GBM using first-generation adenoviral vectors (Ads) to deliver a combination of therapeutic transgenes into the tumor mass (2, 7, 9, 12, 14, 17, 48) which is slated to begin phase I clinical testing this year. In our strategy, rather than vaccinating against tumor antigens, we aim to reconstruct an immune circuit that is absent from the normal brain. Our gene therapy strategy consists of the conditionally cytotoxic herpes simplex virus type 1 thymidine kinase (TK) (2, 12), which kills proliferating tumor cells in the presence of the prodrug ganciclovir (GCV), used in combination with human soluble Fms-like tyrosine kinase 3 ligand (Flt3L), which recruits bone marrow-derived dendritic cells (DCs) to the normal brain or brain tumor microenvironment in mice (11, 12) and rats (1, 13). We previously showed that codelivery of Ad-Flt3L and Ad-TK into the brain tumor milieu induces an anti-GBM-specific immune response (9, 12, 17, 27, 48), leading to long-term survival of rats bearing intracranial CNS1, 9L, and F98 tumors (2, 17, 27) and mice bearing intracranial GL26, GL261, and B16-F10 tumors (12, 48). In addition, the combination of Ad-Flt3L plus Ad-TK induces GBM-specific immunological memory that improves survival in intracranial multifocal and recurrent models of GBM in both rats and mice (9, 12, 17, 20, 22, 27).First-generation adenoviral vectors have been used in clinical trials to deliver HSV1-TK (31). Ad-TK delivered into the margins of the tumor cavity after surgical resection of GBM was well tolerated in over 70 patients in six early clinical trials (19, 34). Publication of final results from a large, multicenter phase III trial involving 251 patients is awaited; preliminary analysis indicates small but statistically significant benefits from adding gene therapy to standard care. Final analysis will have to wait for the detailed publications (3, 16).To provide stable, long-term therapeutic transgene expression, “gutless,” high-capacity adenoviral (HC-Ad) vectors have been engineered in which all viral encoding genes have been eliminated and replaced with inert, noncoding stuffer DNA sequences (26, 30). In the presence of an anti-adenoviral immune response, transgene expression in the brain from first-generation Ads is reduced, while expression from HC-Ad vectors remains stable for at least 1 year (4, 25, 28). Therefore, we engineered two HC-Ad vectors to either constitutively express TK (HC-Ad-TK) (21, 27) or express Flt3L under the control of the tightly regulatable mCMV-TetOn expression system (HC-Ad-TetOn-Flt3L) (6, 8, 27, 47). Intratumoral administration of HC-Ad-TK and HC-Ad-TetOn-Flt3L led to high levels of therapeutic efficacy, and the therapy was well tolerated, with no overt toxicity and no spread of vector genomes outside the injected brain hemisphere in treated Lewis rats bearing syngeneic intracranial brain tumors (27).To reduce vector dose and facilitate GMP manufacturing of the clinical product, we engineered a novel, bicistronic HC-Ad vector that encodes, for the first time, both constitutively expressed HSV1-TK and inducible Flt3L from a single HC-Ad vector genome. Herein we demonstrate therapeutic efficacy of the bicistronic HC-Ad in naïve rats bearing intracranial tumors and also in tumor-bearing rats which had been exposed to adenoviruses and therefore exhibited anti-Ad immunity. The safety profile of this approach was assessed by evaluating the biodistribution of HC-Ad vector genomes, systemic and neurological toxicity, and behavioral abnormalities over the course of 1 year posttreatment. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.     

Both cell-surface and secreted CSF-1 expressed by tumor cells metastatic to bone can contribute to osteoclast activation

Tumors metastatic to the bone produce factors that cause massive bone resorption mediated by osteoclasts in the bone microenvironment. Colony stimulating factor (CSF-1) is strictly required for the formation and survival of active osteoclasts, and is frequently produced by tumor cells. Here we hypothesize that the CSF-1 made by tumor cells contributes to bone destruction in osteolytic bone metastases. We show that high level CSF-1 protected osteoclasts from suppressive effects of transforming growth factor β (TGF-β). r3T cells, a mouse mammary tumor cell line that forms osteolytic bone metastases, express abundant CSF-1 in vitro as both a secreted and a membrane-spanning cell-surface glycoprotein, and we show that both the secreted and the cell-surface form of CSF-1 made by r3T cells can support osteoclast formation in co-culture experiments in the presence of RankL. Mice with r3T bone metastases have elevated levels of both circulating and bone-associated CSF-1, and the majority of CSF-1 found in bone metastases is associated with the tumor cells. These results support the idea that tumor-cell produced CSF-1 contributes to osteoclast development and survival in bone metastasis.     

A Review on Epigenetic Inheritance of Experiences in Humans

Biochemical Genetics - If genetics defines the inheritance of DNA, epigenetics aims to regulate and make it adaptable. Epigenetic alterations include DNA methylation, chromatin remodelling,...     

A potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins.

An antimicrobial protein of about 10 kD, called Ace-AMP1, was isolated from onion (Allium cepa L.) seeds. Based on the near-complete amino acid sequence of this protein, oligonucleotides were designed for polymerase chain reaction-based cloning of the corresponding cDNA. The mature protein is homologous to plant nonspecific lipid transfer proteins (nsLTPs), but it shares only 76% of the residues that are conserved among all known plant nsLTPs and is unusually rich in arginine. Ace-AMP1 inhibits all 12 tested plant pathogenic fungi at concentrations below 10 micrograms mL-1. Its antifungal activity is either not at all or is weakly affected by the presence of different cations at concentrations approximating physiological ionic strength conditions. Ace-AMP1 is also active on two Gram-positive bacteria but is apparently not toxic for Gram-negative bacteria and cultured human cells. In contrast to nsLTPs such as those isolated from radish or maize seeds, Ace-AMP1 was unable to transfer phospholipids from liposomes to mitochondria. On the other hand, lipid transfer proteins from wheat and maize seeds showed little or no antimicrobial activity, whereas the radish lipid transfer protein displayed antifungal activity only in media with low cation concentrations. The relevance of these findings with regard to the function of nsLTPs is discussed.     

Identification of AtPIS, a phosphatidylinositol synthase from Arabidopsis.

Phosphatidylinositol synthase is the enzyme responsible for the synthesis of phosphatidylinositol, a key phospholipid component of all eukaryotic membranes and the precursor of messenger molecules involved in signal transduction pathways for calcium-dependent responses in the cell. Using the amino acid sequence of the yeast enzyme as a probe, we identified an Arabidopsis expressed sequence tag potentially encoding the plant enzyme. Sequencing the entire cDNA confirmed the homology between the two proteins. Functional assays, performed by overexpression of the plant cDNA in Escherichia coli, a bacteria which lacks phosphatidylinositol and phosphatidylinositol synthase activity, showed that the plant protein induced the accumulation of phosphatidylinositol in the bacterial cells. Analysis of the enzymatic activity in vitro showed that synthesis of phosphatidylinositol occurs when CDP-diacylglycerol and myo-inositol only are provided as substrates, that it requires manganese or magnesium ions for activity, and that it is at least in part located to the bacterial membrane fraction. These data allowed us to conclude that the Arabidopsis cDNA codes for a phosphatidylinositol synthase. A single AtPIS genetic locus was found, which we mapped to Arabidopsis chromosome 1.