Disruption of mitotic arrest precedes precocious differentiation and transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary

Mammalian sex determination is controlled by antagonistic pathways that are initially co-expressed in the bipotential gonad and subsequently become male- or female-specific. In XY gonads, testis development is initiated by upregulation of Sox9 by SRY in pre-Sertoli cells. Disruption of either gene leads to complete male-to-female sex reversal. Ovarian development is dependent on canonical Wnt signaling through Wnt4, Rspo1 and β-catenin. However, only a partial female-to-male sex reversal results from disruption of these ovary-promoting genes. In Wnt4 and Rspo1 mutants, there is evidence of pregranulosa cell-to-Sertoli cell transdifferentiation near birth, following a severe decline in germ cells. It is currently unclear why primary sex reversal does not occur at the sex-determining stage, but instead occurs near birth in these mutants. Here we show that Wnt4-null and Rspo1-null pregranulosa cells transition through a differentiated granulosa cell state prior to transdifferentiating towards a Sertoli cell fate. This transition is preceded by a wave of germ cell death that is closely associated with the disruption of pregranulosa cell quiescence. Our results suggest that maintenance of mitotic arrest in pregranulosa cells may preclude upregulation of Sox9 in cases where female sex-determining genes are disrupted. This may explain the lack of complete sex reversal in such mutants at the sex-determining stage.     

Oxidized fatty acid analysis by charge-switch derivatization,selected reaction monitoring,and accurate mass quantitation

A highly sensitive, specific, and robust method for the analysis of oxidized metabolites of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was developed using charge-switch derivatization, liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI MS/MS) with selected reaction monitoring (SRM) and quantitation by high mass accuracy analysis of product ions, thereby minimizing interferences from contaminating ions. Charge-switch derivatization of LA, AA, and DHA metabolites with N-(4-aminomethylphenyl)-pyridinium resulted in a 10- to 30-fold increase in ionization efficiency. Improved quantitation was accompanied by decreased false positive interferences through accurate mass measurements of diagnostic product ions during SRM transitions by ratiometric comparisons with stable isotope internal standards. The limits of quantitation were between 0.05 and 6.0 pg, with a dynamic range of 3 to 4 orders of magnitude (correlation coefficient r² > 0.99). This approach was used to quantitate the levels of representative fatty acid metabolites from wild-type (WT) and iPLA₂γ^–/– mouse liver identifying the role of iPLA₂γ in hepatic lipid second messenger production. Collectively, these results demonstrate the utility of high mass accuracy product ion analysis in conjunction with charge-switch derivatization for the highly specific quantitation of diminutive amounts of LA, AA, and DHA metabolites in biologic systems.     

Distributional Evidence for Cospeciation between Neotropical Bats and their Bat Fly Ectoparasites

Distributional evidence bearing on cospeciation of New World bats (superfamily Noctilionoidea) and associated bat flies (Diptera: Streblidae) was analyzed and revealed substantial phylogenetic structure, especially when we focused on "normal" host associations and encoded the data set hierarchically. Brooks' parsimony analyses of bat fly occurrences among hosts recovered generally accepted sets of host relationships. Putatively monophyletic host taxa provide a necessary but insufficient condition for coevolution via cospeciation.     

Configurational assignments of conformationally restricted bis-monoterpene hydroquinones: Utility in exploration of endangered plants

Background

Endangered plant species are an important resource for new chemistry. Lindera melissifolia is native to the Southeastern U.S. and scarcely populates the edges of lakes and ponds. Quantum mechanics (QM) used in combination with NMR/ECD is a powerful tool for the assignment of absolute configuration in lieu of X-ray crystallography.

Methods

The EtOAc extract of L. melissifolia was subject to chromatographic analysis by VLC and HPLC. Spin–spin coupling constant (SSCC) were calculated using DFT at the MPW1PW91/6-31G(d,p) level for all staggered rotamers. ECD calculations employed Amber* force fields followed by PM6 semi-empirical optimizations. Hetero- and homo-nuclear coupling constants were extracted from 1D ¹H, E.COSY and HETLOC experiments.

Results

Two meroterpenoids, melissifolianes A (1) and B (2) were purified and their 2-D structures elucidated using NMR and HRESIMS. The relative configuration of 1 was established using the combination of NOE-based distance restraints and the comparisons of experimental and calculated SSCCs. The comparison of calculated and experimental ECD assigned the absolute configuration of 1. The relative configuration of a racemic mixture, melissifoliane B (2) was established utilizing J-based analysis combined with QM and NMR techniques.Conclusion Our study of the Lindera melissifolia metabolome exemplifies how new chemistry remains undiscovered among the numerous endangered plant species and demonstrates how analysis by ECD and NMR combined with various QM calculations is a sensible approach to support the stereochemical assignment of molecules with conformationally restricted conformations.

General significance

QM–NMR/ECD combined approaches are of utility for unambiguous assignment of 3-D structures, especially with limited plant material and when a molecule is conformationally restricted. Conservation of an endangered plant species can be supported through identification of its new chemistry and utilization of that chemistry for commercial purposes.     

Therapeutic gene modified cell based cancer vaccines

History of cancer immunotherapy lasts for more than 120 years. In 1891 William B. Coley injected bacteria into inoperable cancer (bone sarcoma) and observed tumor shrinkage. He is recognized as the "'"Father of Immunotherapy"'". Cancer immunotherapy is based on the ability of the immune system to recognize cancer cells and to affect their growth and expansion. Beside the fact that, tumor cells are genetically distinct from their normal counterparts, and should be recognized and eliminated by immune system, the tumor associated antigens (TAAs) are often poorly immunogenic due to immunoediting. This process allows tumor to evolve during continuous interactions with the host immune system, and eventually escape from immune surveillance. Furthermore, tumor microenvironment consists of immunosuppressive cells that release immunosuppressive factors including IL-6, IL-10, IDO, TGFβ or VEGF. Interactions between cancer and stroma cells create network of immunosuppressive pathways, while activation of immune defense is inhibited. A key to successful immunotherapy is to overcome the local immunosuppression within tumor microenvironment and activate mechanisms that lead to tumor eradication. There are two clinical approaches of immunotherapy: active and passive. Active immunotherapy involves stimulation of immune response to tumor associated antigens (TAAs), either non-specifically via immunomodulating agents or specifically employing cancer vaccines. This review presents the progress and breakthroughs in design, development and clinical application of selected cell-based tumor vaccines achieved due to the generation and development of gene transfer technologies.     

Use of Genetically Engineered Microorganisms (GEMs) for the Bioremediation of Contaminants

ABSTRACT

This paper presents a critical review of the literature on the application of genetically engineered microorganisms (GEMs) in bioremediation. The important aspects of using GEMs in bioremediation, such as development of novel strains with desirable properties through pathway construction and the modification of enzyme specificity and affinity, are discussed in detail. Particular attention is given to the genetic engineering of bacteria using bacterial hemoglobin (VHb) for the treatment of aromatic organic compounds under hypoxic conditions. The application of VHb technology may advance treatment of contaminated sites, where oxygen availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic pollutants. Despite the many advantages of GEMs, there are still concerns that their introduction into polluted sites to enhance bioremediation may have adverse environmental effects, such as gene transfer. The extent of horizontal gene transfer from GEMs in the environment, compared to that of native organisms including benefits regarding bacterial bioremediation that may occur as a result of such transfer, is discussed. Recent advances in tracking methods and containment strategies for GEMs, including several biological systems that have been developed to detect the fate of GEMs in the environment, are also summarized in this review. Critical research questions pertaining to the development and implementation of GEMs for enhanced bioremediation have been identified and posed for possible future research.     

Production of intersexes and the evolution of androdioecy in the clam shrimp Eulimnadia texana (Crustacea,Branchiopoda, Spinicaudata)

Summary

The production of low numbers of offspring that exhibit a mixture of male and female traits (termed “intersexes”) is commonly reported for crustaceans. The production of intersexes has been ascribed to both genetic and non-genetic (e.g., parasitic infections and environmental pollutants) causes. Herein we report on two observed types of intersexes in the clam shrimp Eulimnadia texana: (1) a “morphological” intersex, possessing secondary male characteristics (e.g., claspers) and an eggproducing gonad, and (2) a “gonadal” intersex, possessing primarily male traits (e.g., male secondary sexual characters and male gamete production) but also producing low levels of abortive female gametes. We propose that these intersexes are likely the products of low frequencies of crossing over between the sex determining chromosomes that result in the array of observed mixed sexual phenotypes. Additionally, we suggest that the low-level production of intersexes, combined with the ephemeral nature of the habitats occupied by these shrimp, may explain the preponderance of androdioecy (mixtures of males and hermaphrodites) found in these clam shrimp, and possibly branchiopods more generally.