Transferability,development of simple sequence repeat (SSR) markers and application to the analysis of genetic diversity and population structure of the African fan palm (Borassus aethiopum Mart.) in Benin

Biological pathways play an important role in the occurrence, development and recovery of complex diseases, such as cancers, which are multifactorial complex diseases that are generally caused by mutation of multiple genes or dysregulation of pathways. We propose a path-specific effect statistic (PSE) to detect the differential specific paths under two conditions (e.g. case VS. control groups, exposure Vs. nonexposure groups). In observational studies, the path-specific effect can be obtained by separately calculating the average causal effect of each directed edge through adjusting for the parent nodes of nodes in the specific path and multiplying them under each condition. Theoretical proofs and a series of simulations are conducted to validate the path-specific effect statistic. Applications are also performed to evaluate its practical performances. A series of simulation studies show that the Type I error rates of PSE with Permutation tests are more stable at the nominal level 0.05 and can accurately detect the differential specific paths when comparing with other methods. Specifically, the power reveals an increasing trends with the enlargement of path-specific effects and its effect differences under two conditions. Besides, the power of PSE is robust to the variation of parent or child node of the nodes on specific paths. Application to real data of Glioblastoma Multiforme (GBM), we successfully identified 14 positive specific pathways in mTOR pathway contributing to survival time of patients with GBM. All codes for automatic searching specific paths linking two continuous variables and adjusting set as well as PSE statistic can be found in supplementary materials.  The proposed PSE statistic can accurately detect the differential specific pathways contributing to complex disease and thus potentially provides new insights and ways to unlock the black box of disease mechanisms.     

Lymphatic and Angiogenic Candidate Genes Predict the Development of Secondary Lymphedema following Breast Cancer Surgery

The purposes of this study were to evaluate for differences in phenotypic and genotypic characteristics in women who did and did not develop lymphedema (LE) following breast cancer treatment. Breast cancer patients completed a number of self-report questionnaires. LE was evaluated using bioimpedance spectroscopy. Genotyping was done using a custom genotyping array. No differences were found between patients with (n = 155) and without LE (n = 387) for the majority of the demographic and clinical characteristics. Patients with LE had a significantly higher body mass index, more advanced disease and a higher number of lymph nodes removed. Genetic associations were identified for four genes (i.e., lymphocyte cytosolic protein 2 (rs315721), neuropilin-2 (rs849530), protein tyrosine kinase (rs158689), vascular cell adhesion molecule 1 (rs3176861)) and three haplotypes (i.e., Forkhead box protein C2 (haplotype A03), neuropilin-2 (haplotype F03), vascular endothelial growth factor-C (haplotype B03)) involved in lymphangiogensis and angiogenesis. These genetic associations suggest a role for a number of lymphatic and angiogenic genes in the development of LE following breast cancer treatment.     

No Evidence That Soluble TACI Induces Signalling via Membrane-Expressed BAFF and APRIL in Myeloid Cells

Myeloid cells express the TNF family ligands BAFF/BLyS and APRIL, which exert their effects on B cells at different stages of differentiation via the receptors BAFFR, TACI (Transmembrane Activator and CAML-Interactor) and/or BCMA (B Cell Maturation Antigen). BAFF and APRIL are proteins expressed at the cell membrane, with both extracellular and intracellular domains. Therefore, receptor/ligand engagement may also result in signals in ligand-expressing cells via so-called “reverse signalling”. In order to understand how TACI-Fc (atacicept) technically may mediate immune stimulation instead of suppression, we investigated its potential to activate reverse signalling through BAFF and APRIL. BAFFR-Fc and TACI-Fc, but not Fn14-Fc, reproducibly stimulated the ERK and other signalling pathways in bone marrow-derived mouse macrophages. However, these effects were independent of BAFF or APRIL since the same activation profile was observed with BAFF- or APRIL-deficient cells. Instead, cell activation correlated with the presence of high molecular mass forms of BAFFR-Fc and TACI-Fc and was strongly impaired in macrophages deficient for Fc receptor gamma chain. Moreover, a TACI-Fc defective for Fc receptor binding elicited no detectable signal. Although these results do not formally rule out the existence of BAFF or APRIL reverse signalling (via pathways not tested in this study), they provide no evidence in support of reverse signalling and point to the importance of using appropriate specificity controls when working with Fc receptor-expressing myeloid cells.     

Targeting Photoreceptors via Intravitreal Delivery Using Novel,Capsid-Mutated AAV Vectors

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.     

Inactivation of Nitric Oxide by Uric Acid

The 1980 identification of nitric oxide (NO) as an endothelial cell-derived relaxing factor resulted in an unprecedented biomedical research of NO and established NO as one of the most important cardiovascular, nervous and immune system regulatory molecule. A reduction in endothelial cell NO levels leading to “endothelial dysfunction” has been identified as a key pathogenic event preceding the development of hypertension, metabolic syndrome, and cardiovascular disease. The reduction in endothelial NO in cardiovascular disease has been attributed to the action of oxidants that either directly react with NO or uncouple its substrate enzyme. In this report, we demonstrate that uric acid (UA), the most abundant antioxidant in plasma, reacts directly with NO in a rapid irreversible reaction resulting in the formation of 6-aminouracil and depletion of NO. We further show that this reaction occurs preferentially with NO even in the presence of oxidants peroxynitrite and hydrogen peroxide and that the reaction is at least partially blocked by glutathione. This study shows a potential mechanism by which UA may deplete NO and cause endothelial dysfunction, particularly under conditions of oxidative stress in which UA is elevated and intracellular glutathione is depleted.     

Functionality of Varroa-resistant honey bees (Hymenoptera: Apidae) when used in migratory beekeeping for crop pollination

Two types of honey bees, Apis mellifera L. (Hymenoptera: Apidae), bred for resistance to Varroa destructor Anderson & Trueman were evaluated for performance when used in migratory crop pollination. Colonies of Russian honey bees (RHB) and outcrossed bees with Varroa-sensitive hygiene (VSH) were managed without miticide treatments and compared with colonies of Italian honey bees that served as controls. Control colonies were managed as groups which either were treated twice each year against V. destructor (CT) or kept untreated (CU). Totals of 240 and 247 colonies were established initially for trials in 2008 and 2009, respectively. RHB and VSH colonies generally had adult and brood populations similar to those of the standard CT group regarding pollination requirements. For pollination of almonds [Prunus dulcis (Mill.) D.A.Webb] in February, percentages of colonies meeting the required six or more frames of adult bees were 57% (VSH), 56% (CT), 39% (RHB), and 34% (CU). RHB are known to have small colonies in early spring, but this can be overcome with appropriate feeding. For later pollination requirements in May to July, 94-100% of colonies in the four groups met pollination size requirements for apples (Malus domestica Borkh.), cranberries (Vaccinium macrocarpon Aiton), and lowbush blueberries (Vaccinium angustifolium Aiton). Infestations with V. destructor usually were lowest in CT colonies and tended to be lower in VSH colonies than in RHB and CU colonies. This study demonstrates that bees with the VSH trait and pure RHB offer alternatives for beekeepers to use for commercial crop pollination while reducing reliance on miticides. The high frequency of queen loss (only approximately one fourth of original queens survived each year) suggests that frequent requeening is necessary to maintain desired genetics.     

Spontaneous mutations in recombinant inbred mice: mutant toll-like receptor 4 (Tlr4) in BXD29 mice

Recombinant inbred (RI) mice are frequently used to identify QTL that underlie differences in measurable phenotypes between two inbred strains of mice. Here we show that one RI strain, C57BL/6J x DBA/2J (BXD29), does not develop an inflammatory response following inhalation of LPS. Approximately 25% of F2 mice [F1(BXD29 x DBA/2J) x F1] are also unresponsive to inhaled LPS, suggesting the presence of a recessive mutation in the BXD29 strain. A genomic scan of these F2 mice revealed that unresponsive animals, but not responsive animals, are homozygous for C57BL/6J DNA at a single locus on chromosome 4 close to the genomic location of Tlr4. All progeny between BXD29 and gene-targeted Tlr4-deficient mice are unresponsive to inhaled LPS, suggesting that the mutation in the BXD29 strain is allelic with Tlr4. Moreover, the intact Tlr4 receptor is not displayed on the cell surface of BXD29 macrophages. Finally, a molecular analysis of the Tlr4 gene in BXD29 mice revealed that it is interrupted by a large insertion of repetitive DNA. These findings explain the unresponsiveness of BXD29 mice to LPS and suggest that data from BXD29 mice should not be included when using BXD mice to study phenotypes affected by Tlr4 function. Our results also suggest that the frequency of such unidentified, spontaneously occurring mutations is an issue that should be considered when RI strains are used to identify QTL.     

Characterization of human mammary cell types in primary culture: Immunofluorescent and immunocytochemical indicators of cellular heterogeneity

Summary Parenchymal organoidal structures that were obtained from collagenase digestion of reduction mammoplasty specimens of apparently normal human breasts have been grown in short-term primary cultures, either on plastic or on floating gels of polymerized rat-tail collagen. Three morphologically distinct major cell types are readily observed in both systems: cuboidal cells, which occupy apical positions on collagen gels; larger, epithelioid, or basal cells on gels; and elongated cells which penetrate into the gel. In addition, a fourth cell type, that of a large, flat cell, is observed less readily by phase contrast microscopy on the surface of cultures grown on plastic. Immunofluorescent and immunocytochemical staining of cultures on plastic or histologic sections of cultures on gels have been undertaken with antisera and other histochemical reagents that stain the different parenchymal cell types in vivo. Thus antisera to epithelial membrane antigen(s), monoclonal antibodies (MABs) to the defatted mammary milk fat globule membrane, peanut lectin, and keratin MAB LE61, which preferentially stain the epithelial cells of ducts in vivo, also stain the cuboidal/apical cells in vitro. The large, flat cells are stained intensely by the first three reagents but not by the last one. Antisera to collagen IV, laminin, fibronectin, actin, keratin MAB LP34, MABs to the common acute lymphoblastic leukemia antigen, and MAB LICR-LON-23.10, which showed enhanced staining for the ductal myoepithelial cells in vivo, also stain the epithelioid/elongated cells in vitro. However, the effect of the last four reagents is reduced considerably in most elongated cells, and MAB LP34 stains the large, flat cells intensely. Heterogeneous cells of intermediate morphologies and staining patterns between the cuboidal/flat cells and large epithelioid cells have also been identified. The results suggest that the cuboidal cells and large, flat cells are related to mammary epithelial cells, whereas the large epithelioid/elongated cells have some characteristics of myoepithelial cells, and that intermediate forms may exist in culture between the two parenchymal cell types. This work was supported in part by the Ludwig Institute for Cancer Research and the Cancer and Polio Research Fund. Dr. M. J. Warburton is supported by the Cancer Research Campaign.     

Epidermal growth factor-conjugated poly(ethylene glycol)-block- poly(delta-valerolactone) copolymer micelles for targeted delivery of chemotherapeutics

Epidermal growth factor (EGF)-conjugated copolymer micelles were prepared from a mixture of diblock copolymers of methoxy poly(ethylene glycol)-block-poly(delta-valerolactone) (MePEG-b-PVL) and EGF-PEG-b-PVL for targeted delivery to EGF receptor (EGFR)-overexpressing cancers. The block copolymers and functionalized block copolymers were synthesized using PEG as the macroinitiator and HCl-diethyl ether as the catalyst. The MePEG-b-PVL and the carboxyl-terminated PEG-b-PVL (HOOC-PEG-b-PVL) copolymers were found to have molecular weights of 5940 and 5900, respectively, as determined by gel permeation chromatography (GPC) analyses. The HOOC-PEG-b-PVL copolymers were then activated by N-hydroxysuccinimide and subsequently reacted with EGF to form the EGF-PEG-b-PVL copolymers. The efficiency for the conjugation of EGF to the copolymer was found to be 60.9%. A hydrophobic fluorescent probe, CM-DiI, was loaded into both the nontargeted MePEG-b-PVL micelles and the targeted EGF-conjugated PEG-b-PVL micelles. The effective mean diameters of the CMDiI-loaded nontargeted and the CMDiI-loaded targeted micelles were found to be 32 +/- 1 nm and 45 +/- 2 nm, respectively, as determined by dynamic light scattering (DLS). The zeta potentials for the nontargeted micelles (no CM-DiI-loaded), CM-DiI-loaded nontargeted micelles, and CM-DiI-loaded targeted micelles were found to be -6.5, -8.7, and - 13.5 mV, respectively. Evaluation of the in vitro release of CM-DiI from the MePEG-b-PVL micelles in phosphate buffer saline (0.01 M, pH = 7.4) containing 10% (v/v) fetal bovine serum at 37 degrees C revealed that approximately 20% of the probe was released within the first 2 h. Confocal laser scanning microscopy (CLSM) analysis revealed that the targeted micelles containing CM-DiI accumulated intracellularly in EGFR-overexpressing MDA-MB-468 breast cancer cells following a 2 h incubation period, while no detectable cell uptake was observed for the nontargeted micelles. Results obtained from the confocal images were confirmed in an independent study by measuring the intracellular CM-DiI fluorescence in cell lysate. In addition, the presence of free EGF was found to decrease the extent of uptake of the targeted micelles. Nuclear staining of the cells with Hoechst 33258 indicated that the targeted micelles mainly localized in the perinuclear region and some of the micelles were localized in the nucleus. These results demonstrate that the EGF-conjugated copolymer micelles developed in this study have potential as vehicles for targeting hydrophobic drugs to EGFR-overexpressing cancers.     

Functional alterations after cardiac sodium-calcium exchanger overexpression in heart failure

The sodium-calcium exchanger (NCX) is discussed as one of the key proteins involved in heart failure. However, the causal role and the extent to which NCX contributes to contractile dysfunction during heart failure are poorly understood. NCX overexpression was induced by infection with an adenovirus coding for NCX, which coexpressed green fluorescence protein (GFP) (AdNCX) by ex vivo gene transfer to nonfailing and failing rabbit cardiomyocytes. Myocardial gene transfer in rabbits in vivo was achieved by adenoviral delivery via aortic cross-clamping. Peak cell shortening of cardiomyocytes was determined photo-optically. Hemodynamic parameters in vivo were determined by echocardiography (fractional shortening) and tip catheter [maximal first derivative of left ventricular (LV) pressure (dP/dt(max)); maximal negative derivative of LV pressure (-dP/dt(max))]. Peak cell shortening was depressed after NCX gene delivery in isolated nonfailing and in failing cardiomyocytes. In nonfailing rabbits in vivo, basal systolic contractility (fractional shortening and dP/dt(max)) and maximum rate of LV relaxation (-dP/dt(max)) in vivo were largely unaffected after NCX overexpression. However, during heart failure, long-term NCX overexpression over 2 wk significantly improved fractional shortening and dP/dt(max) compared with AdGFP-infected rabbits, both without inotropic stimulation and after beta-adrenergic stimulation with isoproterenol. -dP/dt(max) was also improved after NCX overexpression in the failing rabbits group. These results indicate that short-term effects of NCX overexpression impair contractility of isolated failing and nonfailing rabbit cardiomyocytes. NCX overexpression over 2 wk in vivo does not seem to affect myocardial contractility in nonfailing rabbits. Interestingly, in vivo overexpression of NCX decreased the progression of systolic and diastolic contractile dysfunction and improved beta-adrenoceptor-mediated contractile reserve in heart failure in rabbits in vivo.