Aptamer Based,Non-PCR,Non-Serological Detection of Chagas Disease Biomarkers in Trypanosoma cruzi Infected Mice

Chagas disease affects about 5 million people across the world. The etiological agent, the intracellular parasite Trypanosoma cruzi (T. cruzi), can be diagnosed using microscopy, serology or PCR based assays. However, each of these methods has their limitations regarding sensitivity and specificity, and thus to complement these existing diagnostic methods, alternate assays need to be developed. It is well documented that several parasite proteins called T. cruzi Excreted Secreted Antigens (TESA), are released into the blood of an infected host. These circulating parasite antigens could thus be used as highly specific biomarkers of T. cruzi infection. In this study, we have demonstrated that, using a SELEx based approach, parasite specific ligands called aptamers, can be used to detect TESA in the plasma of T. cruzi infected mice. An Enzyme Linked Aptamer (ELA) assay, similar to ELISA, was developed using biotinylated aptamers to demonstrate that these RNA ligands could interact with parasite targets. Aptamer L44 (Apt-L44) showed significant and specific binding to TESA as well as T. cruzi trypomastigote extract and not to host proteins or proteins of Leishmania donovani, a related trypanosomatid parasite. Our result also demonstrated that the target of Apt-L44 is conserved in three different strains of T. cruzi. In mice infected with T. cruzi, Apt-L44 demonstrated a significantly higher level of binding compared to non-infected mice suggesting that it could detect a biomarker of T. cruzi infection. Additionally, Apt-L44 could detect these circulating biomarkers in both the acute phase, from 7 to 28 days post infection, and in the chronic phase, from 55 to 230 days post infection. Our results show that Apt-L44 could thus be used in a qualitative ELA assay to detect biomarkers of Chagas disease.     

The use of inter-specific hybrids in aquaculture and fisheries

Inter-specific hybrid fishes have been produced for aquaculture and stocking programmes to increase growth rate, transfer desirable traits between species, combine desirable traits of two species into a single group of fishes, reduce unwanted reproduction through production of sterile fish or mono-sex offspring, take advantage of sexual dimorphism, increase harvestability, increase environmental tolerances, and to increase overall hardiness in culture conditions. Hybrids constitute a significant proportion of some countries' production for certain taxa; for example, hybrid striped bass in the USA, hybrid clarid catfish in Thailand, hybrid characids in Venezuela, and hybrid tilapia in Israel. Despite its widespread use, there is a general impression that inter-specific hybridization is not a very useful tool for aquaculture. We believe this impression stems from inaccurate reporting of some useful hybrids, limited testing of strains used for hybrids, and from early work on salmonids that did not result in hybrids of commercial advantage.Experimentation with new hybrid fishes is ongoing, especially in marine culture systems where sterile fish may be preferred because of the concern that fish may escape into the marine and coastal environment.Hybridization has been used in tandem with polyploidization to improve developmental stability in hybrid progeny. The results of inter-specific hybridization can be variable and depend on the genetic structure (including the sex) of the parent fish. Inadvertent hybridization and backcrossing can lead to unexpected and undesirable results in hybrid progeny, such as failure to produce sterile fish, loss of color pattern, and reduced viability.Hybridization is only one tool to improve aquaculture production and will require knowledge of the genetic structure of the broodstock, good broodstock management and monitoring of the viability and fertility of the progeny. Hybridization does represent a genetic modification wherein genes are moved between different species; implications for biodiversity conservation and regulation of this type of modification are discussed.     

Comparison of different European strains of Trichogramma aurosum (Hymenoptera: Trichogrammatidae) using fertility life tables

Life table parameters were assessed for seven strains of Trichogramma aurosum Sugonjaev and Sorokina (Hymenoptera: Trichogrammatidae) collected in different European countries, in order to compare their performance when reared on eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) as a potential factitious host for mass-rearing. The average number of progeny per female, cumulative fertility and emergence rate did not differ significantly, whereas female longevity and sex ratio significantly differed between the seven parasitoid strains. The Danish strain survived the longest (6.05 days) and the Dutch strain survived the shortest (2.75 days). Progeny was always female-biased with varying proportions (57.7-96.7%). Survival rates started to decrease after 3 days for some of the strains studied. The mean cohort generation duration (T_c) was 11.40, 10.15, 10.62, 10.63, 9.28, 9.70 and 11.30 days for the Austrian, Luxemburgian, Belgian, French, Dutch, Danish and German strains, respectively. Population doubling time (D_t) was 4.50, 7.96, 3.56, 5.30, 5.23, 7.36 and 3.30 days, respectively. Daily intrinsic rate of increase (r_m) and finite rate of increase (exp. r_m) ranged between 0.087 and 0.210 and 1.091-1.233, respectively. The German strain might be a potential candidate for mass rearing and releases against the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), due to its high net reproduction rate (R₀=10.65 female), a high intrinsic rate of natural increase (r_m=0.210), a high finite rate of increase (exp. r_m=1.23), and a short population doubling time (D_t=3.3 days). The relevance of intra- and interstrain variability as well as the usefulness of fertility life tables for pre-introductory research is discussed.     

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.

Methods

Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.

Results

In vitro binding experiments of ¹²⁵I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.

Conclusions

These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.     

Graphene Doping Induced Tunability of Nanoparticles Plasmonic Resonances

Interest in graphene has been widely increasing since its discovery in 2004. Research on graphene for plasmonic applications has also boomed due to the high potential of these systems. In this article, we discuss the possible interaction between metallic NPs and graphene monolayer. We show how the contact between metallic NPs and graphene results in graphene doping. More importantly, we experimentally put into evidence the possible modulation of the plasmonic resonance of NPs by graphene doping. Understanding and evidencing this interaction is highly important both from a fundamental point of view and for specific applications such as active plasmonic devices.

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Surface functionalization of nanobiomaterials for application in stem cell culture,tissue engineering,and regenerative medicine

Stem cell‐based approaches offer great application potential in tissue engineering and regenerative medicine owing to their ability of sensing the microenvironment and respond accordingly (dynamic behavior). Recently, the combination of nanobiomaterials with stem cells has paved a great way for further exploration. Nanobiomaterials with engineered surfaces could mimic the native microenvironment to which the seeded stem cells could adhere and migrate. Surface functionalized nanobiomaterial‐based scaffolds could then be used to regulate or control the cellular functions to culture stem cells and regenerate damaged tissues or organs. Therefore, controlling the interactions between nanobiomaterials and stem cells is a critical factor. However, surface functionalization or modification techniques has provided an alternative approach for tailoring the nanobiomaterials surface in accordance to the physiological surrounding of a living cells; thereby, enhancing the structural and functional properties of the engineered tissues and organs. Currently, there are a variety of methods and technologies available to modify the surface of biomaterials according to the specific cell or tissue properties to be regenerated. This review highlights the trends in surface modification techniques for nanobiomaterials and the biological relevance in stem cell‐based tissue engineering and regenerative medicine. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:554–567, 2016     

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.     

Differential regulation of glucose transporter 1 and 2 mRNA expression by epidermal growth factor and transforming growth factor-beta in rat hepatocytes.

We have examined by Northern blot analysis the expression of two members of the glucose transporter family of genes (GLUT-1 and GLUT-2) in regenerating liver and in hepatocytes cultured under various conditions. GLUT-1, although thought to be a growth-associated gene, is not expressed in normal or regenerating liver, whereas GLUT-2, a liver-specific gene, is abundant in normal liver and gradually up-regulated during liver regeneration. Conversely, in hepatocytes cultured conventionally on dried rat tail collagen (RTC) in the presence of EGF and insulin, which potentiate proliferation, GLUT-1 mRNA is rapidly and abundantly expressed, whereas GLUT-2 is depressed. To investigate the causes of this "switch" in glucose transporter expression seen when hepatocytes are removed from the liver and cultured under the conventional proliferative conditions, we examined the effects of specific growth factors and extracellular matrices on cultured hepatocytes. EGF, a potent liver mitogen, although causing a threefold induction of GLUT-1, was found to have no effect on GLUT-2 expression, suggesting that the increase in GLUT-2 seen in regenerating liver is not due to EGF. Inhibition of protein synthesis by cycloheximide in cultured hepatocytes does not prevent the induction of GLUT-1 mRNA. In addition, treatment of cells with cycloheximide appears to stabilize the GLUT-2 mRNA, preventing the usual down-regulation of this gene in cultured hepatocytes. The expression of the two glucose transporter mRNAs also differed when the hepatocytes were adherent to particular cell matrices. Culture of hepatocytes on a reconstituted basement membrane gel matrix (EHS) is known to restrain their growth and mediate high levels of differentiated hepatocytic functions that are lost under conventional culture conditions. Unlike cells on RTC, hepatocytes on EHS expressed low levels of GLUT-1 mRNA, and decreased GLUT-2 mRNA. TGF-beta, an attenuator of DNA synthesis, when added to cultures on RTC, substantially down-regulated GLUT-2 but had no effect on GLUT-1. We propose that the effectors, EGF, TGF-beta and basement membrane components, play a significant role in the regulation of expression of GLUT-1 and GLUT-2 in hepatocytes.     

Effect of radiation on some haematological parameters and its modification by vitamin E in chicks.

White leghorn male chicks of 1 and 7 day age groups were studied for acute (2.25 Gy) gamma radiation (with or without vit. E pretreatment) induced haematological changes in the peripheral blood at days 1, 3, 5, 7, 14 and 28 postirradiation. A continuous decrease in the erythrocyte numbers was observed in the animals irradiated without vit. E treatment. The changes in haematocrit, haemoglobin, MCV, MCH and MCHC values were in line with the erythrocytic changes reflecting radiation induced damage to the erythroid elements. Animals pretreated with vit. E show lesser depression in the erythrocytic component at all the stages indicating its radio-protective influence. The significant increase in the immature RBC's in the peripheral blood in vit. E treated animals after irradiation, implies enhanced erythropoiesis.