Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.     

Thrombin binding aptamer analogues containing inversion of polarity sites endowed with antiproliferative and anti-motility properties against Calu-6 cells

Background

Although the thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative properties, it is possible to reduce the first and enhance the second one by suitable chemical modifications.

The Sub-Cellular Localization of WRAP53 Has Prognostic Impact in Breast Cancer

WRAP53 protein controls intracellular trafficking of DNA repair proteins, the telomerase enzyme, and splicing factors. Functional loss of the protein has been linked to carcinogenesis, premature aging and neurodegeneration. The aim of this study was to investigate the prognostic significance of WRAP53 protein expression in breast cancer. A tissue microarray was constructed from primary breast tumors and immunostained by a polyclonal WRAP53 antibody to assess the protein expression pattern. Two different patient cohorts with long term follow-up were studied; a test- and a validation set of 154 and 668 breast tumor samples respectively. Breast cancer patients with tumor cells lacking the expression of WRAP53 in the nucleus had a significantly poorer outcome compared to patients with tumor cells expressing this protein in the nuclei (HR = 1.95, 95%CI = 1.09–3.51, p = 0.025). Nuclear localization of WRAP53 was further shown to be an independent marker of prognosis in multivariate analysis (HR = 2.57, 95%CI = 1.27–5.19, p = 0.008), and also significantly associated with better outcome in patients with TP53 mutation. Here we show that the sub-cellular localization of the WRAP53 protein has a significant impact on breast cancer survival, and thus has a potential as a clinical marker in diagnostics and treatment.     

Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation

Background

Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation.

Results

To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells.

Conclusions

We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors.

     

A trypsin and chymotrypsin inhibitor from seeds of Bauhenia purpurea

A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other.     

Mutations to azaguanine resistance induced in cultured diploid human fibroblasts by the carcinogen, N-acetoxy-2-Acetylaminofluorene

The ability of the carcinogen, N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), to induce mutations to azaguanine resistance in diploid human cells was quantitatively investigated and shown to be dose-dependent. The 8-azaguanine (AG) resistance was shown to be heritable in the absence of mutagen or selective agent and the cells of the mutant clones were shown to retain normal sensitivity to N-AcO-AAF.     

L-leucine stimulation of glucose uptake and utilization involves modulation of glucose – lipid metabolic switch and improved bioenergetic homeostasis in isolated rat psoas muscle ex vivo

Amino Acids - The antidiabetic effect of l-leucine has been attributed to its modulatory effect on glucose uptake and lipid metabolism in muscles. However, there is a dearth on its effect on...