Development of hydrogels for regenerative engineering

The aim of regenerative engineering is to restore complex tissues and biological systems through convergence in the fields of advanced biomaterials, stem cell science, and developmental biology. Hydrogels are one of the most attractive biomaterials for regenerative engineering, since they can be engineered into tissue mimetic 3D scaffolds to support cell growth due to their similarity to native extracellular matrix. Advanced nano‐ and micro‐technologies have dramatically increased the ability to control properties and functionalities of hydrogel materials by facilitating biomimetic fabrication of more sophisticated compositions and architectures, thus extending our understanding of cell‐matrix interactions at the nanoscale. With this perspective, this review discusses the most commonly used hydrogel materials and their fabrication strategies for regenerative engineering. We highlight the physical, chemical, and functional modulation of hydrogels to design and engineer biomimetic tissues based on recent achievements in nano‐ and micro‐technologies. In addition, current hydrogel‐based regenerative engineering strategies for treating multiple tissues, such as musculoskeletal, nervous and cardiac tissue, are also covered in this review. The interaction of multiple disciplines including materials science, cell biology, and chemistry, will further play an important role in the design of functional hydrogels for the regeneration of complex tissues.     

Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol.     

Extracting Curvature Preferences of Lipids Assembled in Flat Bilayers Shows Possible Kinetic Windows for Genesis of Bilayer Asymmetry and Domain Formation in Biological Membranes

Studies on the assembly of pure lipid components allow mechanistic insights toward understanding the structural and functional aspects of biological membranes. Molecular dynamic (MD) simulations on membrane systems provide molecular details on membrane dynamics that are difficult to obtain experimentally. A large number of MD studies have remained somewhat disconnected from a key concept of amphipathic assembly resulting in membrane structures—shape parameters of lipid molecules in those structures in aqueous environments. This is because most of the MD studies have been done on flat lipid membranes. With the above in view, we analyzed MD simulations of 26 pure lipid patches as a function of (1) lipid type(s) and (2) time of MD simulations along with 35–40 ns trajectories of five pure lipids. We report, for the first time, extraction of curvature preferences of lipids from MD simulations done on flat bilayers. Our results may lead to mechanistic insights into the possible origins of bilayer asymmetries and domain formation in biological membranes.     

In vitro and in vivo characterization of designed immunogens derived from the CD‐helix of the stem of influenza hemagglutinin

The conserved “stem” domain of influenza virus hemagglutinin (HA) is a target for broadly neutralizing antibodies and a potential vaccine antigen for induction of hetero‐subtypic protection. The epitope of 12D1, a previously reported bnAb neutralizing several H3 subtype influenza strains, was putatively mapped to residues 76–106 of the CD‐helix, also referred to as long alpha helix (LAH) of the HA stem. A peptide derivative consisting of wt‐LAH residues 76–130 conjugated to keyhole limpet hemocyanin was previously shown to confer robust protection in mice against challenge with influenza strains of subtypes H3, H1, and H5 which motivated the present study. We report the design of multiple peptide derivatives of LAH with or without heterologous trimerization sequences and show that several of these are better folded than wt‐LAH. However, in contrast to the previous study immunization of mice with wt‐LAH resulted in negligible protection against a lethal homologous virus challenge, while some of the newly designed immunogens could confer weak protection. Combined with structural analysis of HA, our data suggest that in addition to LAH, other regions of HA are likely to significantly contribute to the epitope for 12D1 and will be required to elicit robust protection. In addition, a dynamic, flexible conformation of isolated LAH peptide may be required for eliciting a functional anti‐viral response. Proteins 2013; 81:1759–1775. © 2013 Wiley Periodicals, Inc.     

Differential remodeling of cadherins and intermediate cytoskeletal filaments influence microenvironment of solid and ascitic sarcoma

Different forms of sarcoma (solid or ascitic) often pose a critical medical situation for pediatric or adolescent group of patients. To date, predisposed genetic anomalies and related changes in protein expression are thought to be responsible for sarcoma development. However, in spite of genetic abnormality, role of tumor microenvironment is also indispensable for the evolving neoplasm. In our present study, we characterized the deferentially remodeled microenvironment in solid and ascitic tumors by sequential immunohistochemistry and flowcytometric analysis of E-cdaherin, N-cadherin, vimentin, and cytokeratin along with angiogenesis and metastasis. In addition, we considered flowcytometric apoptosis and CD133 positive cancer stem cell analysis. Comparative hemogram was also considered as a part. Our investigation revealed that both types of tumor promoted neovascularization over time with sign of local inflammation. Invasion of neighboring skeletal muscle by solid sarcoma was more frequent than its ascitic counterpart. In contrary, rapid and earlier cadherin switching (E-cadherin to N-cadherin) in ascitic sarcoma made them more aggressive than that of solid sarcoma and helped to early metastasize distant tissue like liver through the hematogenous route. Differential cadherin switching and infidelity of cytokeratin expression in Vimentin positive sarcoma also influenced the behavior of ascitic CD133+ cancer initiating cell pool with respect to CD133+ cells housed in solid sarcoma. Therefore our study concludes that differential cadherin switching program and infidelity of intermediate filaments in part, sharply discriminate the severity and metastatic potentiality of either type of sarcoma accompanying with CD133+ cellular repertoire. Besides, tumor phenotype-based dichotomous cadherin switching program could be exploited as a future drug target to manage decompensated malignant ascitic and solid sarcoma.     

Drosophila Clueless Is Highly Expressed in Larval Neuroblasts,Affects Mitochondrial Localization and Suppresses Mitochondrial Oxidative Damage

Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu’s specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu’s role in mitochondrial function is not critical during larval development, but is important for pupae and adults.     

Quantification of photonic localization properties of targeted nuclear mass density variations: Application in cancer‐stage detection

Light localization is a phenomenon which arises due to the interference effects of light waves inside a disordered optical medium. Quantification of degree light localization in optical media is widely used for characterizing degree of structural disorder in that media. Recently, this light localization approach was extended to analyze structural changes in biological cell like heterogeneous optical media, with potential application in cancer diagnostics. Confocal fluorescence microscopy was used to construct “optical lattices,” which represents 2‐dimensional refractive index map corresponding to the spatial mass density distribution of a selected molecule inside the cell. The structural disorder properties of the selected molecules were evaluated numerically using light localization strength in these optical lattices, in a single parameter called “disorder strength.” The method showed a promising potential in differentiating cancerous and non‐cancerous cells. In this paper, we show that by quantifying submicron scale disorder strength in the nuclear DNA mass density distribution, a wide range of control and cancerous breast and prostate cells at different hierarchy levels of tumorigenicity were correctly distinguished. We also discuss how this photonic technique can be used in examining tumorigenicity level in unknown prostate cancer cells, and potential to generalize the method to other cancer cells.      

Responses of <Emphasis Type="Italic">Zea mays</Emphasis> L. cultivars ‘Buland’ and ‘Prakash’ to an antiozonant ethylene diurea grown under ambient and elevated levels of ozone

Increase in surface level of ozone (O₃) in last 30 years is one of the major problems for global agriculture. Field experiment was conducted using open top chambers on two Indian maize cultivars (Buland and Prakash) grown under ambient (AO) and elevated (EO) O₃ concentrations to evaluate the effect of an antiozonant ethylene diurea (EDU) given as soil drench. EDU application reduced the ROS production with concomitant decrease in lipid peroxidation. Inductions in activities of enzymatic antioxidants along with increased content of non-enzymatic antioxidants were observed in EDU-treated plants, though the response varied between the cultivars. Photosynthetic proteins (PEP carboxylase and RuBisCO large and small subunits) detected through SDS–PAGE analysis increased with EDU treatment. EDU also led to an increase in jasmonic acid and a decline in salicylic acid contents. The protective effect of EDU was further accompanied by increased pigments (chlorophyll and carotenoids), foliar carbohydrates (starch and total soluble sugars), enhanced biomass, and economic yield. Effectiveness of EDU was more evident at higher O₃ concentration and cultivar Prakash exhibited a more positive response with EDU as compared to Buland.