The Tumor Microenvironment: An Introduction to the Development of Microfluidic Devices.

The tumor microenvironment (TME) is like the Referee of a soccer match who has constant eyes on the activity of all players, such as cells, acellular stroma components, and signaling molecules for the successful completion of the game, that is, tumorigenesis. The cooperation among all the "team members" determines the characteristics of tumor, such as the hypoxic and acidic niche, stiffer mechanical properties, or dilated vasculature. Like in soccer, each TME is different. This heterogeneity makes it challenging to fully understand the intratumor dynamics, particularly among different tumor subpopulations and their role in therapeutic response or resistance. Further, during metastasis, tumor cells can disseminate to a secondary organ, a critical event responsible for approximately 90% of the deaths in cancer patients. The recapitulation of the rapidly changing TME in the laboratory is crucial to improve patients' prognosis for unraveling key mechanisms of tumorigenesis and developing better drugs. Hence, in this chapter, we provide an overview of the characteristic features of the TME and how to model them, followed by a brief description of the limitations of existing in vitro platforms. Finally, various attempts at simulating the TME using microfluidic platforms are highlighted. The chapter ends with the concerns that need to be addressed for designing more realistic and predictive tumor-on-a-chip platforms.

Synthesis and characterization of Cu/Ag nanoparticle loaded mullite nanocomposite system: A potential candidate for antimicrobial and therapeutic applications.

BACKGROUND: Microbial resistance to antibiotics has triggered the development of nanoscale materials as an alternative strategy. To stabilize these particles an inert support is needed. METHOD: Porous nanomullite developed by sol-gel route is loaded with copper and silver nanoparticle by simple adsorption method. These nanocomposites are characterized using XRD, FTIR, TEM, SEM, EDAX and UV-visible spectrophotometer. Antibacterial activity of these nanocomposites against Gram positive and Gram negative bacteria are performed by bactericidal kinetics, flow cytometry and MTT assay. The underlying mechanisms behind the antimicrobial property and cell death are also investigated by EPR spectroscopy, intracellular ROS measurement and ß-galactosidase assay. The cytocompatibility of the nanocomposites is investigated by cell viability (MTT), proliferation (Alamar blue) and wound healing assay of mammalian fibroblast cell line. RESULTS: Nanocomposites show a fairly uniform distribution of metal nanoparticle within mullite matrix. They show excellent antibacterial activity. Metal ions/nanoparticle is found to be released from the materials (CM and SM). Treated cells manifested high intracellular oxidative stress and ß-galactosidase activity in the growth medium. The effect of nanocomposites on mammalian cell line depends on exposure time and concentration. The scratch assay shows normal cell migration with respect to control. CONCLUSION: The fabricated nanoparticles possess diverse antimicrobial mechanism and exhibit good cytocompatibility along with wound healing characteristics in mouse fibroblast cell line (L929). GENERAL SIGNIFICANCE: The newly synthesized materials are promising candidates for the development of antimicrobial ceramic coatings for biomedical devices and therapeutic applications.

Uncommon otological And Head & Neck Manifestations of Wegener's Granulomatosis: A Rare Case Report.

Wegener's granulomatosis is a necrotising vasculitis affecting both arterioles and venules. The classical triad involves acute inflammation of upper airway along with inflammation of lower respiratory tract and renal involvement, however other organ system may also be affected. Our patient presented with severe unilateral earache, ear discharge, hearing loss and ipsilateral facial nerve palsy as the manifestations of the disease, which are rarely reported in medical literature1.

Combining ability analysis in bitter gourd (Momordica charantia L.) for potential quality improvement.

Combining ability analysis provides useful information for the selection of parents, also information regarding the nature and magnitude of involved gene actions. Crops improvement involves strategies for enhancing yield potentiality and quality components. Targeting the improvement of respective characters in bitter gourd, combining ability and genetic parameters for 19 characters were estimated from a 6×6 full diallel analysis technique. The results revealed that the variances due to general combining ability (GCA) and specific combining ability (SCA) were highly significant for most of the important characters. It indicated the importance of both additive and non-additive gene actions. GCA variances were higher in magnitude than SCA variances for all the characters studied indicating the predominance of the additive gene effects in their inheritance. The parent P2 (BG 009) appeared as the best general combiner for earliness; P1 (BG 006) for number of fruits, average single fruit weight and fruit yield; P4 (BG 027) for node number of first female flower and days to seed fruit maturity; P3 (BG 011) for fruit length and thickness of the fruit flesh; P5 (BG 033) for 100-seed weight; and P6 for number of nodes per main vine. The SCA effect as well as reciprocal effect was also significant for most of the important characters in different crosses.

Orientational order in liquid crystals exhibited by some binary mixtures of rod-like and bent-core molecules.

We report measurements of the temperature variations of the optical birefringence in the nematic (N) and partial bilayer SmA (SmA(d)) phases in 4-n-octyloxy 4(')-cyanobiphenyl made of rod-like (R) molecules and five mixtures of this compound with 1,3-phenylene bis[4-(3-methylbenzoyloxy)] 4(')-n-dodecylbiphenyl 4(')-carboxylate, made of bent-core (BC) molecules. The birefringence decreases with the concentration x of the BC molecules but the macroscopic order parameter initially decreases upto 11 mol% of BC molecules and subsequently increases with x. This is attributed to the possible formation of polar clusters of BC molecules. Orientation of BC molecules changes between the N and SmA(d) phases and the birefringence data in the two phases imply that the kink angle of the BC molecules is approximately 90 degrees rather than approximately 110 degrees as obtained from calculations which minimize the energy of the molecule. IR spectroscopic measurements on the mixture with 11 mol% of BC molecules have been used to estimate the molecular order parameter S of the R molecules, and to provide additional support for a relatively small kink angle of BC molecules.

Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus.

We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K(m) of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.

Orthopaedic applications of bone graft & graft substitutes: a review.

Treatment of delayed union, malunion, and nonunion is a challenge to the orthopaedic surgeons in veterinary and human fields. Apart from restoration of alignment and stable fixation, in many cases adjunctive measures such as bone-grafting or use of bone-graft substitutes are of paramount importance. Bone-graft materials usually have one or more components: an osteoconductive matrix, which acts as scaffold to new bone growth; osteoinductive proteins, which support mitogenesis of undifferentiated cells; and osteogenic cells, which are capable of forming bone in the appropriate environment. Autologous bone remains the "gold standard" for stimulating bone repair and regeneration, but its availability may be limited and the procedure to harvest the material is associated with complications. Bone-graft substitutes can either substitute autologous bone graft or expand an existing amount of autologous bone graft. We review the currently available bone graft and graft substitutes for the novel therapeutic approaches in clinical setting of orthopaedic surgery.

Development of porous HAp and ß-TCP scaffolds by starch consolidation with foaming method and drug-chitosan bilayered scaffold based drug delivery system.

The inability to maintain high concentrations of antibiotic at the site of infection for an extended period of time along with dead space management is still the driving challenge in treatment of osteomyelitis. Porous bioactive ceramics such as hydroxyapatite (HAp) and beta-tri calcium phosphate (ß-TCP) were some of the alternatives to be used as local drug delivery system. However, high porosity and high interconnectivity of pores in the scaffolds play a pivotal role in the drug release and bone resorption. Ceftriaxone is a cephalosporin that has lost its clinical popularity. But has recently been reported to exhibit better bactericidal activity in vitro and reduced probability of resistance development, in combination with sulbactam, a ß-lactamase inhibitor. In this article, a novel approach of forming HAp and pure ß-TCP based porous scaffolds by applying together starch consolidation with foaming method was used. For the purpose, pure HAp and ß-TCP were prepared in the laboratory and after thorough characterization (including XRD, FTIR, particle size distribution, etc.) the powders were used for scaffold fabrication. The ability of these scaffolds to release drugs suitably for osteomyelitis was studied in vitro. The results of the study indicated that HAp exhibited better drug release profile than ß-TCP when drug was used alone indicating the high influence of the carrier material. However, this restriction got relaxed when a bilayered scaffold was formed using chitosan along with the drug. SEM studies along with EDAX on the drug-chitosan bilayered scaffold showed closest apposition of this combination to the calcium phosphate surface.

Biodiversity of phosphate solubilizing bacteria in rhizosphere of chickpea, mustard and wheat grown in different regions of Haryana.

The native population of phosphate solubilizing bacteria (PSB) was studied in the rhizosphere of chickpea, mustard and wheat grown in different regions of Haryana. A total of 193 PSB were isolated from 245 rhizospheric samples collected from south-west and north-east zones. The PSB count showed large variations (3-67 × 10(5)cfu/g) and biodiversity within the crop and place of sampling. Using biochemical analysis, the isolates were tentatively identified as belonging to four genera, Pseudomonas, Aeromonas, Klebsiella and Enterobacter. Phosphate solubilization of these isolates varied from 5.9 to 123.8% and 2.2 to 227.2 µg/ml in solid and liquid Pikovskaya's medium, respectively. Based on their morphological traits, all the isolates were placed into 20 groups, majority of them falling in the group having white, round and gummy colonies, irrespective of the crop or the region. The intrinsic antibiotic resistance pattern showed large variations among the isolates and most of the isolates were resistant to streptomycin, ampicillin and penicillin. The highest PSB number and greatest variability were found in the rhizosphere of chickpea, followed by wheat and then mustard.

Mechanical Property of Hydrogels and the Presence of Adipose Stem Cells in Tumor Stroma Affect Spheroid Formation in the 3D Osteosarcoma Model.

Osteosarcoma is one of the most common metastatic bone cancers, which results in significant morbidity and mortality. Unfolding of effectual therapeutic strategies against osteosarcoma is impeded because of the absence of adequate animal models, which can truly recapitulate disease biology of humans. Tissue engineering provides an opportunity to develop physiologically relevant, reproducible, and tunable in vitro platforms to investigate the interactions of osteosarcoma cells with its microenvironment. Adipose-derived stem cells (ASCs) are detected adjacent to osteosarcoma masses and are considered to have protumor effects. Hence, the present study focuses on investigating the role of reactive ASCs in formation of spheroids of osteosarcoma cells (Saos 2) within a three-dimensional (3D) niche, which is created using gellan gum (GG)-silk fibroin. By modifying the blending ratio of GG-silk, the optimum stiffness of the resultant hydrogels such as GG and GG75: S25 is obtained for cancer spheroid formation. This work indicates that the co-existence of cancer and stem cells can form a spheroid, the hallmark of cancer, only in particular microenvironment stiffness. The incorporation of fibrillar silk fibroin within the hydrophilic network of GG in GG75: S25 spongy-like hydrogels closely mimics the stiffness of commercially established cancer biomaterials (e.g., Matrigel, HyStem). The GG75: S25 hydrogel maintains the metabolically active construct for a longer time with elevated expression of osteopontin, osteocalcin, RUNX 2, and bone sialoprotein genes, the biomarkers of osteosarcoma, compared to GG. The GG75: S25 construct also exhibits intense alkaline phosphatase expression in immunohistochemistry compared to GG, indicating itspotentiality to serve as biomimetic niche to model osteosarcoma. Taken together, the GG-silk fibroin-blended spongy-like hydrogel is envisioned as an alternative low-cost platform for 3D cancer modeling.

Template mediated protein self-assembly as a valuable tool in regenerative therapy.

The assembly of natural proteinaceous biopolymers into macro-scale architectures is of great importance in synthetic biology, soft-material science and regenerative therapy. The self-assembly of protein tends to be limited due to anisotropic interactions among protein molecules, poor solubility and stability. Here, we introduce a unique platform to self-immobilize diverse proteins (fibrous and globular, positively and negatively charged, low and high molecular weight) using silicon surfaces with pendant -NH2 groups via a facile one step diffusion limited aggregation (DLA) method. All the experimental proteins (type I collagen, bovine serum albumin and cytochrome C) self-assemble into seaweed-like branched dendritic architectures via classical DLA in the absence of any electrolytes. The notable differences in branching architectures are due to dissimilarities in protein colloidal sub-units, which is typical for each protein type, along with the heterogeneous distribution of surface -NH2 groups. Fractal analysis of assembled structures is used to explain the underlying route of fractal deposition; which concludes how proteins with different functionality can yield similar assembly. Further, the nano-micro-structured surfaces can be used to provide functional topographical cues to study cellular responses, as demonstrated using rat bone marrow stem cells. The results indicate that the immobilization of proteins via DLA does not affect functionality, instead serving as topographical cues to guide cell morphology. This indicates a promising design strategy at the tissue-material interface and is anticipated to guide future surface modifications. A cost-effective standard templating strategy is therefore proposed for fundamental and applied particle aggregation studies, which can be used at multiple length scales for biomaterial design and surface reformation.

A novel dodecapeptide from a combinatorial synthetic library exhibits potent antifungal activity and synergy with standard antimycotic agents.

There has been a marked expansion in the discovery of new antifungal peptides. This paper describes a novel dodecapeptide, H-Arg-Trp-Trp-Arg-D-Trp-D-Phe-Ile-D-Phe-His-Trp-Arg-Trp-NH(2), derived from a previously described nonapeptide and synthesized by the combinatorial approach. Further, interaction of this peptide with antifungals such as amphotericin B, flucytosine and fluconazole was studied by checkerboard analysis and time-kill assay to obtain the dynamic picture with respect to time. The best synergistic activity was observed with a combination of peptide and fluconazole, followed by peptide and flucytosine.

Towards determination of the structure of the Saccharomyces cerevisiae a-factor: an acylated pentadecapeptide blocks a-factor activity.

Putative a-factor peptides YIIKGVFWADP, YIIKGVFWANP, YIIKGLFWADP, YIIKGLFWANP, YIIKGVFWDPA, and YIIKGVFWDPACVIA and several peptide derivatives were synthesized and were found to be inactive in growth arrest assays, yet they blocked the activity of biological a-factor. Antagonism was greatest with YIIKGVFWDPAC(palmitoyl)VIA. Thus, the structure of a-factor may be a lipopeptide resembling this palmitoylated pentadecapeptide.

Paddy straw as substrate for ethanol production.

Pretreatment of paddy straw with 2% sodium hydroxide at 15 psi for 1 h resulted in 83% delignification. The hydrolysis of alkali treated paddy straw with a commercial preparation of cellulase for 2 h at 50°C resulted in release of 65% total reducing sugars. Maximum sugars were released at enzyme loading of 1.5% (v/v). The fermentation of hydrolysate supplemented with nutrients by S. cerevisiae resulted in the production of 20-30 g L(-1) ethanol after 48 h incubation which was further improved with addition of yeast nitrogen base and inoculated with 1% (w/v) yeast cells.

Silk fibroin hydrogel as physical barrier for prevention of post hernia adhesion.

BACKGROUND: Adhesion formation remains a major complication following hernia repair surgery. Physical barriers though effective for adhesion prevention in clinical settings are associated with major disadvantages, therefore, needs further investigation. This study evaluates silk fibroin hydrogel as a physical barrier on polypropylene mesh for the prevention of adhesion following ventral hernia repair. STUDY DESIGN: Peritoneal explants were cultured on silk fibroin scaffold to evaluate its support for mesothelial cell growth. Full thickness uniform sized defects were created on the ventral abdominal wall of rabbits, and the defects were covered either with silk hydrogel coated polypropylene mesh or with plain polypropylene mesh as a control. The animals were killed after 1 month, and the adhesion formation was graded; healing response of peritoneum was evaluated by immunohistochemistry with calretinin, collagen staining of peritoneal sections, and expression of PCNA, collagen-I, TNFα, IL6 by real time PCR; and its adverse effect if any was determined. RESULTS: Silk fibroin scaffold showed excellent support for peritoneal cell growth in vitro and the cells expressed calretinin. A remarkable prevention of adhesion formation was observed in the animals implanted with silk hydrogel coated mesh compared to the control group; in these animals peritoneal healing was complete and predominantly by mesothelial cells with minimum fibrotic changes. Expression of inflammatory cytokines decreased compared to control animals, histology of abdominal organs, haematological and blood biochemical parameters remained normal. CONCLUSION: Therefore, silk hydrogel coating of polypropylene mesh can improve peritoneal healing, minimize adhesion formation, is safe and can augment the outcome of hernia surgery.

Identification of a novel antifungal nonapeptide generated by combinatorial approach.

It is becoming clear that antimicrobial peptides are important components of the innate defences of all species of life. They kill very rapidly, do not easily select resistant mutants and are synergistic with potentially toxic conventional therapeutic agents against microbes. This paper describes an attempt to expand a lead hexapeptide motif synthesized through combinatorial approach. A cationic peptide H-Arg-Trp-Trp-Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH2 was found to be active with a therapeutic index of >17. I was proposed that the combination of peptide with known antifungal agents may identify synergistic combinations that would ideally reduce the dosage of conventional antifungals as well as their associated toxicity. Nine different pathogenic strains and species of Candida and two of Cryptococcus neoformans were employed in chequerboard method and in time kill assays to evaluate the synergistic effect of the lead peptide in combination with amphotericin B, 5-flucytosine, ketoconazole and fluconazole. We found synergistic interaction between the peptide and all four drugs against Cryptococcus isolates whilst both synergistic and additive combinations occurred when Candida isolates were used.

Identification of enzyme inhibitors using combinatorial libraries.

Potent enzyme inhibitors have long been recognized as powerful tools for assessing the physiological roles of enzymes and have led to the therapeutic drugs able to modulate their activities in vivo. However, to be valuable tools such inhibitors should be selective so that they do not interfere with other members of the particular enzyme family. Combinatorial chemistry has proven to be a novel approach for the identification of molecules with a desired selectivity profile from the libraries of several million compounds. In recent years it has been extensively used in conjunction with computational methods for the development of potent inhibitors of therapeutically interesting targets. This review describes the various structurally diverse enzyme inhibitors identified by screening combinatorial libraries of peptides and small organic molecules.

Structure--activity relationships of the didemnins.

Bioactivities of 42 didemnin congeners, either isolated from the marine tunicates Trididemnun solidum and Aplidium albicans or prepared synthetically and semisynthetically, have been compared. The growth inhibition of various murine and human tumor cells and plaque reduction of HSV-1 and VSV grown on cultured mammalian cells were used to assess cytotoxicity and antiviral activity. Biochemical assays for macromolecular synthesis (protein, DNA, and RNA) and enzyme inhibition (dihydrofolate reductase, thymidylate synthase, DNA polymerase, RNA polymerase, and topoisomerases I and II) were also performed to specify the mechanisms of action of each analogue. Immunosuppressive activity of the didemnins was determined using a mixed lymphocyte reaction (MLR) assay. These assays revealed that the native cyclic depsipeptide core is an essential structural requirement for most of the bioactivites of the didemnins, especially for cytotoxicities and antiviral activities. The linear side-chain portion of the peptide can be altered with a gain, in some cases, of bioactivities. In particular, dehydrodidemnin B, tested against several types of tumor cells and in in vivo studies in mice, as well as didemnin M, tested for the mixed lymphocyte reaction and graft vs host reaction in murine systems, showed remarkable gains in their in vitro and in vivo activities compared to didemnin B.

Molecular mechanism of host specificity in legume-rhizobium symbiosis.

Rhizobium - legume symbiosis is a highly specific interaction between the two partners. Host specificity is evident at early stages of infection and results from multiple interactions involving signalling among bacteria and host plants. Host specific plant signals (flavanoids) convert the NodD protein to an active form and its binding with nod box initiates the transcription of inducible nod operons. Common nod genes (nodABC) code for an extracellular mitogenic Nod factor which is required for nodule organogenesis. Host specific genes (hsn) modify the Nod factor to induce root hair deformation on specific hosts. The structure of Nod factor controls host range distinction between species and biovars of rhizobia. Interactions of lectins and Exopolysaccharide/Lipopolysaccharide result in host specific attachment of Rhizobium and its subsequent invasion. Change in Expopolysaccharide structure by the transfer of hsn genes enables the Rhizobium to bind with heterologous host lectins. Conversely, changes in root lectins via gene manipulation enables the heterologous rhizobia to bind and initiate nodulation on heterologous hosts. Finally, host specific signals are required to initiate nitrogen fixation in nodules that are formed.

Identification of novel alpha-glucosidase inhibitors by screening libraries based on N- [4-(benzyloxy) benzoyl] alanine derivatives.

A library of 72 compounds related to N- [4-(benzyloxy) benzoyl]alanine (I) was synthesized, prepared and screened for alpha-glucosidase inhibitory activity. Four compounds showed potent inhibition, six compounds moderate inhibition, and 16 were weak inhibitors. One compound, N- [4-(benzyloxy) benzoyl] serine, was found to be a potent inhibitor of alpha-glucosidase with 100% inhibition at 1 micro M. This inhibitor was at least five times more potent than the lead compound I.