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Supplementary Data from Systemic Administration of Polymeric Nanoparticle-Encapsulated Curcumin (NanoCurc) Blocks Tumor Growth and Metastases in Preclinical Models of Pancreatic Cancer

Supplementary Figure 2 from Systemic Administration of Polymeric Nanoparticle-Encapsulated Curcumin (NanoCurc) Blocks Tumor Growth and Metastases in Preclinical Models of Pancreatic Cancer

Curcumin or diferuloylmethane is a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa). A large volume (several hundreds) of published reports has established the anticancer and chemopreventative properties of curcumin in preclinical models of every known major cancer type. Nevertheless, the clinical translation of curcumin has been significantly hampered due to its poor systemic bioavailability, which mandates that patients consume up to 8 to 10 g of the free drug orally each day to achieve detectable levels in circulation. We have engineered a polymeric nanoparticle encapsulated curcumin formulation (NanoCurc) that shows remarkably higher systemic bioavailability in plasma and tissues compared with free curcumin upon parenteral administration. In xenograft models of human pancreatic cancer established in athymic mice, administration of parenteral NanoCurc significantly inhibits primary tumor growth in both subcutaneous and orthotopic settings. The combination of parenteral NanoCurc with gemcitabine results in enhanced tumor growth inhibition versus either single agent, suggesting an additive therapeutic influence in vivo. Furthermore, this combination completely abrogates systemic metastases in orthotopic pancreatic cancer xenograft models. Tumor growth inhibition is accompanied by significant reduction in activation of nuclear factor-κB, as well as significant reduction in expression of matrix metalloproteinase-9 and cyclin D1, in xenografts treated with NanoCurc and gemcitabine. NanoCurc is a promising new formulation that is able to overcome a major impediment for the clinical translation of curcumin to cancer patients by improving systemic bioavailability, and by extension, therapeutic efficacy. Mol Cancer Ther; 9(8); 2255–64. ©2010 AACR.

Supplementary Figure 1 from Systemic Administration of Polymeric Nanoparticle-Encapsulated Curcumin (NanoCurc) Blocks Tumor Growth and Metastases in Preclinical Models of Pancreatic Cancer

Supplementary Figure 3 from Systemic Administration of Polymeric Nanoparticle-Encapsulated Curcumin (NanoCurc) Blocks Tumor Growth and Metastases in Preclinical Models of Pancreatic Cancer

Supplementary Figure 5 from Systemic Administration of Polymeric Nanoparticle-Encapsulated Curcumin (NanoCurc) Blocks Tumor Growth and Metastases in Preclinical Models of Pancreatic Cancer

Supplementary Figure 4 from Systemic Administration of Polymeric Nanoparticle-Encapsulated Curcumin (NanoCurc) Blocks Tumor Growth and Metastases in Preclinical Models of Pancreatic Cancer

Magnolia bark extract (MBE) is a natural product used as an anti-inflammatory, anti platelet, and chemo-preventive agent. Here, we investigate the effects of MBE on the self perceived freshness of breath evaluated in ten human volunteers, who chewed gum with and without MBE added, as a functional food. Furthermore, the effects of exposing oral bacteria to MBE on cell-surface hydrophobicity were determined and bactericidal effects of MBE in human saliva were assessed. Volunteers perceived freshness of breath similar directly after chewing a gum with or without MBE added, but 60 min after chewing MBE gum, freshness was perceived significantly better. Also, cell-surface hydrophobicity of Gram-negative, but not of Gram-positiye bacteria, increased dose-responsively after exposure to MBE, while spiking of saliva with MBE did not affect bacterial viability. Taken together, MBE gum improves self-perceived freshness of breath likely due to an increase in cell-surface hydrophobicity of Gram-negative bacteria, instead of a bactericidal mechanism. (C) 2016 Elsevier Ltd. All rights reserved.

Introduction: Over the years, chewing gum has developed from a candy towards an oral health-promoting nutraceutical. This review summarizes evidence for the oral health benefits of chewing gum, emphasizing identification of active ingredients in gum that facilitate prevention and removal of oral biofilm. Areas covered: Chewing of sugar-free gum yields oral health benefits that include clearance of food debris, reduction in oral dryness, increase of biofilm pH and remineralization of enamel. These basic effects of chewing gum are attributed to increased mastication and salivation. Active ingredients incorporated in chewing gums aim to expand these effects to inhibition of extrinsic tooth stain and calculus formation, enhanced enamel remineralization, reduction of the numbers of bacteria in saliva and amount of oral biofilm, neutralization of biofilm pH, and reduction of volatile sulfur compounds. Expert opinion: Evidence for oral-health benefits of chewing gum additives is hard to obtain due to their relatively low concentrations and rapid wash-out. Clinical effects of gum additives are overshadowed by effects of increased mastication and salivation due to the chewing of gum and require daily chewing of gum for prolonged periods of time. Future studies on active ingredients should focus on specifically targeting pathogenic bacteria, whilst leaving the healthy microbiome unaffected.

Inflammatory diseases of the periodontal tissues are known health problems worldwide. Therefore, anti-inflammatory active compounds are used in oral care products to reduce long-term inflammation. In addition to inducing inflammation, pathogen attack leads to an increased production of reactive oxygen species (ROS), which may lead to oxidative damage of macromolecules. Magnolia officinalis L. bark extract (MBE) has been shown to possess antioxidant and anti-inflammatory potential in vitro. In the present study, the influence of MBE-fortified chewing gum on the resistance against lipopolysaccharide (LPS)-induced inflammation and oxidative stress of oral epithelial cells was investigated in a four-armed parallel designed human intervention trial with 40 healthy volunteers. Ex vivo stimulation of oral epithelial cells with LPS from Porphyromonas gingivalis for 6[Formula: see text]h increased the mRNA expression and release of the pro-inflammatory cytokines IL-1[Formula: see text], IL-[Formula: see text], IL-8, MIP-1[Formula: see text], and TNF[Formula: see text]. Chewing MBE-fortified gum for 10[Formula: see text]min reduced the ex vivo LPS-induced increase of IL-8 release by 43.8 [Formula: see text] 17.1% at the beginning of the intervention. In addition, after the two-week intervention with MBE-fortified chewing gum, LPS-stimulated TNF[Formula: see text] release was attenuated by 73.4 [Formula: see text] 12.0% compared to chewing regular control gum. This increased resistance against LPS-induced inflammation suggests that MBE possesses anti-inflammatory activity in vivo when added to chewing gum. In contrast, the conditions used to stimulate an immune response of oral epithelial cells failed to induce oxidative stress, measured by catalase activity, or oxidative DNA damage.

The micromolar calcium-activated neutral protease (CAPN1) gene encodes μ-calpain that degrades myofibril proteins under the post-mortem conditions which appears to be the primary enzyme in the postmortem tenderization process, hence affecting the meat quality. Objective of this study was to analyze genetic polymorphism of the CAPN1 gene in seven diverse Indian goat breeds. Direct DNA sequencing was carried out to identify the genetic polymorphisms in 84 unrelated goats representing seven different goat breeds (Barbari, Beetal, Ganjam, Sirohi, Black Bengal, Osmanabadi and Malabari). Six single nucleotide polymorphisms (SNPs) were identified in CAPN1 gene intron 17. The observed heterozygosity (Ho) was highest (0.50) in Sirohi and Osmanabadi (0.42) breeds and lowest in Black Bengal (0.06) breed. The expected heterozygosity (He) was highest (0.45) in Sirohi followed by Osmanabadi (0.37), Barbari (0.34) and Beetal (0.37). The allele frequencies ranged from 0.083 to 0.917 and the major allele frequencies ranged from 0.595 (locus 6499 T/C) to 0.917 (locus 6529 T/C). In Black Bengal, polymorphism at all theloci was fixed with the exception of 6417 T/G and 6533C/G. The identity values oscillated from 0.7866 to 0.9593. The Nei's genetic distances were below 0.0294. Genetic diversity among different goat meat breeds indicates possible role in meat quality trait. Allelic diversity at this locus could be used in further for the association between gene polymorphism and enzyme activity as well as caprine meat quality traits.

Reproductive traits are extremely important to the goat industry because moderate increase in litter size can lead to large profit. Traditional selection for improving litter size is difficult due to sex-limited nature and low heritability of the trait (5–10%). In addition, the lack of knowledge on the number of the genes controlling this trait and the possible gene interactions are the other limitations for this trait. Molecular genetics can overcome these limitations offering new opportunities for the improvement of reproductive traits, as it supplies tools to analyze genetic variability directly at the DNA level with the possibility of detecting the individual genes influencing the reproductive capability. For the last decade, molecular genetics has led to the discovery of individual genes or candidate genes with substantial effects on the reproductive trait viz. BMPR1B , GDF9 , BMP15 , FSHβ , FSHR , POU1F1 , PRLR , KiSS-1 , GPR54 , GH , INH , CART , GnRH , GnRHR , LHβ , BMP4 , KITLG , MT2 , CYP21 and AA-NAT . Despite the progress made in goat fecundity studies, it's hard to satisfy the actual application because the reproductive traits are complex quantitative traits involving multiple genes, loci and interactions. So it is important to analyze the combined effect of multiple genes or loci on reproductive traits. Little progress has been made on understanding interaction between genes, gene and environment, and genetic effect analysis. Thus the need of the hour is to identify more functional genes, clarify molecular mechanism of action and regulatory network and to resort to more holistic approaches like genomic selection which can tremendously accelerate the goat improvement.

New, quick, and inexpensive methods for genotyping novel caprine Fec gene polymorphisms through tetra-primer ARMS PCR were developed in the present investigation. Single nucleotide polymorphism (SNP) genotyping needs to be attempted to establish association between the identified mutations and traits of economic importance. In the current study, we have successfully genotyped three new SNPs identified in caprine fecundity genes viz. T(-242)C (BMPR1B), G1189A (GDF9) and G735A (BMP15). Tetra-primer ARMS PCR protocol was optimized and validated for these SNPs with short turn-around time and costs. The optimized techniques were tested on 158 random samples of Black Bengal goat breed. Samples with known genotypes for the described genes, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the tetra-primer ARMS PCR assays and the sequencing results. These results highlight the ability of tetra-primer ARMS PCR in genotyping of mutations in Fec genes. Any associated SNP could be used to accelerate the improvement of goat reproductive traits by identifying high prolific animals at an early stage of life. Our results provide direct evidence that tetra-primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of mutations in caprine Fec genes., Highlights • Tetra-primer ARMS-PCR protocols were developed for genotyping of novel mutations in caprine Fec genes. • The protocols were optimized and tested on 158 random samples of Black Bengal goat breed. • The results were validated by direct DNA sequencing. • Complete concordance was observed between the tetra-primer ARMS PCR assays and sequencing results.

The efficacy of pEGFP (plasmid expressing enhanced green fluorescent protein)-encapsulated PEGylated (meaning polyethylene glycol coated) magnesium phosphate nanoparticles (referred to as MgPi-pEGFP nanoparticles) for the induction of immune responses was investigated in a mouse model. MgPi-pEGFP nanoparticles induced enhanced serum antibody and antigen-specific T-lymphocyte responses, as well as increased IFN-? and IL-12 levels compared to naked pEGFP when administered via intravenous, intraperitoneal or intramuscular routes. A significant macrophage response, both in size and activity, was also observed when mice were immunized with the nanoparticle formulation. The response was highly specific for the antigen, as the increase in interaction between macrophages and lymphocytes as well as lymphocyte proliferation took place only when they were re-stimulated with recombinant green fluorescence protein (rGFP). Thus the nanoparticle formulation elicited both humoral as well as cellular responses. Cytokine profiling revealed the induction of Th-1 type responses. The results suggest DNA-encapsulated magnesium phosphate (MgPi) nanoparticles may constitute a safer, more stable and cost-efficient DNA vaccine formulation., Graphical abstract

The calpains and calpastatin (CAST) make up a major cytosolic proteolytic system, the calpain-calpastatin system, found in mammalian tissues. The relative levels of the components of the calpain-calpastatin system determine the extent of meat tenderization during postmortem storage. Calpastatin (CAST) is a protein inhibitor of the ubiquitous calcium-dependent proteases-micro-calpain and m-calpain. Polymorphisms in the bovine, ovine and pig CAST gene have been associated with meat tenderness but little is known about how caprine CAST gene may affect goat meat quality traits. In this study we selected different parts of the CAST gene: 1) that have been previously reported to be polymorphic, intron 5 and 12 and 3'UTR; 2) first time explored (exon 3, 7 and 8 and part of intron 7 and 8) to investigate polymorphic status of caprine CAST gene. Using comparative sequencing ten novel SN Ps located in exon 3 and intron 5, 7 and 8 were identified. Previously reported SNPs in intron 5, 3'UTR and intron 12 were absent. Sequence analysis revealed a non synonymous amino acid variation in exon 3, which would result in Lys/Arg substitution in the corresponding protein sequence. Considerable variation was detected in intronic regions. Twenty-four InDel were also recognized in intronic regions (15) and 3'UTR (9). All the sequences shared high homology with published bovine and ovine sequences. Three PCR-RFLP loci have been established for further analyzing genetic polymorphism in indigenous goats.

Cattle are the most important livestock in India and play a pivotal role in agrarian economy. There are 34 recognized breeds of cattle and number of unexplored lesser known populations. The present study is a contribution towards determining genetic Variation and understanding the relationship among four lesser known populations. A total of 194 unrelated DNA samples from three cattle populations of Orissa (Binjharpuri, Ghumsuri, Motu) and Hill cattle of Kumaun (Kumauni) were collected from respective breeding tracts. Genotyping was done with 23 bovine microsatellite markers as suggested by International Society for Animal Genetics (ISAG) and FAO (DAD-IS) on automated sequencer. The average observed heterozygosity in the four populations lie within the narrow range of 0.623 ± 0.04 in Binjharpuri to 0.664 ± 0.03 in Kumauni. Mean estimates of observed and expected heterozygosity over all loci and breeds were 0.651 ± 0.02 and 0.720 ± 0.01, respectively. In the overall population, the homozygote excess (FIT) of 0.132 ± 0.03, was partly due to the genetic differentiation among breeds (FST = 0.044 ± 0.01) and, to a larger extent, to a significant homozygote excess within breeds (FIS = 0.094 ± 0.03). The phylogenetic reconstruction from a UPGMA clustering based on Nei’s Standard genetic distance yielded a tree with Binjharpuri and Ghumsuri on a single node and Motu and Kumauni on separate nodes. The most probable clustering detected by STRUCTURE in population was three. Binjharpuri and Ghumsuri animals were assigned to one cluster with high proportion of membership.

This paper is being retracted by the authors in response to a misconduct investigation carried out by the Research and Publication Ethics Committee of Jamia Hamdard.

IL-17, the hallmark cytokine of the Th17 population, mediates immunity to extracellular pathogens and promotes autoimmune immunopathology. The signaling mechanisms triggered by the IL-17 receptor (IL-17RA) and related receptors are strikingly different from other cytokine subclasses. Namely, IL-17Rs contain a conserved SEF/IL-17R (SEFIR) subdomain that engages Act1, leading to activation of TRAF6, NF-κB, and other events. Although the SEFIR is critical for signaling, the molecular details of the functional subdomains within IL-17RA remain poorly characterized. Here, we provide a detailed structure-function analysis delineating the C-terminal boundary of the SEFIR-containing region of IL-17RA. We show that functionality of this domain requires a large extension to the previously identified SEFIR motif. In contrast to the SEFIR, this extension is not conserved among IL-17R family members. Surprisingly, Act1 recruitment is not sufficient for downstream signaling activation, whereas ubiquitination of TRAF6 correlates tightly with functional receptors. We further demonstrate that IL-17RA exhibits signaling properties that are nonredundant with other IL-17R family members. Finally, we report that IL-17 signals synergistically with lymphotoxin-α3, using the same signaling motifs within IL-17RA. These studies provide new insight into the structure-function relationships of IL-17RA and reveal distinct signaling differences among IL-17R family members.

Chemotherapy is a major therapeutic approach for the treatment of localized and metastasized cancers. Whereas potent chemotherapeutic agents seem promising in the test tube, clinical trials often fail due to unfavorable pharmacokinetics, poor delivery, low local concentrations, and limited accumulation in the target cell. The pathophysiology of the tumor vasculature and stromal compartment presents a major obstacle to effective delivery of agents to solid tumors. Poor perfusion of the tumor, arterio-venous shunting, necrotic and hypoxic areas, as well as a high interstitial fluid pressure work against favorable drug uptake. Thus, targeted drug delivery using long-circulating particulate drug carriers such as hydrogels of controlled size (

IL-17 is the hallmark cytokine of the newly described “Th17” lymphocyte population. The composition, subunit dynamics, and ligand contacts of the IL-17 receptor are poorly defined. We previously demonstrated that the IL-17RA subunit oligomerizes in the membrane without a ligand. In this study, computational modeling identified two fibronectin-III-like (FN) domains in IL-17RA connected by a nonstructured linker, which we predicted to mediate homotypic interactions. In yeast two-hybrid, the membrane-proximal FN domain (FN2), but not the membrane-distal domain (FN1), formed homomeric interactions. The ability of FN2 to drive ligand-independent multimerization was verified by coimmunoprecipitation and fluorescence resonance energy transfer microscopy. Thus, FN2 constitutes a “pre-ligand assembly domain” (PLAD). Further studies indicated that the FN2 linker domain contains the IL-17 binding site, which was never mapped. However, the FN1 domain is also required for high affinity interactions with IL-17. Therefore, although the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker and FN1.

The condensation of iodobenzene to biphenyl is an industrially important reaction due to its sig- nificant role in organic synthesis as drug intermediates. The reaction takes place in the presence of copper powder as catalyst. We have shown in this paper that the size of the copper nanoparticles as well as its ex- posed surface area is responsible for the yield of chemical reaction. The uncapped copper powder showed a 43% conversion of iodobenzene to biphenyl in 5 h under our experimental conditions. Same amount of copper nanoparticles (size, ~ 66 nm diameter) prepared by citrate capping showed 88% conversion of iodobenzene to biphenyl, which increased to about 95% when 8 nm diameter capped copper nanoparticles are used. Surpris- ingly, 5 nm size copper nanoparticles showed no change in the yield of about 95%.

The IL-17 cytokine family plays a major role in promoting autoimmune and inflammatory diseases. The original member of this family, IL-17 (CTLA-8 or IL-17A), has been the center of intense research, whereas IL-17F is much less understood. In many respects, IL-17F functions similarly to IL-17A: both are produced by a unique subset of CD4+ T helper cells (termed TH17) and both bind a common receptor subunit (IL-17RA). Hence, IL-17F is implicated in the diseases where IL-17A clearly plays a key role. This patent describes antagonists that can directly or indirectly inhibit IL-17F signaling. The authors also report the identification of new receptor subunits, heteromeric combinations of IL-17A and IL-17F, and that IL-21 may be a stimulus for production of IL-17F. The invention of these antagonists may provide reagents to enable further research on this intriguing molecule, as well as establish a basis for treating IL-17F-mediated diseases.

Single nucleotide polymorphisms (SNPs) are the most abundant form of DNA polymorphismwhich can be used as simple genetic markers for many breeding applications as well as for population studies. SNPs in fecundity (Fec) genes viz. BMPR1B, GDF9 and BMP15 were genotyped in Indian goat breeds to assess their basic population parameters and genetic structuring, using simple and economical methods of PCR-RFLP and Tetra-primer ARMS PCR. Three hundred and ninety one animals of seven different goat breeds differing in prolificacy, utility and geographic distribution were employed for diversity assessment at six loci in these Fec genes. Four loci were polymorphic in all the breeds with varied allelic frequencies. Statistical analysis revealed that breeds differed with respect to genetic variability as observed heterozygosity varied from 0.163 (Ganjam) to 0.359 (Jakhrana). Sangamneri and Osmanabadi were least differentiated (0.003) whereas Jakhrana and Black Bengal were most differentiated (0.048) on the basis of genetic distance. The genetic distance as well as PCA analysis indicated that breeds are grouped according to their geographical distribution. The genetic diversity based on fecundity genes presented here is an essential step towards the future exploitation of the available goat genetic resources in research and breeding programs.

Sortase A (SrtA) from Gram positive pathogens is an attractive target for inhibitors due to its role in the attachment of surface proteins to the cell wall. We found that the plant natural product trans-chalcone inhibits Streptococcus mutans SrtA in vitro and also inhibited S. mutans biofilm formation. Mass spectrometry revealed that the trans-chalcone forms a Michael addition adduct with the active site cysteine. The X-ray crystal structure of the SrtA H139A mutant provided new insights into substrate recognition by the sortase family. Our study suggests that chalcone flavonoids have potential as sortase-specific oral biofilm inhibitors.

Inhibition of Corrosion of Nickel in Different Acids by Organic Compounds

Random copolymeric micelles composed of N-isopropylacrylamide (NIPAAM) and N-vinylpyrrolidone (VP) cross-linked with N,N'-methylenebisacrylamide (MBA) have been used as nanogel carriers to encapsulate N-hexylcarbamoyl-5-fluorouracil (HCFU), a prodrug of 5-FU, and have been targeted to brain tissue across blood-brain barrier (BBB) after coating with polysorbate 80. Accumulation of nanogel particles in the brain and other tissues of "strain A" mice had been monitored by radiolabeling of nanogels with (99m)Tc. Gamma Scintigraphic technique was also performed to see the distribution of (99m)Tc labeled nanogels in the brain. The retention time in blood appeared to be slightly longer for coated nanogels than that of uncoated nanogels though the accumulation of coated nanogels in the RES was more or less same as that of uncoated nanogels. The blood however had almost double accumulation of polysorbate 80 coated nanogels in the initial 5 min compared to that shown by uncoated nanogels. We speculate that coating of nanogels with polysorbate 80 alters the surface properties of nanogels, which results in relatively higher uptake in the brain tissue. The studies revealed that a large portion of (99m)Tc labeled HCFU loaded nanogels are accumulated in the RES (lung, liver and spleen). The accumulation of the labeled nanogels in the brain, however, is much less compared to RES and it has been found that while an amount of uncoated labeled nanogels was found to be 0.18% of the injected dose, it increased to 0.52% on coating with polysorbate 80. The optimal amount of polysorbate 80 added to nanogels for the maximum delivery of particles to brain was found to be 1% w/w. These results were further supported by the gamma scintigrams of New Zealand rabbits. Thus, the present nanogel system has opened a new avenue for poorly soluble drugs to be targeted to brain by coating the particles with polysorbate 80.

Adverse effects of viral vectors, instability of naked DNA, cytotoxicity and low transfection of cationic lipids, cationic polymers and other synthetic vectors are currently severe limitations in gene therapy. In addition to targeting a specific cell type, an ideal nonviral vector must manifest an efficient endosomal escape, render sufficient protection of DNA in the cytosol and help provide an easy passage of cytosolic DNA to the nucleus. Virus-like size calcium phosphate nanoparticles have been found to overcome many of these limitations in delivering genes to the nucleus of specific cells. This review has focused on some applications of DNA-loaded calcium phosphate nanoparticles as nonviral vectors in gene delivery, and their potential use in gene therapy, as well as highlighting the mechanistic studies to probe the reason for high transfection efficiency of the vector. It has been demonstrated that calcium ions play an important role in endosomal escape, cytosolic stability and enhanced nuclear uptake of DNA through nuclear pore complexes. The special role of exogenous calcium ions to overcome obstacles in practical realization of this field suggests that calcium phosphate nanoparticles are not 'me too' synthetic vectors and can be designated as second-generation nonviral vectors for gene therapy.

Hollow nanoshells of gold entrapping an enzyme, horseradish peroxidase (HRP), in the cavity of the nanoshell have been prepared in the reverse micelles by leaching out silver chloride (AgCl) from Au(shell)AgCl(core) nanoparticles with dilute ammonia solution. The particles have been characterised by dynamic laser light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), and electron diffraction. The particle size is below 100 nm diameter, depending upon the size of the aqueous core of reverse micelles in which these particles have been prepared. This soft-chemical method for the preparation of such particles allows the entrapped enzyme to remain active inside the hollow gold nanoparticles. Small substrate molecules such as o-dianisidine can easily enter through the pores of the nanoshell and can undergo enzymatic oxidation by H2O2. The enzyme kinetics follows Michaelis-Menten mechanism. When the substrate is chemically conjugated with dextran molecule (10 kDa), the enzymatic reaction is practically completely prevented perhaps by the inability of dextran-o-dianisidine conjugate to penetrate the pores of the nanoshells. However, HRP did not show any activity when trapped inside solid gold nanoparticles.

Due to limitations of the existing preparative methods of hollow nanoparticles by either heating at high temperature (>600 °C) or by using strong acid, alkali, or an organic solvent, it was not possible up till now to encapsulate any sensitive organic molecule like enzyme or others inside the cavity of hollow nanoparticles. We have demonstrated a much softer method of preparing hollow silica nanoparticles with horseradish peroxidase (HRP) inside the cavity by synthesizing HRP-doped core-shell silica-coated silver chloride nanoparticles and finally leaching out silver chloride with dilute ammonia at low temperatures. TEM pictures showed the hollow cavity inside the nanoparticles. The enzyme entrapped in these particles was active. The turnover number of HRP entrapped into these hollow particles and dispersed in aqueous buffer (pH 7.2) ( k cat = 2.56 × 10 6 s −1 ) was found to be less than that of free enzyme in aqueous buffer ( k cat = 6.133 × 10 7 s −1 ) but higher than that of HRP entrapped in solid-core silica nanoparticles and dispersed in aqueous buffer ( k cat = 1.05 × 10 5 s −1 ). The result showed that hollow nanoparticles could be prepared using soft chemical methods and sensitive chemicals like active enzyme could be entrapped in the cavities and it retains its activity.

Summary Guanosine tetraphosphate (ppGpp) is a signal of nutritional stress that regulates transcription. An RNA polymerase rudder mutant rpoC (Δ 312–315) is found to suppress ppGpp deficiency phenotypes by restoring both negative and positive activities of promoter fusions in vivo, as if ppGpp were present. Measurements of defects in transcription of the P argT tRNA promoter with mutant RNA polymerase reveal that the mutant enzyme quantitatively mimics the presence of added ppGpp. DNaseI footprints and mobility shifts under RNA polymerization conditions reveal that the promoter-specific transcription defect of the mutant enzyme can be ascribed to the presence of inactive dead-end promoter complexes with features similar to those of a stable closed complex. We propose that formation of such inactive complexes represents an alternative explanation of "stringent RNA polymerase" mutant behavior to those currently published, and it represents a newly discovered mode of action of ppGpp.

The interference of colloidal tin oxides on the biodistribution of 99m Technetium radiolabeled chitosan nanoparticles has been overcome by using sodium borohydride instead of commonly used stannous salts as reducing agent for the reduction of 99m Tc (VII) to lower valency states. Biodistribution of radiolabeled chitosan nanoparticles prepared by using stannous chloride method revealed localization of the radioactivity mainly in the liver and spleen while that of radiolabeled chitosan nanoparticles prepared by using sodium borohydride method manifested the presence of radioactivity in blood up to an extent of 10% even after 2 h. Interestingly, the reduction of radioactivity in the latter case with the progress of time was not manifested through an increase in activity in the liver. Rather, a time dependent increased accumulation of radioactive materials was observed in the stomach. From the results it has been concluded that the biodistribution is strongly influenced by the presence of colloidal particles of tin oxides and 99m Tc labeled chitosan nanoparticles are RES evading and long circulating in blood when Tc (VII) is reduced by sodium borohydride and not by stannous chloride during radiolabeling process.

The blood-brain barrier (BBB) represents an insurmountable obstacle for delivery of a large number of drugs, including antibacterials, anticancer agents and neuropeptides. One approach to overcoming this barrier has been drug delivery to the brain using appropriately modified nanoparticles. Since polysorbate80 is known to facilitate uptake by brain endothelial cells, nanoparticles coated with polysorbate80 hold great promise for the transport of agents across the BBB. Since chitosan nanoparticles have extended circulation time in the blood and decreased uptake by the reticuloendothelial system, we decided to evaluate the efficiency of polysorbate80-coated chitosan nanoparticles as brain-delivery carriers. Polysorbate80 (1% w/w)-coated, ultra-fine, crosslinked chitosan nanoparticles (10% crosslinking with glutaraldehyde)

Nanoparticles of calcium phosphate encapsulating plasmid DNA (pDNA) of size 100-120 nm in diameter were prepared. XRD studies of these nanoparticles showed them to be crystalline in nature having hydroxyapatite structure. The maximum loading of pDNA and its release from nanoparticles were studied using gel electrophoresis. The time dependent size measurement of these particles demonstrated that these particles show strong aggregational behaviour in aqueous dispersion. Calcium phosphate nanoparticles were found to be dissolved even in low acidic buffer (pH 5.0) releasing the pDNA, which suggested that DNA release from these particles in the endosomal compartment was possible. In vitro transfection efficiency of these calcium phosphate nanoparticles was found to be higher than that of the commercial transfecting reagent Polyfect.

Organically modified silanes (ORMOSIL) such as vinyl triethoxysilane readily aggregate in the aqueous cores of reverse micelles where the triethoxysilane moieties are hydrolyzed to form a hydrated silica network and the vinyl groups protruded out from the surface of the nanoparticles toward the hydrophobic side of the micellar interface. These particles are spherical and the size distribution of the particles is relatively narrow, with an average diameter of 87 nm. Surface vinyl silica nanoparticles so formed have been oxidized to surface carboxylic silica nanoparticles, followed by chemical conjugation with polyethyleneglycol amine (PEG amine) through the ethyl-3-(3-dimethylaminopropyl) (EDCI) carbodiimide reaction. The characteristic surface groups have been identified by Fourier transform infrared spectroscopy, while the size and the morphology of the particles have been studied by dynamic light scattering and transmission electron microscopy. It has been found that about 80-85% of the carboxylic groups are PEGylated during the EDCI reaction.

The aggregation behavior of sodium salicylate (SS) in aqueous solutions has been studied by positronium annihilation technique. From the change of the pick-off life time as well as the intensity of the ortho -positronium component, it has been concluded that the minimal hydrotropic concentration (MHC) of SS lies in the range 0.9–1.0 M concentration. To support the above contention, the molecular association of SS has also been investigated by the changes in light scattering intensity, conductivity as well as NMR data. These experiments conjointly infer that the stacking of SS molecules appears to start beyond 0.9 M concentration.

Titanium dioxide nanoparticles prepared in water-in-oil microemulsion droplets by controlled hydrolysis of TiCl(4)-generated crystalline nanoparticles of sizes from 115 nm down to 6 nm diameter depending on the size of the aqueous core of the micellar droplets. Powder X-ray diffraction of the vacuum-dried product (without sintering) indicated the presence of an unusual type of orthorhombic crystal structure nearly similar to titanium dioxide crystals prepared at high pressure. On gradual heating up to 900 degrees C these metastable crystals are converted into relatively more stable nanorods perhaps through making and breaking of the Ti-O-Ti bonds. It has been concluded that chemical pressure generated within the constrained volume of aqueous core of the reverse micellar droplets is responsible for the unusual crystal structure of TiO(2) nanoparticles.

Ormosil (organically modified silane) such as n-octyl triethoxy silane has been found to aggregate in the form of normal micelles as well as reverse micelles in which the triethoxy silane moeities are hydrolyzed to form a hydrated silica network while the n-octyl groups are held together through hydrophobic interaction. These nanoparticles are spherical in shape and are nearly monodispersed with an average diameter of below 100 nm. The nanoparticles originating from the micellar aggregate have an hydrophobic core with a layer of the hydrated silica network at the surface. The hydrophobic core can host hydrophobic molecules such as tetraphenyl porphyrin, which is leached out of the particles extremely slowly compared to that in Triton X-100 micelles. The nanoparticles originating from the reverse micelles have a hydrated silica network in the core surrounded by the hydrophobic n-octyl chains on the particle surface. The hydrophilic silica cores of these nanoparticles have been used to encapsulate horseradish peroxidase (HRP) and the enzyme shows its activity and follows Michaelis-Menten kinetics.

Hydrogel nanoparticles of cross-linked polyvinylpyrrolidone (PVP-NP) (35–50 nm in diameter) containing fluoresceinated dextran (FITC-Dx) were encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F-virosomes11Process for Producing a Targeted Gene (1997) US Patent 5, 683, 866).). Incubation of these loaded F-virosomes with human hepatoblastoma cells (HepG2) in culture resulted in membrane-fusion-mediated delivery of NPs to the cell cytoplasm, as inferred from the ability of cells to internalize FITC-Dx loaded PVP-NP (PVPf-NP) in the presence of azide (an inhibitor of the endocytotic process). Introduction of PVPf-NP into the HepG2 cells was assured by selective accumulation of FITC fluorescence in the cytosolic compartment. The structural integrity of the internalized PVPf-NP was also confirmed by fluorescence microscopy and ultracentrifugation analysis. The potential usefulness of PVP-NP-mediated cytosolic release of water soluble drugs both in vitro and in vivo has been established for the first time.

Studies of the Escherichia coli RNA polymerase subunit sigma-70 employing limited proteolytic digestion and binding by monoclonal antibodies indicate that conserved region 3 is solvent accessible in the free protein and in the RNA polymerase holoenzyme. Conversely, when sigma-70 binds to core RNA polymerase, proteolytic cleavage of region 3 is dramatically reduced. The former set of results seems to indicate the physical presence of region 3 on or near the surface of the holoenzyme while the latter of these results suggest that region 3 is sequestered in a direct protein-protein contact within the RNA holoenzyme which alters its protease sensitivity. To further investigate these possibilities we inserted an internal histidine-tag within region 3 of sigma(70) (sigma(70)-R3-His6) between amino acids 508 and 509. Confirmation that the internal His-tag insertion does not disrupt normal sigma(70) function was verified by genetic complementation. His-tagged protein was immobilized on nickel-agarose and core RNAP was tethered via the sigma-core interaction. Our results are consistent with the localization of region 3 on or near the surface both of free sigma(70) and of RNA polymerase holoenzyme. Furthermore, we find that the sigma(70)-core interaction is resistant to high ionic conditions but is completely disrupted by the presence of the low-molecular-weight hydrophobic amino acids phenylalanine and leucine free in solution. These results demonstrate the general usefulness of this approach to the disruption of protein-protein interactions and its potential application for protein purification.

Differential radiomodification induced by 2-deoxy-D-glucose (2-DG) is proving to be a feasible modality for optimizing tumor radiotherapy. Our earlier work on Ehrlich ascites tumor cells has shown that pretreatment with hematoporphyrin derivatives increases the uptake and phosphorylation of 2-DG. Moreover, the alteration induced in bioenergetic profile was more drastic and less reversible. The promising combination of hematoporphyrin derivatives and 2-DG has been further evaluated in the Ehrlich ascites tumor bearing mice for determining the effects on radiotherapeutic response.

Novel, injectable, and sprayable nanometer size hydrated silica particles encapsulating high molecular weight compounds such as [125I]tyraminylinulin (mol wt 5 kD), FITC-dextran (mol wt 19.6 kD), and horse radish peroxidase (mol wt 40 kD) have been prepared and characterized. The size of these particles is always below 100 nm in diameter and the entrapment efficiency was found to be as high as 80%. The entrapped compounds show practically zero leachability for more than 45 days. Enzymes entrapped in these particles show Michaelis−Menten kinetics and the catalytic reaction takes place only after the diffusion of substrate molecules into the particles through the pores of the silica matrix. Peroxidase entrapped into silica nanoparticles shows higher stability toward temperature and pH changes compared to free enzyme molecules.

We describe the synthesis and characterization of ultrafine TiO2 particles (in both anatase as well as rutile form) produced by a chemical reaction within the aqueous core of a water-in-oil microemulsion. The microemulsion was stabilized and the Ti4+ ions provided by a functionalized surfactant derived from the commercially available Aerosol-OT, i.e., sodium bis (2-ethylhexyl) sulfosuccinate (Na-DEHSS). The Na+ ions in Aerosol-OT were completely replaced by Ti4+ through an ion-exchange reaction in nonaqueous solvents. Ultrafine TiO2 particles were produced by the hydrolysis of the Ti-containing surfactant in the water droplets. The dependence of the size of the precipitated TiO2 · xH2O particles on various structure parameters of the microemulsion was studied in detail.

Ultrafine monodisperse polyvinylpyrrolidone nanoparticles (PVP np) entrapping allergens/antigens of A. fumigatus were prepared by reverse micelles method. The entrapment efficiency of various formulations of PVP np varied from 74 to 92%. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), enzyme linked immunosorbent assay (ELISA) and western blot analysis showed intact integrity and immunoreactivity of the allergens/antigens after entrapment in nanoparticles. The effect of various factors such as size of the particles, ratio of monomer to antigen concentration, degree of cross-linking and schedule of immunisation were examined on the in vitro release rate and production of specific IgG and IgE antibodies in the mice. The studies showed that the formulation with 1% cross-linking agent with a mean particle size of 100 nm and a monomer: antigen ratio of 0.09:1 released encapsulated antigens in 9 weeks. The same formulation showed a sustained IgG level (>0.400 Abs) for approximately 12 weeks in comparison to IgG level (>0.400 Abs) for 7 days with free antigen. Increase in particle size or change in monomer concentration resulted in accelerated release rate. A sustained level of IgG antibodies achieved with three divided doses of entrapped antigen could also be achieved with same amount of entrapped antigens administered in single dose. The A. fumigatus specific IgE levels of nanoparticle entrapped allergens/antigens were lower than obtained with free allergens/antigens. It is concluded that PVP np provide a promising delivery system for proteins as it is biocompatible, conserves the integrity and biological activities of proteins, augments antibody response and provide a sustained antibody level with single step immunisation. The formulation may also be useful for hyposensitisation therapy for various allergens.

Polyacrylamide nanoparticles of different size (100 nm) were prepared in reverse micelles under various dynamic conditions of micellar systems. The nanoparticles were always larger than the aqueous core in which they were formed; however, the size of the nanoparticles could be controlled if the interdroplet interaction and coalescence rate in reverse micelles were regulated. Factors such as interfacial rigidity of the droplets, size of the aqueous core, temperature, and concentrations of acrylamide (monomer) and N ,N '-methylenebisacrylamide (crosslinking agent) have a profound effect on the size of nanoparticles. By adjustment of these parameters in reverse micelles, nanoparticles ranging from about 10 to more than 100 nm have been prepared.

This study was designed to compare the anti-inflammatory potential of a Magnolia officinalis L. bark extract solely or in combination with extracts prepared from either Polygonum aviculare L., Sambucus nigra L., or Isodon japonicus L. in bacterial lipopolysaccharide (LPS) stimulated human gingival fibroblasts (HGF-1) and human U-937 monocytes, as cell models of periodontal disease. HGF-1 and U-937 cells were incubated with LPS from either Porphyromonas gingivalis or Escherichia coli together with the four plant extracts alone or in combination. Secretion of anti-inflammatory cytokines from HGF-1 and U-937 cells was measured by means of a multiplexed bead assay system. Magnolia officinalis L. bark extract, at concentrations of 1 μg/mL and 10 μg/mL, reduced interleukin 6 (IL-6) and interleukin-8 (IL-8) secretion from HGF-1 cells to 72.5 ± 28.6% and reduced matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) secretion from U-937 cells to 8.87 ± 7.97% compared to LPS-treated cells (100%). The other three extracts also reduced secretion of these inflammatory markers but were not as effective. Combination of 9 μg/mL Magnolia officinalis L. extract with 1 μg/mL of each of the other extracts maintained the anti-inflammatory effect of Magnolia officinalis L. extract. Combination of 5 μg/mL Magnolia officinalis L. extract with 5 μg/mL Isodon japonicus L. extract also maintained the anti-inflammatory potential of the Magnolia officinalis L. extract, whereas increasing concentrations of any of the other plant extracts in the combination experiments reduced the Magnolia officinalis L. extract efficacy in U-937 cells.