Application of the Dynamical Network Biomarker Theory to Raman Spectra.

The dynamical network biomarker (DNB) theory detects the early warning signals of state transitions utilizing fluctuations in and correlations between variables in complex systems. Although the DNB theory has been applied to gene expression in several diseases, destructive testing by microarrays is a critical issue. Therefore, other biological information obtained by non-destructive testing is desirable; one such piece of information is Raman spectra measured by Raman spectroscopy. Raman spectroscopy is a powerful tool in life sciences and many other fields that enable the label-free non-invasive imaging of live cells and tissues along with detailed molecular fingerprints. Naïve and activated T cells have recently been successfully distinguished from each other using Raman spectroscopy without labeling. In the present study, we applied the DNB theory to Raman spectra of T cell activation as a model case. The dataset consisted of Raman spectra of the T cell activation process observed at 0 (naïve T cells), 2, 6, 12, 24 and 48 h (fully activated T cells). In the DNB analysis, the F-test and hierarchical clustering were used to detect the transition state and identify DNB Raman shifts. We successfully detected the transition state at 6 h and related DNB Raman shifts during the T cell activation process. The present results suggest novel applications of the DNB theory to Raman spectra ranging from fundamental research on cellular mechanisms to clinical examinations.

Haloarcula mannanilytica sp. nov., a galactomannan-degrading haloarchaeon isolated from commercial salt.

A mannan-degrading halophilic archaeal strain, MD130-1T, was isolated from a commercial salt sample. Cells were motile, rod-shaped, and stained Gram-negative. Colonies were pink pigmented. Strain MD130-1T was able to grow at 1.5-4.6 M NaCl (optimum, 3.6 M) at pH 6.0-8.0 (optimum, pH 7.0) and at 25-50 °C (optimum, 40 °C). The DNA G+C content was 62.1 mol% (genome). The orthologous 16S rRNA gene sequence showed the highest similarity (99.4 %) to those of Haloarcula japonica JCM 7785T and Haloarcula hispanica JCM 8911T. The values of genome relatedness between strain MD130-1T and Haloarcula species were 84.33-85.96 % in ANIb and 30.4-32.9 % using GGDC formula 2. The polar lipids of strain MD130-1T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and triglycosyl diether-2. Based on the results of phenotypic and phylogenetic analyses, the strain represents a new species of the genus Haloarcula, for which the name Haloarcula mannanilytica sp. nov. is proposed. The type strain is MD130-1T (=JCM 33835T=KCTC 4287T) isolated from commercial salt made in Ishikawa prefecture, Japan.

Huge mucinous carcinoma of the breast in which doubling time was observed by computed tomography during 3 years without treatment.

Here we report the case of a 77-year-old woman with a huge cancer of the right breast for which size was measured on computed tomography in our hospital preoperatively and in other hospital 3 years earlier. During the 3-year untreated interval, the tumor grew from 4 cm to 13 cm in maximum diameter, and the tumor-volume doubling time (TVDT) was calculated as 209 days. The patient underwent mastectomy with axillary lymph node dissection, with the large skin defect covered by autologous skin graft. The pathological diagnosis was pure mucinous carcinoma (MC) of the breast with a low MIB-1 index, no vessel invasion, and no lymph node metastasis. Breast MC has been known to show a slow growth rate, but the TVDT of this current tumor was not markedly different from that of common breast cancers described in previous reports. This short TVDT notwithstanding the low aggressiveness may be due to abundant mucin occupying the majority of the tumor volume. To the best of our knowledge, no previous reports have provided accurate TVDTs for breast MC.

Exploring an Artificial Metabolic Route in Fusarium sporotrichioides: Production and Characterization of 7-Hydroxy T-2 Toxin.

An artificial metabolic route to an unnatural trichothecene was designed by taking advantage of the broad substrate specificities of the T-2 toxin biosynthetic enzymes of Fusarium sporotrichioides. By feeding 7-hydroxyisotrichodermin, a shunt pathway metabolite of F. graminearum, to a trichodiene synthase-deficient mutant of F. sporotrichioides, 7-hydroxy T-2 toxin (1) was obtained as the final metabolite. Such an approach may have future applications in the metabolic engineering of a variety of fungal secondary metabolites. The toxicity of 7-hydroxy T-2 toxin was 10 times lower than that of T-2 toxin in HL-60 cells.

Salinarchaeum chitinilyticum sp. nov., a chitin-degrading haloarchaeon isolated from commercial salt.

Two chitin-degrading halophilic archaeal strains, MC-74T and MC-23, were isolated from commercial salt samples. Cells were motile, rod-shaped and stained Gram-negative. Colonies were vermillion-pigmented. Strains MC-74T and MC-23 were able to grow with 1.5-5.1 M NaCl (optimum, 2.6-3.1 M) at pH 6.0-10.0 (optimum, pH 7.0) and at 20-50 °C (optimum, 40 °C). The orthologous 16S rRNA gene sequence similarity between the two strains was 99.8 %, and the closest phylogenetic relative was Salinarchaeum laminariae JCM 17267T with 99.3-99.5 % similarity. The level of DNA-DNA relatedness between the two strains was 93 and 94 % (reciprocally), and those between the two strains and Salinarchaeumlaminariae JCM 17267T were 35-36 % and 38-39 % (reciprocally). The polar lipids of both strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Glycolipids were not detected. Based on the phenotypic and phylogenetic analyses, the strains represent a novel species of the genus Salinarchaeum, for which the name Salinarchaeum chitinilyticum sp. nov. is proposed. The type strain is MC-74T (=JCM 19597T=KCTC 4262T), isolated from solar salt produced in France. Strain MC-23, isolated from a commercial solar salt sample produced in China, is an additional strain of the species.

Haloparvum alkalitolerans sp. nov., alkali-tolerant haloarchaeon isolated from commercial salt.

A Gram-stain-negative, rod-pleomorphic, aerobic, halophilic archaeon, strain MK62-1T, was isolated from commercial salt made from seawater in the Philippines. Strain MK62-1T was able to grow at 2.1-4.7 M NaCl (with optimum at 2.1-2.6 M NaCl), pH 6.5-9.5 (optimum, pH 7.0-7.5) and 20-55 °C (optimum, 45-50 °C). Based on the orthologous 16S rRNA gene sequence, the closest relative was Haloparvum sedimenti JCM 30891T with 99.2 % similarity. The RNA polymerase subunit B' gene sequence also showed the highest similarity (97.4 %) to that of Haloparvum sedimenti DYS4T. The DNA G+C content of MK62-1T was 70.1 mol%, while that of Haloparvum sedimenti JCM 30891T was 69.5 mol% by the HPLC method. The levels of DNA-DNA relatedness between MK62-1T and Haloparvum sedimenti JCM 30891T were 60.6 and 60.8 % (reciprocally). The major polar lipids of the isolate were C20C20 archaeol derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Based on the phenotypic and phylogenetic analyses, it is proposed that the isolate represents species within the genus Haloparvum, for which the name Haloparvum alkalitolerans sp. nov. is proposed. The type strain is MK62-1T (=JCM 30442T =KCTC 4214T).

Plasmonic fluorescent CdSe/Cu2S hybrid nanocrystals for multichannel imaging and cancer directed photo-thermal therapy.

A simple, crude Jatropha curcas (JC) oil-based synthesis approach, devoid of any toxic phosphine and pyrophoric ligands, to produce size and shape tuned CdSe QDs and a further copper sulfide (Cu2S) encasing is presented. The QDs exhibited excellent photoluminescent properties with narrow band gap emission. Furthermore, the Cu2S shell rendered additional cytocompatibility and stability to the hybrid nanomaterial, which are major factors for translational and clinical applications of QDs. The nanocomposites were PEGylated and folate conjugated to augment their cytoamiability and enhance their specificity towards cancer cells. The nanohybrids possess potentials for visible, near infrared (NIR), photoacoustic (PA) and computed tomography (µCT) imaging. The diverse functionality of the composite was derived from the multi-channel imaging abilities and thermal competence on NIR laser irradiation to specifically actuate the photo-thermal ablation of brain cancer cells.

Upregulation of TGF-ß1 and basic FGF in elastofibroma: an immunohistochemical analysis.

Elastofibroma is a rare tumour that occurs in the subscapular space, and it typically presents in middle-aged and older individuals. The aetiology of elastofibroma remains unknown. Recent, sporadic reports have shown, immunohistologically, that fibroblasts in elastofibroma may produce abnormal elastic and collagen fibres through the action of transforming growth factor-beta (TGF-ß), a factor that promotes fibroblast proliferation. However, that finding lacked quantitative measurements and controls. Therefore, in this study, we performed quantitative, immunohistochemical analyses of TGF-ß1 and basic fibroblast growth factor (bFGF) in three elastofibromas, and we compared them to ten dermatofibromas and keloids, and five normal skin. In elastofibroma specimens, 16-59 % fibroblasts were positive for TGF-ß1 in the cytoplasm, compared to 96 % in dermatofibroma, 93 % in keloid and 2 % in normal dermis specimens. Also, in elastofibroma specimens, 26-67 % of fibroblasts were positive for bFGF in the cytoplasm, compared to 97 % in dermatofibroma, 97 % in keloid, and 22 % in normal dermis specimens. Intriguingly, the tumour size and growth rate were proportional to the percentage of cells positive for bFGF. Finally, greater levels of bFGF expressions in fibroblasts were associated with larger sized elastofibromas. These results suggested that elastofibroma development depended on high expression of TGF-ß1 and bFGF.

Halorubrum gandharaense sp. nov., an alkaliphilic haloarchaeon from commercial rock salt.

A Gram-stain-negative, non-motile, pleomorphic rod-shaped, orange-red-pigmented, facultatively aerobic and haloalkaliphilic archaeon, strain MK13-1T, was isolated from commercial rock salt imported from Pakistan. The NaCl, pH and temperature ranges for growth of strain MK13-1T were 3.0-5.2 M NaCl, pH 8.0-11.0 and 15-50 °C, respectively. Optimal growth occurred at 3.2-3.4 M NaCl, pH 9.0-9.5 and 45 °C. Addition of Mg2+ was not required for growth. The major polar lipids of the isolate were C20C20 and C20C25 archaeol derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Glycolipids were not detected. The DNA G+C content was 64.1 mol%. The 16S rRNA gene sequence of strain MK13-1T was most closely related to those of the species of the genus Halorubrum, Halorubrum luteum CECT 7303T (95.9% similarity), Halorubrum alkaliphilum JCM 12358T (95.3%), Halorubrum kocurii JCM 14978T (95.3%) and Halorubrum lipolyticum JCM 13559T (95.3%). The rpoB' gene sequence of strain MK13-1T had < 90% sequence similarity to those of other members of the genus Halorubrum. Based on the phylogenetic analysis and phenotypic characterization, strain MK13-1T may represent a novel species of the genus Halorubrum, for which the name Halorubrum gandharaense sp. nov. is proposed, with the type strain MK13-1T ( = JCM 17823T = CECT 7963T).

Multi-stimuli responsive Cu2S nanocrystals as trimodal imaging and synergistic chemo-photothermal therapy agents.

A size and shape tuned, multifunctional metal chalcogenide, Cu2S-based nanotheranostic agent is developed for trimodal imaging and multimodal therapeutics against brain cancer cells. This theranostic agent was highly efficient in optical, photoacoustic and X-ray contrast imaging systems. The folate targeted NIR-responsive photothermal ablation in synergism with the chemotherapeutic action of doxorubicin proved to be a rapid precision guided cancer-killing module. The multi-stimuli, i.e., pH-, thermo- and photo-responsive drug release behavior of the nanoconjugates opens up a wider corridor for on-demand triggered drug administration. The simple synthesis protocol, combined with the multitudes of interesting features packed into a single nanoformulation, clearly demonstrates the competing role of this Cu2S nanosystem in future cancer treatment strategies.

Structurally distinct hybrid polymer/lipid nanoconstructs harboring a type-I ribotoxin as cellular imaging and glioblastoma-directed therapeutic vectors.

A nanoformulation composed of a ribosome inactivating protein-curcin and a hybrid solid lipid nanovector has been devised against glioblastoma. The structurally distinct nanoparticles were highly compatible to human endothelial and neuronal cells. A sturdy drug release from the particles, recorded upto 72 h, was reflected in the time-dependent toxicity. Folate-targeted nanoparticles were specifically internalized by glioma, imparting superior toxicity and curbed an aggressively proliferating in vitro 3D cancer mass in addition to suppressing the anti-apoptotic survivin and cell matrix protein vinculin. Combined with the imaging potential of the encapsulated dye, the nanovector emanates as a multifunctional anti-cancer system.

Bacterial exopolysaccharide based magnetic nanoparticles: a versatile nanotool for cancer cell imaging, targeted drug delivery and synergistic effect of drug and hyperthermia mediated cancer therapy.

Microbial exopolysaccharides (EPSs) are highly heterogeneous polymers produced by fungi and bacteria that have garnered considerable attention and have remarkable potential in various fields, including biomedical research. The necessity of biocompatible materials to coat and stabilize nanoparticles is highly recommended for successful application of the same in biomedical regime. In our study we have coated magnetic nanoparticles (MNPs) with two bacterial EPS-mauran (MR) and gellan gum (GG). The biocompatibility of EPS coated MNPs was enhanced and we have made it multifunctional by attaching targeting moiety, folate and with encapsulation of a potent anticancerous drug, 5FU. We have conjugated an imaging moiety along with nanocomposite to study the effective uptake of nanoparticles. It was also observed that the dye labeled folate targeted nanoparticles could effectively enter into cancer cells and the fate of nanoparticles was tracked with Lysotracker. The biocompatibility of EPS coated MNPs and synergistic effect of magnetic hyperthermia and drug for enhanced antiproliferation of cancer cells was also evaluated. More than 80% of cancer cells was killed within a period of 60 min when magnetic hyperthermia (MHT) was applied along with drug loaded EPS coated MNPs, thus signifying the combined effect of drug loaded MNPs and MHT. Our results suggests that MR and GG coated MNPs exhibited excellent biocompatibility with low cell cytotoxicity, high therapeutic potential, and superparamagnetic behavior that can be employed as prospective candidates for bacterial EPS based targeted drug delivery, cancer cell imaging and for MHT for killing cancer cells within short period of time.

Curcumin and 5-fluorouracil-loaded, folate- and transferrin-decorated polymeric magnetic nanoformulation: a synergistic cancer therapeutic approach, accelerated by magnetic hyperthermia.

The efficient targeting and therapeutic efficacy of a combination of drugs (curcumin and 5-Fluorouracil [5FU]) and magnetic nanoparticles encapsulated poly(D,L-lactic-co-glycolic acid) nanoparticles, functionalized with two cancer-specific ligands are discussed in our work. This multifunctional, highly specific nanoconjugate resulted in the superior uptake of nanoparticles by cancer cells. Upon magnetic hyperthermia, we could harness the advantages of incorporating magnetic nanoparticles that synergistically acted with the drugs to destroy cancer cells within a very short period of time. The remarkable multimodal efficacy attained by this therapeutic nanoformulation offers the potential for targeting, imaging, and treatment of cancer within a short period of time (120 minutes) by initiating early and late apoptosis.

Type 1 ribotoxin-curcin conjugated biogenic gold nanoparticles for a multimodal therapeutic approach towards brain cancer.

BACKGROUND: Gliomas have been termed recurrent cancers due to their highly aggressive nature. Their tendency to infiltrate and metastasize has posed significant roadblocks to in attaining fool proof treatment solutions. An initiative to curb such a scenario was successfully demonstrated in vitro, utilizing a multi-conceptual gold nanoparticle based photo-thermal and drug combination therapy. METHODS: Gold nanoparticles (Au NPs) were synthesized with a highly environmentally benign process. The Au NPs were PEGylated and conjugated with folate and transferrin antibody to achieve a dual targeted nano-formulation directed towards gliomas. Curcin, a type 1 ribosome inactivating protein, was attached to the Au NPs as the drug candidate, and its multifarious toxic aspects analyzed in vitro. NIR photo-thermal properties of the Au nano-conjugates were studied to selectively ablate the glioma cancer colonies. RESULTS: Highly cyto-compatible, 10-15nm Au NP conjugates were synthesized with pronounced specificity towards gliomas. Curcin was successfully conjugated to the Au NPs with pH responsive drug release. Prominent toxic aspects of curcin, such as ROS generation, mitochondrial and cytoskeletal destabilization were witnessed. Excellent photo-thermal ablation properties of gold nanoparticles were utilized to completely disrupt the cancer colonies with significant precision. CONCLUSION: The multifunctional nanoconjugate projects its competence in imparting complete arrest of the future proliferation or migration of the cancer mass. GENERAL SIGNIFICANCE: With multifunctionality the essence of nanomedicine in recent years, the present nanoconjugate highlights itself as a viable option for a multimodal treatment option for brain cancers and the like.

Fluorogenic probes using 4-substituted-2-nitrobenzenesulfonyl derivatives as caging groups for the analysis of human glutathione transferase catalyzed reactions.

We have synthesized a series of 4-substituted-2-nitrobenzene-sulfonyl compounds for caged fluorogenic probes and conducted a Hammett plot analysis using the steady-state kinetic parameters. The results revealed that the glutathione transferase (GST) alpha catalyzed reaction was dependent on the σ value in the same way as the non-enzymatic reaction, whereas the dependence of the σ value of the GST mu and pi was not as pronounced as that of GST alpha.

Accelerated killing of cancer cells using a multifunctional single-walled carbon nanotube-based system for targeted drug delivery in combination with photothermal therapy.

The photothermal effect of single-walled carbon nanotubes (SWCNTs) in combination with the anticancer drug doxorubicin (DOX) for targeting and accelerated destruction of breast cancer cells is demonstrated in this paper. A targeted drug-delivery system was developed for selective killing of breast cancer cells with polyethylene glycol biofunctionalized and DOX-loaded SWCNTs conjugated with folic acid. In our work, in vitro drug-release studies showed that the drug (DOX) binds at physiological pH (pH 7.4) and is released only at a lower pH, ie, lysosomal pH (pH 4.0), which is the characteristic pH of the tumor environment. A sustained release of DOX from the SWCNTs was observed for a period of 3 days. SWCNTs have strong optical absorbance in the near-infrared (NIR) region. In this special spectral window, biological systems are highly transparent. Our study reports that under laser irradiation at 800 nm, SWCNTs exhibited strong light-heat transfer characteristics. These optical properties of SWCNTs open the way for selective photothermal ablation in cancer therapy. It was also observed that internalization and uptake of folate-conjugated NTs into cancer cells was achieved by a receptor-mediated endocytosis mechanism. Results of the in vitro experiments show that laser was effective in destroying the cancer cells, while sparing the normal cells. When the above laser effect was combined with DOX-conjugated SWCNTs, we found enhanced and accelerated killing of breast cancer cells. Thus, this nanodrug-delivery system, consisting of laser, drug, and SWCNTs, looks to be a promising selective modality with high treatment efficacy and low side effects for cancer therapy.

In vitro evaluation of antioxidant defense mechanism and hemocompatibility of mauran.

Mauran (MR), a highly polyanionic sulfated exopolysaccharide was extracted from moderately halophilic bacterium; Halomonas maura and characterized using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. Purified MR was evaluated for antioxidant defense mechanisms under in vitro conditions using L929, mouse fibroblast cell line and mice liver homogenate. It was demonstrated that MR could impart protective effect against oxidative stress in both cells and tissue up to a concentration of 500 µg, which is found to be safe under laboratory conditions. Various enzymatic and non-enzymatic parameters of antioxidant mechanisms were evaluated and concluded that MR has the tendency to maintain a balance of antioxidative enzymes with in the test systems studied. Also, hemocompatibility assay performed revealed that MR has a lesser hemolytic index and exhibited a prolonged clotting time, which shows both antihemolytic, and antithrombogenic nature respectively. Furthermore, absorption studies performed using fluorescent-labeled MR confirmed that MR accumulated within the cell cytoplasm neither induced cellular lysis nor affected the cell integrity.

Biocompatible nanofibers based on extremophilic bacterial polysaccharide, Mauran from Halomonas maura.

Extremophilic bacterial polysaccharide based biocompatible nanofibers were produced for the first time via electrospinning technique. Mauran (MR), an extremophilic sulfated exopolysaccharide was extracted from moderately halophilic bacterium, Halomonas maura and characterized for the application of nanofiber synthesis. Thin-uniform MR nanofibers were produced using homogenous solutions of poly (vinyl alcohol) (PVA) blended with different concentrations of MR. Characterization of complex MR/PVA nanofibers were performed using scanning electron microscope and analyzed for the cytotoxicity using mouse fibroblast cells as well as mesenchymal stem cells. An average of 120 nm sized nanofibers were produced and tested for an enhanced cell growth under in vitro conditions in comparison with control. MR and MR/PVA nanofibers were found to be an excellent biomaterial for the migration, proliferation and differentiation of mammalian cells, which was confirmed by cell adhesion studies and confocal microcopy. Interestingly, biological and physicochemical properties of MR hasten the application of MR based nanofibers for various biomedical applications like tissue engineering and drug delivery.