Abiotic elicitation of gymnemic acid in the suspension cultures of Gymnema sylvestre

Elicitation is one of the few strategies that find commercial application in the enhancement of secondary metabolite production from plants as well as cell culture systems. Due to their immense medicinal value, production of saponins in suspension cultures has been attempted by many researchers. Gymnema sylvestre is a rich source of gymnemic acids (saponins) that find application in the treatment of diabetes. The present study is an attempt to evaluate the effect of various metal salts (cadmium chloride, mercuric chloride, silver nitrate, cupric chloride, cobaltous chloride and calcium chloride) in eliciting the response from G. sylvestre suspension cultures. The maximum gymnemic acid production in the suspensions was achieved on day 12 of culture, though the maximum biomass was obtained on day 16. Among the different salts, CdCl₂ gave maximum response (59.97 mg/gDCW) at 2 mM concentration after a 24 h time period, while, AgNO₃ gave the least response (18.35 mg/gDCW) on incubation of 48 h at 1 mM concentration, in terms of gymnemic acid accumulation. The accumulation of gymnemic acid was found to be dependent on treatment time and concentration of the elicitor. The enhanced gymnemic acid production shown by the suspensions in response to the metal salts indicates their role in evoking the plant defense mechanisms. These elicitation studies help in providing a platform for improved commercial supply of bioactive gymnemic acids.     

Reassessing the folding of the KIX domain: Evidence for a two‐state mechanism

The debate about the presence and role of intermediates in the folding of proteins has been a critical issue, especially for fast folders. One of the classical methodologies to identify such metastable species is the “burst-phase analysis,” whereby the observed signal amplitude from stopped-flow traces is determined as a function of denaturant concentration. However, a complication may arise when folding is sufficiently fast to jeopardize the reliability of the stopped-flow technique. In this study, we reassessed the folding of the KIX domain from cAMP Response Element-Binding (CREB)-binding protein, which has been proposed to involve the formation of an intermediate that accumulates in the dead time of the stopped flow. By using an in-house-built capillary continuous flow with a 50-μs dead time, we demonstrate that this intermediate is not present; the problem arose because of the instrumental limitation of the standard stopped flow to assess very fast refolding rate constants (e.g., ≥500 s⁻¹).     

Identifying new therapeutic targets via modulation of protein corona formation by engineered nanoparticles

Background

We introduce a promising methodology to identify new therapeutic targets in cancer. Proteins bind to nanoparticles to form a protein corona. We modulate this corona by using surface-engineered nanoparticles, and identify protein composition to provide insight into disease development.

Methods/Principal Findings

Using a family of structurally homologous nanoparticles we have investigated the changes in the protein corona around surface-functionalized gold nanoparticles (AuNPs) from normal and malignant ovarian cell lysates. Proteomics analysis using mass spectrometry identified hepatoma-derived growth factor (HDGF) that is found exclusively on positively charged AuNPs (⁺AuNPs) after incubation with the lysates. We confirmed expression of HDGF in various ovarian cancer cells and validated binding selectivity to ⁺AuNPs by Western blot analysis. Silencing of HDGF by siRNA resulted s inhibition in proliferation of ovarian cancer cells.

Conclusion

We investigated the modulation of protein corona around surface-functionalized gold nanoparticles as a promising approach to identify new therapeutic targets. The potential of our method for identifying therapeutic targets was demonstrated through silencing of HDGF by siRNA, which inhibited proliferation of ovarian cancer cells. This integrated proteomics, bioinformatics, and nanotechnology strategy demonstrates that protein corona identification can be used to discover novel therapeutic targets in cancer.     

Characteristics of Bacterial Isolates from the Gut of Freshwater Fish, Labeo rohita that May be Useful as Potential Probiotic Bacteria

In this study, we aimed to evaluate the in vitro probiotic characteristics of three bacteria, Lactobacillus plantarum VSG3, Pseudomonas aeruginosa VSG2, and Bacillus subtilis VSG1, isolated from the gut of Labeo rohita. The bacterial isolates tolerated low pH and high bile concentrations in the fish well. The bacterial adhesion capacity to fish intestinal mucosa revealed that the three potential probiotic isolates had a significantly higher adhesion capacity compared to the pathogenic strains tested. L. plantarum VSG3 exhibited the best adhesion capacity (19.1?%) to the intestinal mucosa. Among the isolates, L. plantarum VSG3 and P. aeruginosa VSG2 showed strong antibacterial activities against fish pathogens as measured in spent culture liquids. Moreover, all the isolates were susceptible to each tested antibiotic, which ensured their inability to exhibit antibiotic-resistance properties. Considering these promising results, selected strains should be further studied to determine their probiotic effects in vivo in fish.     

Role of vitamin E-acetate on cisplatin induced genotoxicity: An <Emphasis Type="Italic">in vivo</Emphasis> analysis

Vitamin E has generated immense interest because of its potential of being an antioxidant, a neuroprotector, and a protector against atherosclerosis, carcinogenesis and cardiovascular disease. However, the prooxidant chemistry of vitamin E cannot be ignored since it is related to the generation of peroxyl radicals. In the present study, 125, 250 and 500 mg/kg of vitamin E-acetate (VE) administered intraperitoneally (i.p.) to Balb/C mice significantly induced 6%, 8% and 11.33% (control value=2.33%) of chromosome aberrations (CA) and 0.88%, 1.39% and 1.81% (control value=0.61%) of micronucleus (MN), following 24 hour of treatment in the bone marrow cells. In the germ cells, VE did not induce any sperm head abnormality (SHA) after 35 days of exposure. Most importantly, it has been observed that pre-treatment with VE significantly reduces CA, MN, and SHA induction by chemotherapeutic drug cisplatin (CIS). Our findings suggest that lone treatment with VE induce genotoxicity in somatic cells after 24 and 48 hours of exposure but not in germ cells after 35 days of exposure, whereas pre-treatment with VE reduces CIS induced genotoxicity as well as cytotoxicity. There exists a thin line of difference on the behavioral transition of VE when acting alone and when acting with a drug.     

Low plasma albumin levels are associated with increased plasma protein glycation and HbA1c in diabetes

Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC-MS(E). In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins.     

Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection

The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4-6 months later. Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals. Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post-dissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.     

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides¹. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously¹. This combinatorial platform has been validated with conventional methods² and the polyanhydride film and nanoparticle libraries have been characterized with ¹H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion^1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.