Rapid and sensitive approaches for detecting food fraud: A review on prospects and challenges.

Precise and reliable analytical techniques are required to guarantee food quality in light of the expanding concerns regarding food safety and quality. Because traditional procedures are expensive and time-consuming, quick food control techniques are required to ensure product quality. Various analytical techniques are used to identify and detect food fraud, including spectroscopy, chromatography, DNA barcoding, and inotrope ratio mass spectrometry (IRMS). Due to its quick findings, simplicity of use, high throughput, affordability, and non-destructive evaluations of numerous food matrices, NI spectroscopy and hyperspectral imaging are financially preferred in the food business. The applicability of this technology has increased with the development of chemometric techniques and near-infrared spectroscopy-based instruments. The current research also discusses the use of several multivariate analytical techniques in identifying food fraud, such as principal component analysis, partial least squares, cluster analysis, multivariate curve resolutions, and artificial intelligence.

Advanced oxidation process for the treatment of industrial wastewater: A review on strategies, mechanisms, bottlenecks and prospects.

Due to its complex and, often, highly contaminated nature, treating industrial wastewater poses a significant environmental problem. Many of the persistent pollutants found in industrial effluents cannot be effectively removed by conventional treatment procedures. Advanced Oxidation Processes (AOPs) have emerged as a promising solution, offering versatile and effective means of pollutant removal and mineralization. This comprehensive review explores the application of various AOP strategies in industrial wastewater treatment, focusing on their mechanisms and effectiveness. Ozonation (O3): Ozonation, leveraging ozone (O3), represents a well-established AOP for industrial waste water treatment. Ozone's formidable oxidative potential enables the breakdown of a broad spectrum of organic and inorganic contaminants. This paper provides an in-depth examination of ozone reactions, practical applications, and considerations involved in implementing ozonation. UV/Hydrogen Peroxide (UV/H2O2): The combination of ultraviolet (UV) light and hydrogen peroxide (H2O2) has gained prominence as an AOP due to its ability to generate hydroxyl radicals (ȮH), highly efficient in pollutant degradation. The review explores factors influencing the efficiency of UV/H2O2 processes, including H2O2 dosage and UV radiation intensity. Fenton and Photo-Fenton Processes: Fenton's reagent and Photo-Fenton processes employ iron ions and hydrogen peroxide to generate hydroxyl radicals for pollutant oxidation. The paper delves into the mechanisms, catalyst selection, and the role of photoactivation in enhancing degradation rates within the context of industrial wastewater treatment. Electrochemical Advanced Oxidation Processes (EAOPs): EAOPs encompass a range of techniques, such as electro-Fenton and anodic oxidation, which employ electrode reactions to produce ȮH radicals. This review explores the electrochemical principles, electrode materials, and operational parameters critical for optimizing EAOPs in industrial wastewater treatment. TiO2 Photocatalysis (UV/TiO2): Titanium dioxide (TiO2) photocatalysis, driven by UV light, is examined for its potential in industrial wastewater treatment. The review investigates TiO2 catalyst properties, reaction mechanisms, and the influence of parameters like catalyst loading and UV intensity on pollutant removal. Sonolysis (Ultrasonic Irradiation): High-frequency ultrasound-induced sonolysis represents a unique AOP, generating ȮH radicals during the formation and collapse of cavitation bubbles. This paper delves into the physics of cavitation, sonolytic reactions, and optimization strategies for industrial wastewater treatment. This review offers a critical assessment of the applicability, advantages, and limitations of these AOP strategies in addressing the diverse challenges posed by industrial wastewater. It emphasizes the importance of selecting AOPs tailored to the specific characteristics of industrial effluents and outlines potential directions for future research and practical implementation. The integrated use of these AOPs, when appropriately adapted, holds the potential to achieve sustainable and efficient treatment of industrial wastewater, contributing significantly to environmental preservation and regulatory compliance.

Preparation, characterization, and performance evaluation of composite films of polyvinyl alcohol/ cellulose nanofiber extracted from Imperata cylindrica.

In recent years, production of cellulose nanofiber (CNF) from waste materials has achieved great interest owing to their renewable nature, biodegradability, high mechanical properties, economic value, and low density. Because Polyvinyl alcohol (PVA) is a synthetic biopolymer with good water solubility and biocompatibility, the composite material formed of CNF and PVA, is a sustainable way of monetizing to address environmental and economic issues. In this work pure PVA, PVA/CNF0.5, PVA/CNF1.0, PVA/CNF1.5, and PVA/CNF2.0 nanocomposite films were produced using the solvent casting approach with the addition of 0, 0.5, 1.0, 1.5, and 2.0 wt% of CNF concentrations respectively. The strongest water absorption behaviour was found as 25.82% for pure PVA membrane, followed by PVA/CNF0.5 (20.71%), PVA/CNF1.0 (10.26%), PVA/CNF1.5 (9.63%), and PVA/CNF2.0 (4.35%). The water contact angle of 53.1°, 47.8°, 43.4°, 37.7°, and 32.3° was formed between water droplet and the solid-liquid interface of pure PVA, PVA/CNF0.5, PVA/CNF1.0, PVA/CNF1.5, PVA/CNF2.0 composite films respectively. The SEM image clearly shows that a network structure like a tree form at the PVA/CNF0.5 composite film, where the sizes and number of pores are apparent. XRD analysis suggested that unique peaks found at 2θ = 17.5°, 28.1°, 33.4°, and 38° for nanocomposites indicating new crystal plane generated upon cross-linking in presence of malic acid. The maximum loss rate temperature (Td,max) for PVA/CNF0.5, PVA/CNF1.0, PVA/CNF1.5 was determined by TG analysis to be around 273.4 °C. FTIR studies suggested that PVA/CNF0.5 composite film showed the highest peak at 1428 cm-1 as compared to other PVA/CNF composite films representing the presence of higher crystalline band in the composite film matrix. PVA/CNF0.5 composite film was found to have a surface porosity and mean pore size of 27.35% and 0.19 µm respectively, classifying it in the MF membrane category. The maximum tensile strength (TS) of 5.27 MPa was found for PVA/CNF0.5, followed by PVA/CNF1.0, PVA/CNF1.5, pure PVA, and PVA/CNF2.0. The maximum young's modulus (111 MPa) was found for PVA/CNF1.0, followed by PVA/CNF0.5, PVA/CNF2.0, PVA/CNF1.5, and pure PVA, which could be attributed to the cyclization of the molecular structures by cross-linking. PVA/CNF0.5 exhibits greater elongation at break (21.7) than the other polymers, indicating a material's ability to undergo significant deformation before failure. Performance evaluation of the PVA/CNF0.5 composite film showed that 46.3% and 92.8% yield were found in the retentate for 200 mg/L of BSA, and 5 × 107 CFU/mL respectively. However, more than 90% E. coli was retained by PVA/CNF0.5 composite film, therefore absolute rating of this membrane is 0.22 µm. The size of this composite film may be therefore considered in the range of MF.

Identification of Novel Protein Targets of Prodigiosin for Breast Cancer Using Inverse Virtual Screening Methods.

Prodigiosin (PG) is chemically formulated as 4-methoxy-5-[(5-methyl-4-pentyl-2H-pyrrol-2ylidene)methyl]-2,2'-bi-1H-pyrrole and it is an apoptotic agent. Only a few protein targets for PG have been identified so far for regulating various diseases; nevertheless, finding more PG targets is crucial for novel drug discovery research. A bioinformatics method was applied in this work to find additional potential PG targets. Initially, a text mining analysis was conducted to determine the relationship between PG and a variety of metabolic processes. One hundred sixteen proteins from the KEGG pathway were selected for the docking study. Inverse virtual screening was performed by Discovery Studio software 4.1 using CHARMm-based docking tool. Twelve proteins are screened out of 116 because their CDOCKER interaction energy is larger than - 40.22 kcal/mol. The best docking score with PG was reported to be - 44.25 kcal/mol, - 44.99 kcal/mol, and - 40.91 kcal/mol for three novel proteins, such as human epidermal growth factor-2 (HER-2), mitogen-activated protein kinase (MEK), and S6 kinase protein (S6K) respectively. The interactions in the S6K/PG complex are predominantly hydrophobic; however, hydrogen bond interactions can be identified in the MEK/PG and HER-2/PG complexes. The root-mean-square deviation (RMSD) and key interaction score system (KISS) were further used to validate the docking approach. The docking approach employed in this work has a low RMSD value (2.44 Å) and a high KISS score (0.5), indicating that it is significant.

Cloning, improved expression and purification of invasion plasmid antigen D (IpaD): an effector protein of enteroinvasive Escherichia coli (EIEC).

The widespread increase in broad-spectrum antimicrobial resistance is making it more difficult to treat gastrointestinal infections. Enteroinvasive Escherichia coli is a prominent etiological agent of bacillary dysentery, invading via the fecal-oral route and exerting virulence on the host via the type III secretion system. IpaD, a surface-exposed protein on the T3SS tip that is conserved among EIEC and Shigella, may serve as a broad immunogen for bacillary dysentery protection. For the first time, we present an effective framework for improving the expression level and yield of IpaD in the soluble fraction for easy recovery, as well as ideal storage conditions, which may aid in the development of new protein therapies for gastrointestinal infections in the future. To achieve this, uncharacterized full length IpaD gene from EIEC was cloned into pHis-TEV vector and induction parameters were optimized for enhanced expression in the soluble fraction. After affinity-chromatography based purification, 61% pure protein with a yield of 0.33 mg per litre of culture was obtained. The purified IpaD was retained its secondary structure with a prominent α-helical structure as well as functional activity during storage, at 4°C, -20°C and -80°C using 5% sucrose as cryoprotectants, which is a critical criterion for protein-based treatments.

Downstream Process Development for Extraction of Prodigiosin: Statistical Optimization, Kinetics, and Biochemical Characterization.

Prodigiosin is natural red colourant derived from Serratia marcescens. However, the high cost of prodigiosin restricts its use in food and pharmaceutical industries, which can be addressed with the design of a suitable extraction procedure. Therefore, the present study aims to use Taguchi methodology to optimize various process parameters during ultrasound-assisted extraction (UAE) to get a higher prodigiosin extraction yield. The most significant contribution comes from the solid-to-liquid ratio (36.66%), followed by sonication of duty cycle (34.82%), medium pH (15.7%), and acoustic intensity (12.82%). The Taguchi technique predicts the highest optimal yield using the solid-liquid ratio (0.3 g/mL), duty cycle sonication (75%), acoustic intensity (12.5 w/cm2), and medium pH (3) as parameters. When the extraction conditions were optimized, the yield of prodigiosin increased by 4166.89 mg/L. In the future, the above extraction conditions determined using Taguchi approach will be applied for large-scale extraction of prodigiosin. Finally, a second-order kinetic model is used to suit the batch extraction investigation and the second-order rate constant (k) has a value of 4 × 10-5 L/mg/min. In the future, the rate constant, which is reported for the first time, will be used to create a batch extractor for commercial extraction of prodigiosin. Prodigiosin has also been shown to have substantial antioxidant and scavenging properties, which increase in a dose-dependent way with prodigiosin concentration. Because of its antioxidant and scavenging properties, prodigiosin can be used as food additives or pharmaceutical ingredients in industries.

Bi-Functional Gold Nanorod-Protein Conjugates with Biomimetic BSA@Folic Acid Corona for Improved Tumor Targeting and Intracellular Delivery of Therapeutic Proteins in Colon Cancer 3D Spheroids.

Gold nanorods (AuNRs) remain well-developed inorganic nanocarriers of small molecules for a plethora of biomedical and therapeutic applications. However, the delivery of therapeutic proteins using AuNRs with high protein loading capacity (LC), serum stability, excellent target specificity, and minimal off-target protein release is not known. Herein, we report two bi-functional AuNR-protein nanoconjugates, AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA, supramolecularly coated with folic acid-modified BSA (BSAFA) acting as biomimetic protein corona to demonstrate targeted cytosolic delivery of enhanced green fluorescent protein (EGFP) and therapeutic ribonuclease A enzyme (RNase A) in their functional forms. AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA exhibit high LCs of ∼42 and ∼54%, respectively, increased colloidal stability, and rapid protein release in the presence of biological thiols. As a nanocarrier, AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA show resistance to corona formation in high-serum media even after 24 h, guaranteeing a greater circulation lifetime. Folate receptor-targeting BSAFA on the AuNR surface facilitates the receptor-mediated internalization, followed by the release of EGFP and RNase A in HT29 cells. The green fluorescence dispersed throughout the cell's cytoplasm indicates successful cytosolic delivery of EGFP by AuNR@EGFP-BSAFA. AuNR@RNaseA-BSAFA-mediated therapeutic RNase A delivery in multicellular 3D spheroids of HT29 cells exhibits a radical reduction in the cellular RNA fluorescence intensity to 38%, signifying RNA degradation and subsequent cell death. The versatile nanoformulation strategy in terms of the anisotropic particle morphology, protein type, and ability for targeted delivery in the functional form makes the present AuNR-protein nanoconjugates a promising platform for potential application in cancer management.

Exploration of binding mechanism of triclosan towards cancer markers using molecular docking and molecular dynamics.

The molecule 5-chloro-2-(2,4-dichlorophenoxy) phenol is well-known as Triclosan (TCS), which is also a potential endocrine disrupting synthetic chemical. TCS exposure has been connected to the control of the human enoyl-acyl carrier protein-reductase (hER), which has been linked to a range of life threatening diseases. However, other than hER, the new protein targets for TCS that are responsible for a variety of cancers are yet unclear. The goal of this work is to investigate into the protein binding patterns of TCS and proteins from various cancer signaling pathways. Discovery Studio 4.1 was used to perform molecular docking and molecular dynamics (MD) on the protein-triclosan complex. The proteins were first screened using CHARMM-based docking with a CDOCKER energy greater than -21.40 kcal/mol. The CDOCKER energies of Fas-associated death domain (FADD), Receptor-interacting protein 1 (RIP1), F-κB-inducing kinase (NIK), c-Jun N-terminal kinase (JNK), Apoptosis signal-regulating kinase 1 (ASK1), B-cell lymphoma 2 (Bcl-2), Apoptosis-inducing factor (AIF), α-tubulin, and Actin were -20.68 kcal/mol, -26.88 kcal/mol, -23.43 kcal/mol, -22.21 kcal/mol, -20.40 kcal/mol, -21.10 kcal/mol, -20.98 kcal/mol, -24.67 kcal/mol, and -23.09 kcal/mol respectively. MD was performed on the screened proteins by standard dynamics cascade tool using CHARMM Force field. The MD results were accessed using the energy-time graph, root-mean-square deviation (RMSD), and root mean square fluctuations (RMSF). The 100 conformers of α-tubulin, NIK, FADD, and RIP1 were found to have a trend of increasing RMSD, whereas Bcl-2, ASK1, AIF, Actin, and JNK proteins had lower RMSD values. In compared to FADD, AIF, and JNK, the RMSF variations of the Bcl-2, ASK1, α-tubulin, Actin, NIK, and RIP1 residues were shown to be high. Similar patterns were seen in the energy variations, which range from 1000 kcal/mol to 2000 kcal/mol. RIP1 and Bcl-2 showed more variation in the sidechain RMSF in comparison to FADD, ASK1, AIF, Actin, α-tubulin, NIK and JNK. Thus, it can be postulated that AIF and JNK proteins of apoptosis signaling pathway are pivotal in the TCS mediated reactions.

Development of 6-Thioguanine conjugated PLGA nanoparticles through thioester bond formation: Benefits of electrospray mediated drug encapsulation and sustained release in cancer therapeutic applications.

Polymeric nanoparticle-based successful delivery of hydrophobic drugs is highly desirable for its controlled and sustained release at the disease site, which is a challenge with the current synthesis methods. In the present study, an electrospray mediated facile one-step synthesis approach is explored in which a solution mixture of a hydrophobic drug, 6-thioguanine (Tg) and a biocompatible FDA approved polymer, Poly (d, l-lactide-co-glycolide) (PLGA) is injected in an applied electric field of suitable intensity to prepare drug encapsulated PLGA nanoparticles, PLGA-Tg with high yield. In order to explore the effect of external electric field on Tg loading and delivery applications, the nanoparticles are characterized using EDX, AFM, FESEM, TEM, FTIR, Raman, fluorescence, and mass spectroscopy techniques. The characterization studies indicate that the electric field mediated synthesis exhibits spherical nanoparticles with a homogenous core size distribution of ~60 nm, high encapsulation (~97.22%) and stable conjugation of Tg (via thioester linkages) with PLGA molecules in the presence of the applied electric field. The kinetic study demonstrates the 'anomalous diffusion' (non-Fickian diffusion) release mechanism in which Tg escapes from PLGA matrix with a slow, but steady diffusion rate and the sustained drug release profile continues for 60 days. To check the biological activity of the encapsulated Tg, in-vitro cell studies of the PLGA-Tg are performed on HeLa cells. The MTT assay shows significant cell death after 48 h of treatment, and the cellular internalization of the drug-loaded nanoparticles occurs through pinocytosis mediated uptake, which is established by the AFM analysis. The Raman and mass spectroscopy studies suggest that the PLGA-Tg nanoparticles are rapidly hydrolyzed inside cell cytoplasm to release Tg which initiates apoptosis-mediated cell death confirmed by as DNA fragmentation and membrane blebbing studies. The results clearly emphasize the benefits of electrospray based synthesis of polymeric nanodrug formulation through the formation of chemical bonds between polymer and drug molecules that could be easily implemented in the design and development of an effective nanotherapeutic platform with no typical 'burst effect,' prolonged release profile, and significant toxicity to the cancer cells.

Ethylenediamine assisted functionalization of self-organized poly (d, l-lactide-co-glycolide) patterned surface to enhance cancer cell isolation.

Protein functionalized micro-scale patterned structures are developed using a biocompatible polymer PLGA (poly (d, l-lactide-co-glycolide)) via thin film dewetting and by step-wise chemical conjugations with EDA (ethylenediamine) and anti-EpCAM (Epithelial Cell Adhesion Molecule) antibodies to target the epithelial cell adhesion molecules of cancer cells. The effectiveness of such protein functionalized patterned surface is checked through cell isolation process using blood samples spiked with different cancer cells such as MCF-7, A549, MDA-MB-231. An efficient capture yield of 92% is obtained with MCF-7 cells over a two hour incubation time. The study demonstrates the effects of cell concentration and incubation time on the binding of cancer cells to the modified patterned surfaces. For the first time, a simple and inexpensive method is reported to fabricate functionalized PLGA patterned surface for an efficient isolation of cancer cells from diluted blood samples. The method shows the potential to be used as an effective platform for the development of an improved circulating tumor cell (CTC) isolation device from the clinical blood sample.

Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide.

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.

Outer membrane protein A (OmpA) of Shigella flexneri 2a, induces protective immune response in a mouse model.

BACKGROUND: In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant. KEY RESULTS: In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPO® vector and expressed using induction with isopropyl thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines. CONCLUSION: These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associated with the marked protective efficacy of immunized mice after intranasal shigellae infection.

Thermostable direct hemolysin downregulates human colon carcinoma cell proliferation with the involvement of E-cadherin, and ß-catenin/Tcf-4 signaling.

BACKGROUND: Colon cancers are the frequent causes of cancer mortality worldwide. Recently bacterial toxins have received marked attention as promising approaches in the treatment of colon cancer. Thermostable direct hemolysin (TDH) secreted by Vibrio parahaemolyticus causes influx of extracellular calcium with the subsequent rise in intracellular calcium level in intestinal epithelial cells and it is known that calcium has antiproliferative activity against colon cancer. KEY RESULTS: In the present study it has been shown that TDH, a well-known traditional virulent factor inhibits proliferation of human colon carcinoma cells through the involvement of CaSR in its mechanism. TDH treatment does not induce DNA fragmentation, nor causes the release of lactate dehydrogenase. Therefore, apoptosis and cytotoxicity are not contributing to the TDH-mediated reduction of proliferation rate, and hence the reduction appears to be caused by decrease in cell proliferation. The elevation of E-cadherin, a cell adhesion molecule and suppression of ß-catenin, a proto-oncogene have been observed in presence of CaSR agonists whereas reverse effect has been seen in presence of CaSR antagonist as well as si-RNA in TDH treated cells. TDH also triggers a significant reduction of Cyclin-D and cdk2, two important cell cycle regulatory proteins along with an up regulation of cell cycle inhibitory protein p27(Kip1) in presence of CaSR agonists. CONCLUSION: Therefore TDH can downregulate colonic carcinoma cell proliferation and involves CaSR in its mechanism of action. The downregulation occurs mainly through the involvement of E-cadherin-ß-catenin mediated pathway and the inhibition of cell cycle regulators as well as upregulation of cell cycle inhibitors.

34 kDa MOMP of Shigella flexneri promotes TLR2 mediated macrophage activation with the engagement of NF-kappaB and p38 MAP kinase signaling.

The 34 kDa major outer membrane protein (MOMP) of Shigella flexneri 2a induces combinatorial expression of TLR2 and TLR6 on peritoneal macrophages of BALB/c mice. Between the two best-characterized TLRs, to date, TLR2 and TLR4, which are chiefly responsible for recognizing majority of bacterial products, TLR2 alone participates in recognition of 34 kDa MOMP. In addition to TLRs, MOMP enhances the mRNA expression of MyD88 and TRAF6 and induces the nuclear translocation of NF-kappaB as well as activates p38 MAP kinase, suggesting the involvement of these molecules in the mechanism of action of MOMP. 34 kDa MOMP also stimulates macrophages, up regulates the surface expression of MHC-II and B7-1 and enhances the production of different cytokines (such as ILp70, TNF-alpha, Il-6) and chemokines (like MIP-1 alpha, MIP-1 beta and RANTES). The ability of the protein in the activation of macrophages, i.e. the induction of nuclear translocation of NF-kappaB and secretion of cytokines are dependent on TLR2 expression as demonstrated by the lack of response by macrophages pre-treated with inhibitory TLR2 mAb. Moreover, it has been found that MOMP induced regulation of TLR2 gene expression is dependent on NF-kappaB and p38 MAP kinase in murine macrophages for the first time. The MOMP induced cytokines and chemokines profile reflect that the protein has the ability to translate innate towards type-1 adaptive response. In conclusion MOMP recognizes by and activates macrophages which may be an initiating event in the antibacterial host response.

Reorganization of cytoskeletal proteins by Escherichia coli heat-stable enterotoxin (STa)-mediated signaling cascade.

BACKGROUND: IP(3)-mediated calcium mobilization from intracellular stores activates and translocates PKC-alpha from cytosol to membrane fraction in response to STa in COLO-205 cell line. The present study was undertaken to determine the involvement of cytoskeleton proteins in translocation of PKC-alpha to membrane from cytosol in the Escherichiacoli STa-mediated signaling cascade in a human colonic carcinoma cell line COLO-205. METHODS: Western blots and consequent densitometric analysis were used to assess time-dependent redistribution of cytoskeletal proteins. This redistribution was further confirmed by using confocal microscopy. Pharmacological reagents were applied to colonic carcinoma cells to disrupt the microfilaments (cytochalasin D) and microtubules (nocodazole). RESULTS: STa treatment in COLO-205 cells showed dynamic redistribution and an increase in actin content in the Triton-insoluble fraction, which corresponds to an increase in polymerization within 1min. Moreover, pharmacological disruption of actin-based cytoskeleton greatly disturbed PKC-alpha translocation to the membrane. CONCLUSIONS: These results suggested that the organization of actin cytoskeleton is rapidly rearranged following E. coli STa treatment and the integrity of the actin cytoskeleton played a crucial role in PKC-alpha movement in colonic cells. Depolymerization of tubulin had no effect on the ability of the kinase to be translocated to the membrane. GENERAL SIGNIFICANCE: In the present study, we have shown for the first time that in colonic carcinoma cells, STa-mediated rapid changes of actin cytoskeleton arrangement might be involved in the translocation of PKC-alpha to membrane.

Purification and characterization of an immunogenic outer membrane protein of Shigella flexneri 2a.

In the present study we purified 34 kDa major outer membrane protein (MOMP) of Shigella flexneri 2a for the first time, which was cross-reactive and antigenically conserved among Shigella spp. and the epitope was surface exposed on the intact bacterium. The purified antigen was found to be glycosylated, which aids in binding to macrophages and up-regulated the production of nitric oxide, granulocyte-colony stimulating factor and IL-12p70, indicating that the MOMP is immunogenic and has the ability to commence protective immune responses against intracellular pathogens, thereby it may be considered as a potential vaccine candidate.