Quorum-sensing regulation of virulence factors in bacterial biofilm.

Chronic polymicrobial wound infections are often characterized by the presence of bacterial biofilms. They show considerable structural and functional heterogeneity, which influences the choice of antimicrobial therapy and wound healing dynamics. The hallmarks of biofilm-associated bacterial infections include elevated antibiotic resistance and extreme pathogenicity. Biofilm helps bacteria to evade the host defense mechanisms and persist longer in the host. Quorum-sensing (QS)-mediated cell signaling primarily regulates biofilm formation in chronic infections and plays a major role in eliciting virulence. This review focuses on the QS mechanisms of two major bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa and explains how they interact in the wound microenvironment to regulate biofilm development and virulence. The review also provides an insight into the treatment modalities aimed at eradicating polymicrobial biofilms. This information will help us develop better diagnostic modalities and devise effective treatment regimens to successfully manage and overcome severe life-threatening bacterial infections.

Photodynamic therapy to control microbial biofilms.

Microorganisms thrive in well-organized biofilm ecosystems. Biofilm-associated cells typically show increased resistance to antibiotics and contribute significantly to treatment failure. This has prompted investigations aimed at developing advanced and novel antimicrobial approaches that could effectively overcome the shortcomings associated with conventional antibiotic therapy. Studies are ongoing to develop effective curative strategies ranging from the use of peptides, small molecules, nanoparticles to bacteriophages, sonic waves, and light energy targeting various structural and physiological aspects of biofilms. In photodynamic therapy, a light source of a specific wavelength is used to irradiate non-toxic photosensitizers such as tetrapyrroles, synthetic dyes or, naturally occurring compounds to generate reactive oxygen species that can exert a lethal effect on the microbe especially by disrupting the biofilm. The photosensitizer preferentially binds to and accumulates in the microbial cells without causing any damage to the host tissue. Currently, photodynamic therapy is increasingly being used for the treatment of oral caries and dental plaque, chronic wound infections, infected diabetic foot ulcers, cystic fibrosis, chronic sinusitis, implant device-associated infections, etc. This approach is recognized as safe, as it is non-toxic and minimally invasive, making it a reliable, realistic, and promising therapeutic strategy for reducing the microbial burden and biofilm formation in chronic infections. In this review article, we discuss the current and future potential strategies of utilizing photodynamic therapy to extend our ability to impede and eliminate biofilms in various medical conditions.

Retinoblastoma tumor suppressor gene expression determines the response to sequential flavopiridol and doxorubicin treatment in small-cell lung carcinoma.

PURPOSE: Small-cell lung cancers (SCLC) are defective in many regulatory mechanisms that control cell cycle progression, i.e., functional retinoblastoma protein (pRb). Flavopiridol inhibits proliferation and induces apoptosis in SCLC cell lines. We hypothesized that the sequence flavopiridol followed by doxorubicin would be synergistic in pRb-deficient SCLC cells. EXPERIMENTAL DESIGN: A H69 pRb-deficient SCLC cell line, H865, with functional pRb and H865 pRb small interfering RNA (siRNA) knockdown cells were used for in vitro and in vivo experiments. The in vivo efficiencies of various sequential combinations were tested using nude/nude athymic mice and human SCLC xenograft models. RESULTS: Flavopiridol then doxorubicin sequential treatment was synergistic in the pRB-negative H69 cell line. By knocking down pRb with specific siRNA, H865 clones with complete pRb knockdown became sensitive to flavopiridol and doxorubicin combinations. pRb-deficient SCLC cell lines were highly sensitive to flavopiridol-induced apoptosis. pRb-positive H865 cells arrested in G0-G1 with flavopiridol exposure, whereas doxorubicin and all flavopiridol/doxorubicin combinations caused a G2-M block. In contrast, pRb-negative SCLC cells did not arrest in G0-G1 with flavopiridol exposure. Flavopiridol treatment alone did not have an in vivo antitumor effect, but sequential flavopiridol followed by doxorubicin treatment provided tumor growth control and a survival advantage in Rb-negative xenograft models, compared with the other sequential treatments. CONCLUSIONS: Flavopiridol and doxorubicin sequential treatment induces potent in vitro and in vivo synergism in pRb-negative SCLC cells and should be clinically tested in tumors lacking functional pRB.

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 potently stimulates gene-specific DNA binding of the vitamin D receptor in osteoblasts.

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD) is a highly potent analog of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) whose actions are mediated through the vitamin D receptor (VDR). In this report, we have replicated this increased potency of 2MD in vitro using osteoblastic cells and explored its underlying molecular mechanism. 2MD stimulates the expression of several vitamin D-sensitive genes including 25-hydroxyvitamin D3-24 hydroxylase (Cyp24), osteopontin and receptor activator of NF kappa B ligand and suppresses osteoprotegerin at concentrations two logs lower than that for 1,25(OH)2D3. 2MD is also more potent in stimulating transfected chimeric reporter genes under either Cyp24 or the osteocalcin promoter control. Enhanced potency is retained regardless of medium serum content. Interestingly, the uptake of both 1,25(OH)2D3 and 2MD into cells is similar, as is their rapid association with the VDR. This indicates that comparable levels of occupied VDR do not elicit equivalent levels of transactivation. Using chromatin immunoprecipitation (ChIP), however, we observed a strong correlation between DNA-bound receptor and the level of induced transcription suggesting a 2MD-induced increase in affinity of the VDR for DNA. Additional studies using a mammalian two-hybrid system and ChIP indicate that 2MD is also more potent in promoting interaction with RXR and the coactivators SRC-1 and DRIP205. Finally, protease digestion studies revealed a unique VDR conformation in the presence of 2MD. These studies suggest that the molecular mechanism of 2MD potency is due to its ability to promote enhanced levels of specific DNA binding by the VDR and could suggest possible explanations for the tissue- and gene-selective actions of 2MD.