Co-Culture of P. gingivalis and F. nucleatum Synergistically Elevates IL-6 Expression via TLR4 Signaling in Oral Keratinocytes.

Periodontitis, characterized by persistent inflammation in the periodontium, is intricately connected to systemic diseases, including oral cancer. Bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, play a pivotal role in periodontitis development because they contribute to dysbiosis and tissue destruction. Thus, comprehending the interplay between these bacteria and their impacts on inflammation holds significant relevance in clinical understanding and treatment advancement. In the present work, we explored, for the first time, their impacts on the expressions of pro-inflammatory mediators after infecting oral keratinocytes (OKs) with a co-culture of pre-incubated P. gingivalis and F. nucleatum. Our results show that the co-culture increases IL-1ß, IL-8, and TNF-α expressions, synergistically augments IL-6, and translocates NF-kB to the cell nucleus. These changes in pro-inflammatory mediators-associated with chronic inflammation and cancer-correlate with an increase in cell migration following infection with the co-cultured bacteria or P. gingivalis alone. This effect depends on TLR4 because TLR4 knockdown notably impacts IL-6 expression and cell migration. Our study unveils, for the first time, crucial insights into the outcomes of their co-culture on virulence, unraveling the role of bacterial interactions in polymicrobial diseases and potential links to oral cancer.

Bacterial Synthesis of Ternary CdSAg Quantum Dots through Cation Exchange: Tuning the Composition and Properties of Biological Nanoparticles for Bioimaging and Photovoltaic Applications.

In this study, we introduce a biological method for the production of ternary Quantum Dots (QDs): complex nanostructures with tunable optical and structural properties that utilizes post-synthesis modifications through cation exchange. This versatile in-situ cation exchange method being reported for the first time shows great potential for extending the scope of microbial synthesis. By using this bacterial-based method, we easily synthesize and purify CdS, CdSAg, and Ag2S nanocrystals of a size below 15 nm and with variable morphologies that exhibit fluorescence emissions covering a broad spectral range (from 400 to 800 nm). Energy-dispersive X-ray spectroscopy (EDS) results indicate the partial replacement of Cd2+ by Ag+ when AgNO3 concentration is increased. This replacement produces CdSAg ternary QDs hetero-structures with high stability, fluorescence in the NIR-I (700 - 800 nm), and 36.13% quantum yield. Furthermore, this reaction can be extended for the production of soluble Ag2S nanoparticles (NPs) without any traces of Cd. QDs biosynthesized through this cation exchange process display very low toxicity when tested in bacterial or human cell lines. Biosynthesized ternary hetero-structures were used as red fluorescent dyes to label HeLa cells in confocal microscopy studies, which validates its use in bioimaging applications in the near infrared region. In addition, the application of biologically-produced cadmium NPs in solar cells is reported for the first time. The three biosynthesized QDs were successfully used as photosensitizers, where the CdSAg QDs show the best photovoltaic parameters. Altogether, obtained results validate the use of bacterial cells for the controlled production of nanomaterials with properties that allow their application in diverse technologies. We developed a simple biological process for obtaining tunable Quantum Dots (QDs) with different metal compositions through a cation exchange process. Nanoparticles (NPs) are produced in the extracellular space of bacterial cells exposed to cysteine and CdCl2 in a reaction that depends on S2- generation mediated by cysteine desulfhydrase enzymes and uses cellular biomolecules to stabilize the nanoparticle. Using this extracellular approach, water-soluble fluorescent CdS, CdSAg, and Ag2S Quantum Dots with a tunable emission ranging from 400 to 800 nm were generated. This is the first study reporting the use of microorganisms to produce tunable ternary QDs and the first time that a cation exchange process mediated by cells is described. Obtained results validate the use of biological synthesis to produce NPs with new characteristics and opens a completely new research field related to the use of microorganisms to synthesize complex NPs that are difficult to obtain with regular chemical methods.

Variability in Genomic and Virulent Properties of Porphyromonas gingivalis Strains Isolated From Healthy and Severe Chronic Periodontitis Individuals.

Porphyromonas gingivalis has been extensively associated with both the onset and progression of periodontitis. We previously isolated and characterized two P. gingivalis strains, one from a patient exhibiting severe chronic periodontitis (CP3) and another from a periodontally healthy individual (H3). We previously showed that CP3 and H3 exhibit differences in virulence since H3 showed a lower resistance to cationic peptides compared with CP3, and a lower ability to induce proliferation in gingival epithelial cells. Here, we aimed to determine whether differences in virulence between these two strains are associated with the presence or absence of specific genes encoding virulence factors. We sequenced the whole genomes of both P. gingivalis CP3 and H3 and conducted a comparative analysis regarding P. gingivalis virulence genetic determinants. To do so, we performed a homology search of predicted protein sequences in CP3 and H3 genomes against the most characterized virulence genes for P. gingivalis available in the literature. In addition, we performed a genomic comparison of CP3 and H3 with all the 62 genomes of P. gingivalis found in NCBI's RefSeq database. This approach allowed us to determine the evolutionary relationships of CP3 and H3 with other virulent and avirulent strains; and additionally, to detect variability in presence/absence of virulence genes among P. gingivalis genomes. Our results show genetic variability in the hemagglutinin genes. While CP3 possesses one copy of hagA and two of hagC, H3 has no hagA and only one copy of hagC. Experimentally, this finding is related to lower in vitro hemmaglutination ability of H3 compared to CP3. Moreover, while CP3 encodes a gene for a major fimbrium subunit FimA type 4 (CP3_00160), H3 possess a FimA type 1 (H3_01400). Such genetic differences are in agreement with both lower biofilm formation ability and less intracellular invasion to oral epithelial cells exhibited by H3, compared with the virulent strain CP3. Therefore, here we provide new results on the genome sequences, comparative genomics analyses, and phenotypic analyses of two P. gingivalis strains. The genomics comparison of these two strains with the other 62 genomes included in the analysis provided relevant results regarding genetic determinants and their association with P. gingivalis virulence.

Biomimetic quantum dot-labeled B16F10 murine melanoma cells as a tool to monitor early steps of lung metastasis by in vivo imaging.

BACKGROUND: Numerous studies have proposed the use of fluorescent semiconductor nanoparticles or quantum dots (QDs) as novel tools to label cells and tumors. However, QD applications are limited by their toxicity in biological systems and little is known about whether QDs affect the capacity of cancer cells to metastasize. Previously, we described the "biomimetic" synthesis of CdTe-QDs (QDs-glutathione [GSH]) with increased biocompatibility and the potential utility in labeling cells. PURPOSE: In order to determine the feasibility of using QDs-GSH as a tool for tracking tumor cells during early metastasis, we characterized here for the first time, the in vitro and in vivo effects of the incorporation of green or red biomimetic QDs-GSH into B16F10 cells, a syngeneic mouse melanoma line for metastasis assays in C57BL/6 mice. METHODS: B16F10 cells were labeled with green or red biomimetic QDs-GSH in the presence or absence of n-acetylcysteine. Then, migration, invasion and proliferation of labeled B16F10 were evaluated in vitro. Finally, the B16F10 cells labeled with red QDs-GSH were used to monitor in vivo lung metastasis at early time points (5 minutes to 24 hours) or after 21 days in C57BL/6 mice. RESULTS: We developed a methodology that allows obtaining QDs-GSH-labeled B16F10 cells (nearly 100% viable labeled cells), which remained viable for at least 5 days and migrated similarly to control cells. However, proliferation, invasion, and the capacity to form metastatic nodules in the lungs were severely attenuated. Fluorescence imaging revealed that distribution/accumulation of QDs-GSH-labeled B16F10 cells could be tracked following injection into C57BL/6 mice (syngeneic preclinical metastasis model) and that these cells preferentially accumulated in the perialveolar area in lungs as early as 5 minutes post-injection. CONCLUSION: The methodology described here represents a useful alternative for monitoring initial events during tumor cell metastasis.

Changes in lipopolysaccharide profile of Porphyromonas gingivalis clinical isolates correlate with changes in colony morphology and polymyxin B resistance.

Virulence factors on the surface of Porphyromonas gingivalis constitute the first line of interaction with host cells and contribute to immune modulation and periodontitis progression. In order to characterize surface virulence factors present on P. gingivalis, we obtained clinical isolates from healthy and periodontitis subjects and compared them with reference strains. Colony morphology, aggregation in liquid medium, surface charge, membrane permeability to bactericidal compounds, novobiocin and polymyxin B resistance, capsule presence and lipopolysaccharide (LPS) profiles were evaluated. By comparing isolates from healthy and periodontitis subjects, differences in colony morphology and aggregation in liquid culture were found; the latter being similar to two reference strains. These differences were not a consequence of variations in bacterial surface charge. Furthermore, isolates also presented differences in polymyxin B and novobiocin resistance; isolates from healthy subjects were susceptible to polymyxin B and resistant to novobiocin and, in contrast, isolates from periodontitis subjects were resistant to polymyxin B and susceptible to novobiocin. These changes in antimicrobial resistance levels correlate with variations in LPS profiles, since -unlike periodontitis isolates-isolates from healthy samples synthesize LPS molecules lacking both O-antigen moieties and anionic polysaccharide. Additionally, this phenotype correlated with the absence of O-antigen ligase activity. Altogether, our results reveal novel variations on surface components of P. gingivalis isolates obtained from healthy and periodontitis subjects that could be associated with differences in bacterial virulence and periodontitis progression.

Use of titanium dioxide nanoparticles biosynthesized by Bacillus mycoides in quantum dot sensitized solar cells.

BACKGROUND: One of the major challenges of nanotechnology during the last decade has been the development of new procedures to synthesize nanoparticles. In this context, biosynthetic methods have taken hold since they are simple, safe and eco-friendly. RESULTS: In this study, we report the biosynthesis of TiO2 nanoparticles by an environmental isolate of Bacillus mycoides, a poorly described Gram-positive bacterium able to form colonies with novel morphologies. This isolate was able to produce TiO2 nanoparticles at 37 ° C in the presence of titanyl hydroxide. Biosynthesized nanoparticles have anatase polymorphic structure, spherical morphology, polydisperse size (40-60 nm) and an organic shell as determined by UV-vis spectroscopy, TEM, DLS and FTIR, respectively. Also, conversely to chemically produced nanoparticles, biosynthesized TiO2 do not display phototoxicity. In order to design less expensive and greener solar cells, biosynthesized nanoparticles were evaluated in Quantum Dot Sensitized Solar Cells (QDSSCs) and compared with chemically produced TiO2 nanoparticles. Solar cell parameters such as short circuit current density (ISC) and open circuit voltage (VOC) revealed that biosynthesized TiO2 nanoparticles can mobilize electrons in QDSSCs similarly than chemically produced TiO2. CONCLUSIONS: Our results indicate that bacterial extracellular production of TiO2 nanoparticles at low temperatures represents a novel alternative for the construction of green solar cells.