Characterization of the Thermostable Biosurfactant Produced by Burkholderia thailandensis DSM 13276.

Biosurfactants synthesized by microorganisms represent safe and sustainable alternatives to the use of synthetic surfactants, due to their lower toxicity, better biodegradability and biocompatibility, and their production from low-cost feedstocks. In line with this, the present study describes the physical, chemical, and functional characterization of the biopolymer secreted by the bacterium Burkholderia thailandensis DSM 13276, envisaging its validation as a biosurfactant. The biopolymer was found to be a glycolipopeptide with carbohydrate and protein contents of 33.1 ± 6.4% and 23.0 ± 3.2%, respectively. Galactose, glucose, rhamnose, mannose, and glucuronic acid were detected in the carbohydrate moiety at a relative molar ratio of 4:3:2:2:1. It is a high-molecular-weight biopolymer (1.0 × 107 Da) with low polydispersity (1.66), and forms aqueous solutions with shear-thinning behavior, which remained after autoclaving. The biopolymer has demonstrated a good emulsion-stabilizing capacity towards different hydrophobic compounds, namely, benzene, almond oil, and sunflower oil. The emulsions prepared with the biosurfactant, as well as with its autoclaved solution, displayed high emulsification activity (>90% and ~50%, respectively). Moreover, the almond and sunflower oil emulsions stabilized with the biosurfactant were stable for up to 4 weeks, which further supports the potential of this novel biopolymer for utilization as a natural bioemulsifier.

Bioconversion of Terephthalic Acid and Ethylene Glycol Into Bacterial Cellulose by Komagataeibacter xylinus DSM 2004 and DSM 46604.

Komagataeibacter xylinus strains DSM 2004 and DSM 46604 were evaluated for their ability to grow and produce bacterial cellulose (BC) upon cultivation on terephthalic acid (TA) and ethylene glycol (EG), which are monomers of the petrochemical-derived plastic polyethylene terephthalate (PET). Both strains were able to utilize TA, EG, and their mixtures for BC synthesis, with different performances. K. xylinus DSM 2004 achieved higher BC production from TA (0.81 ± 0.01 g/L), EG (0.64 ± 0.02 g/L), and TA + EG mixtures (0.6 ± 0.1 g/L) than strain DSM 46604. The latter was unable to utilize EG as the sole carbon source and reached a BC production of 0.16 ± 0.01 g/L and 0.23 ± 0.1 g/L from TA alone or TA + EG mixtures, respectively. Further supplementing the media with glucose enhanced BC production by both strains. During cultivation on media containing TA and EG, rapid pH drop due to metabolization of EG into acidic compounds led to some precipitation of TA that was impregnated into the BC pellicles. An adaptation of the downstream procedure involving BC dissolution in NaOH was used for the recovery of pure BC. The different medium composition tested, as well as the downstream procedure, impacted the BC pellicles' physical properties. Although no variation in terms of the chemical structure were observed, differences in crystallinity degree and microstructure of the produced BC were observed. The BC produced by K. xylinus DSM 2004 had a higher crystallinity (19-64%) than that of the strain DSM 46604 (17-53%). Moreover, the scanning electron microscopy analysis showed a higher fiber diameter for K. xylinus DSM 2004 BC (46-56 nm) than for K. xylinus DSM 46604 (37-49 nm). Dissolution of BC in NaOH did not influence the chemical structure; however, it led to BC conversion from type I to type II, as well as a decrease in crystallinity. These results demonstrate that PET monomers, TA and EG, can be upcycled into a value-added product, BC, presenting an approach that will contribute to lessening the environmental burden caused by plastic disposal in the environment.

Preparation and Characterization of Films Based on a Natural P(3HB)/mcl-PHA Blend Obtained through the Co-culture of Cupriavidus Necator and Pseudomonas Citronellolis in Apple Pulp Waste.

The co-culture of Cupriavidus necator DSM 428 and Pseudomonas citronellolis NRRL B-2504 was performed using apple pulp waste from the fruit processing industry as the sole carbon source to produce poly(3-hydroxybutyrate), P(3HB) and medium-chain length PHA, mcl-PHA, respectively. The polymers accumulated by both strains were extracted from the co-culture's biomass, resulting in a natural blend that was composed of around 48 wt% P(3HB) and 52 wt% mcl-PHA, with an average molecular weight of 4.3 × 105 Da and a polydispersity index of 2.2. Two melting temperatures (Tm) were observed for the blend, 52 and 174 °C, which correspond to the Tm of the mcl-PHA and P(3HB), respectively. P(3HB)/mcl-PHA blend films prepared by the solvent evaporation method had permeabilities to oxygen and carbon dioxide of 2.6 and 32 Barrer, respectively. The films were flexible and easily deformed, as demonstrated by their tensile strength at break of 1.47 ± 0.07 MPa, with a deformation of 338 ± 19% until breaking, associated with a Young modulus of 5.42 ± 1.02 MPa. This study demonstrates for the first time the feasibility of using the co-culture of C. necator and P. citronellolis strains to obtain a natural blend of P(3HB)/mcl-PHA that can be processed into films suitable for applications ranging from commodity packaging products to high-value biomaterials.

Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations.

BACKGROUND: Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds. METHODS: Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot. RESULTS: Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAF(V600E) mutation. In BRAF(V600E) mutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27(Kip1). Specific inhibition of BRAF by RNAi in cells with BRAF(V600E) mutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAF(V600E) mutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2. CONCLUSION: Our results in thyroid cancer cells, namely those harbouring BRAF(V600E) mutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAF(V600E) mutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and harbouring BRAF mutations.

A subset of colorectal carcinomas express c-KIT protein independently of BRAF and/or KRAS activation.

c-KIT is a tyrosine kinase receptor found to be overexpressed in several tumours, namely, GISTs, breast, lung, prostate, ovarian and colorectal carcinomas (CRC). We aimed at determining the frequency of c-KIT expression and mutations in a series of 109 CRC cases (73 primary tumours and 36 lymph node metastases) characterised for KRAS and BRAF mutations. We also aimed at analysing the cellular effects of STI571/Gleevec in CRC-derived cell lines displaying c-KIT expression and KRAS or BRAF mutations. By immunohistochemistry, we found c-KIT overexpression in 15% (11/73) of primary tumours and in 14% (5/36) of metastasis; however, cases showing overexpression did not show c-kit mutations in hotspot regions. The majority (64%) of primary tumours with c-KIT overexpression had mutations at KRAS-BRAF genes. The same was true for 60% of the metastases. We treated CRC cell lines with STI571/Gleevec and verified that it inhibits proliferation and induces apoptosis in all cell lines. In conclusion, overexpression of c-KIT is observed in a subset of primary and CRC metastases in the absence of c-kit mutations. STI571/Gleevec increases apoptosis in CRC cell lines independently of its genetic profile, suggesting that STI571/Gleevec is likely to be an alternative drug for the clinical trials of CRC.

Molecular and genotypic characterization of human thyroid follicular cell carcinoma-derived cell lines.

OBJECTIVE: Our aim was to characterize the molecular and genotypic profile of eight thyroid carcinoma-derived cell lines-TPC1, FB2, B-CPAP, K1, XTC-1, C643, 8505C, and Hth74-in order to use them as in vitro models of thyroid carcinogenesis. DESIGN: We evaluated the expression of five thyroid-specific genes (Tg, TSHr, TPO, PAX8, and TTF-1) to establish the cell lineage and to assess the differentiation status of each of the cell lines. We screened for mutations in the most relevant oncogenes/tumor suppressor genes affected in thyroid carcinogenesis: RAS, BRAF, CTNNB1, and TP53 along with RET/PTC rearrangements. Considering the putative relevance in general carcinogenesis, we have also studied other molecules such as EGFR, PI3K, RAF-1, and THRB. To determine the genetic identity of the cell lines, we performed genotypic analysis. MAIN OUTCOME: The panel of cell lines we have studied displayed activation of several oncogenes (BRAF, RAS, RET/PTC) and inactivation of tumor suppressor genes (TP53) known to be important for thyroid carcinogenesis. Two of the cell lines-TPC1 and FB2-shared the same genotypic profile, probably representing clones of an ancestor cell line (TPC1). CONCLUSION: Due to their different molecular alterations, these cell lines represent a valuable tool to study the molecular mechanisms underlying thyroid carcinogenesis. We suggest that genotypic analyses should be included as a routine procedure to guarantee the uniqueness of each cell line used in research.

BRAF mutations typical of papillary thyroid carcinoma are more frequently detected in undifferentiated than in insular and insular-like poorly differentiated carcinomas.

Somatic mutations of the BRAF gene (BRAFV599E and BRAFK600E) were found to be closely associated with different histotypes of papillary thyroid carcinoma (PTC). The V599E mutation is highly prevalent in PTC with a papillary or mixed papillary follicular growth pattern, and the K600E mutation is apparently restricted to the follicular variant of PTC. It is usually accepted that thyroid malignancies may follow a progression path from well-differentiated to poorly differentiated (PDC) and undifferentiated (UC) carcinomas. One would expect that at least some of the less differentiated carcinomas would harbour the genetic alterations of pre-existing well-differentiated tumours. In order to find the prevalence of BRAF mutations in PDC and UC, we screened a series of 19 PDCs and 17 UCs, as well as 3 UC-derived cell lines, for both mutation types. The group of PDCs was restricted to the so-called insular and insular-like PDCs, thus excluding PTCs with solid, insular or trabecular foci of growth and PDCs displaying typical PTC nuclei. No BRAF mutations were detected in any of the 19 cases of PDC, whereas 6 of the UCs (35%) and one UC-derived cell line presented the BRAFV599E mutation. The BRAFK600E mutation was not detected in any case. We conclude that UC may progress from BRAFV599E-mutated PTC. The absence of BRAF mutations in our series of PDC supports the assumption that pure insular and insular-like PDCs are more closely related to follicular carcinoma than to PTC.

New insight puts CRAF in sight as a therapeutic target.

By selectively depleting components of the RAF-MEK-ERK pathway in transgenic mice, it is now shown in 2 studies that CRAF is critical for signaling to MEK downstream of oncogenic Kras and that BRAF is not required.

Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant chronic myeloid leukemia.

We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.