Economic and environmental assessment of bacterial poly(3-hydroxybutyrate) production from the organic fraction of municipal solid waste.

The management of municipal solid waste is a major logistic and environmental problem worldwide. Nonetheless, the organic fraction of municipal solid waste (OFMSW) is a valuable source of nutrients which can be used for a variety of purposes, according to the Circular Economy paradigm. Among the possible applications, the bioproduction of a biodegradable polyester, poly(3-hydroxybutyrate) [P(3HB)], using OFMSW as carbon platform is a promising strategy. Here, an economic and environmental assessment of bacterial P(3HB) production from OFMSW is presented based on previously published results. The SuperPro Designer® software was used to simulate P(3HB) production under our experimental parameters. Two scenarios were proposed depending on the fermentation medium: (1) enzymatic hydrolysate of OFMSW supplemented with glucose and plum waste juice; and (2) basal medium supplemented with glucose and plum waste juice. According to our results, both scenarios are not economically feasible under our experimental parameters. In Scenario 1, the low fermentation yield, the cost of the enzymes, the labour cost and the energy consumption are the factors that most contribute to that result. In Scenario 2, the cost of the extraction solvent and the low fermentation yield are the most limiting factors. The possibility of using process waste as raw material for the generation of other products must be investigated to enhance economic feasibility. From an environmental viewpoint, the photochemical oxidation potential (derived from the use of anisole as extraction solvent) and the generation of acid rain and global warming effect (caused by the burning of fuels for power generation) are the most relevant impacts associated to P(3HB) production under our experimental parameters.

Giving credit to residual bioresources: From municipal solid waste hydrolysate and waste plum juice to poly (3-hydroxybutyrate).

Municipal solid waste (MSW) is massively generated all over the world. Its organic fraction (OFMSW), which represents a high percentage of MSW, mainly contains biodegradable materials, namely food waste, paper and garden waste. The social cost of OFMSW treatment and/or disposal is a serious and widespread problem, particularly in highly populated areas. Thus, effective and innovative solutions, which include the upgrading of OFMSW, are being currently sought. In fact, the OFMSW abundance, availability and average composition suggest its considerable potential within the circular economy desideratum, paving the way to valorisation approaches. In this context, an OFMSW sugar-rich hydrolysate and its validation as a substrate for the production of the polyester poly(3-hydroxybutyrate) (P(3HB)), to date the only bioplastic easily biodegradable in marine environment, were successfully obtained in a previous study. Based on those results, this work addresses the upscaling of the fermentative production, in fed-batch mode, of P(3HB) by Burkholderia sacchari. The OFMSW hydrolysate was used as cultivation medium due to its balanced nutrient composition, while a plum waste juice, also rich in sugars, was applied as feed to the bioreactor. By implementing this strategy, a maximum P(3HB) production of 30 g·L-1 with an accumulation of 43% g (P(3HB))/g cell dry weight (CDW) after 51 h, was achieved. The use of the hydrolysate as initial medium resulted in higher CDW (71 g·L-1) than that of the simulated hydrolysate (62 g·L-1 in average), probably because the OFMSW hydrolysate favours biomass growth in detriment of P(3HB) production.

Upgrading the organic fraction of municipal solid waste to poly(3-hydroxybutyrate).

The organic fraction of municipal solid waste was studied as feedstock for the production of poly(3-hydroxybutyrate) (P(3HB)). To release the monosaccharides, a diluted acid pre-treatment followed by an enzymatic hydrolysis was applied. A sugar yield of 49% was achieved using a pre-treated waste and an enzyme cocktail of Pentopan 500 BG and Celluclast BG. The addition of Glucoamylase NS 22035 helped to hydrolyze the starch fraction, improving the hydrolysis yield to 56%. The hydrolysate was used as culture medium to produce P(3HB) by Burkholderia sacchari DSM 17165. Assays at shaking flask scale showed that when the hydrolysate was used as substrate, the attained cell concentration was slightly higher than in the control medium. It was necessary to supplement the hydrolysate with extra glucose to increase the C/N ratio and with a mineral solution to overcome the nutritional deficiencies. The P(3HB) accumulation using the supplemented hydrolysate was 58% (g polymer/g biomass).

Heparan sulfate proteoglycans undergo differential expression alterations in left sided colorectal cancer, depending on their metastatic character.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are complex molecules which play a role in the invasion and growth and metastatic properties of cancerous cells. In this work we analyze changes in the patterns of expression of HSPGs in left sided colorectal cancer (LSCRC), both metastatic and non-metastatic, and the results are also compared with those previously obtained for right sided tumors (RSCRCs). METHODS: Eighteen LSCRCs were studied using qPCR to analyze the expression of both the proteoglycan core proteins and the enzymes involved in heparan sulfate chain biosynthesis. Certain HSPGs also carry chondroitin sulfate chains and so we also studied the genes involved in its biosynthesis. The expression of certain genes that showed significant expression differences were also analysed using immunohistochemical techniques. RESULTS: Changes in proteoglycan core proteins were dependent on their location, and the main differences between metastatic and non-metastatic tumors affected cell-surface glypicans, while other molecules were quite similar. Glypicans were also responsible for the main differences between RS- and LS- malignances. Regarding the biosynthesis of heparan sulfate chains, differential alterations in transcription depending on the presence or not of metastasis affected genes involved in the modification of uronic acid (epimerization and 2-O sulfation), and some isoforms responsible for sulfation of glucosamine (NDST1, HS6ST1). Moreover, in RSCRCs differences were preferentially found in the expression of genes involved in C6 and C3 sulfation of glucosamine, but not in NDSTs or SULFs. Finally, synthesis of chondroitin sulfate showed some alterations, which affected various steps, including polimerization and the modification of chains, but the main variations dependent on the presence of metastases were epimerization and 6C sulfation; however, when compared with RSCRCs, the essential divergences affected polymerization of the chains and the 6C sulfation of the galactosamine residue. CONCLUSIONS: We evidenced alterations in the expression of HSPGs, including the expression of cell surface core proteins, many glycosiltransferases and some enzymes that modify the GAG chains in LSCRCs, but this was dependent on the metastatic nature of the tumor. Some of these alterations are shared with RSCRCs, while others, focused on specific gene groups, are dependent on tumor localization.

Upregulated Expression of Heparanase and Heparanase 2 in the Brains of Alzheimer's Disease.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) promote amyloid-ß peptide and tau fibrillization in Alzheimer's disease (AD) and provide resistance against proteolytic breakdown. Heparanase (HPSE) is the only enzyme that cleaves heparan sulfate (HS). Heparanase 2 (HPSE2) lacks HS-degrading activity, although it is able to interact with HS with high affinity. OBJECTIVE: To analyze HPSE and HPSE2 expressions at different stages of AD. METHODS: RT-PCR was used to analyze transcription levels of both heparanases at different stages of AD, and immunohistochemistry was performed to localize each one in different parts of the brain. RESULTS: Both proteins appeared overexpressed at different stages of AD. Immunohistochemistry indicated that the presence of the heparanases was related to AD pathology, with intracellular deposits found in degenerated neurons. At the extracellular level, HPSE was observed only in neuritic plaques with a fragmented core, while HPSE2 appeared in those with compact cores as well. CONCLUSION: Given the involvement of HSPGs in AD pathology, there would seem to be a relationship between the regulation of heparanase expression, the features of the disease, and a possible therapeutic alternative.

Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity toward human cancer cell lines.

BACKGROUND: The objective of this study is to investigate the cytotoxic activity of three isolectins purified from the latex of Euphorbia trigona Miller. HYPOTHESIS: Among lectins are the ribosome-inactivating proteins (RIPs), which are potent inhibitors of protein synthesis in cells and in cell-free systems. RESULTS: Three isolectins, ETR1, ETR2 and ETR3, were purified by anion exchange chromatography. Both ETR1 and ETR3 yielded a single band on SDS-PAGE under reducing conditions, corresponding to a molecular weight of 32 g mol(-1), while ETR2 yielded two bands corresponding to 31 and 33 g mol(-1). When non-reducing conditions were used molecular weight decreased, indicating the presence of intrachain disulfide bonds. Size-exclusion chromatography revealed proteins of apparent molecular weight of 59-63 g mol(-1), suggesting a dimeric nature, with subunits not being held together by disulfide linkage. ETR1, ETR2 and ETR3 hemagglutinated human, sheep and rat erythrocytes and this hemagglutination was specifically inhibited by galactose and its derivatives. The lectins studied were thermostable up to 60 °C and their observed activity was maintained across pH range 5-12. These lectins, from the latex of Euphorbia trigona, are potent inhibitors of eukaryotic protein synthesis in a cell-free system. Flow cytometry analysis revealed the antiproliferative activity of them toward A549, HeLa, H116, HL-60 cell lines. CONCLUSION: Euphorbia trigona isolectins are RIPs with cytotoxic activity toward human cancer cell lines.

Specific genes involved in synthesis and editing of heparan sulfate proteoglycans show altered expression patterns in breast cancer.

BACKGROUND: The expression of a specific set of genes controls the different structures of heparan sulfate proteoglycans (HSPGs), which are involved in the growth, invasion and metastatic properties of cancerous cells. The purpose of this study is to increase knowledge of HSPG alterations in breast cancer. METHODS: Twenty-three infiltrating ductal adenocarcinomas (IDCs), both metastatic and non-metastatic were studied. A transcriptomic approach to the structure of heparan sulfate (HS) chains was used, employing qPCR to analyze both the expression of the enzymes involved in their biosynthesis and editing, as well as the proteoglycan core proteins. Since some of these proteoglycans can also carry chondroitin sulfate chains, we extended the study to include the genes involved in the biosynthesis of these glycosaminoglycans. Histochemical techniques were also used to analyze tissular expression of particular genes showing significant expression differences, of potential interest. RESULTS: No significant change in transcription was detected in approximately 70% of analyzed genes. However, 13 demonstrated changes in both tumor types (40% showing more intense deregulation in the metastatic), while 5 genes showed changes only in non-metastatic tumors. Changes were related to 3 core proteins: overexpression of syndecan-1 and underexpression of glypican-3 and perlecan. HS synthesis was affected by lower levels of some 3-O-sulfotransferase transcripts, the expression of NDST4 and, only in non metastatic tumors, higher levels of extracellular sulfatases. Furthermore, the expression of chondroitin sulfate also was considerably affected, involving both the synthesis of the saccharidic chains and sulfations at all locations. However, the pro-metastatic enzyme heparanase did not exhibit significant changes in mRNA expression, although in metastatic tumors it appeared related to increased levels of the most stable form of mRNA. Finally, the expression of heparanase 2, which displays anti-metastatic features, experienced a strong deregulation in all patients analyzed. CONCLUSIONS: IDCs show alterations in the expression of HSPG genes; principally the expression and localization of proteoglycans and the sulfation patterns of glycosaminoglycan chains, depending on the metastatic nature of the tumor. In addition, the anti-proliferative molecule heparanase 2 experiences strong deregulation, thus highlighting it as a potentially interesting diagnostic factor.

Tobacco as biofactory for biologically active hPL production: a human hormone with potential applications in type-1 diabetes.

Human placental lactogen (hPL) is a peptidic hormone that belongs to the short list of growth factors that could treat type-1 diabetes through pancreatic islet transplantation. Placental lactogen has the capacity to improve islet survival and function before or after transplantation. In this study, transgenic tobacco plants were used as a novel expression system for the production of recombinant hPL protein (rhPL). The expression vector pNEKhPL2 containing hPL cDNA was introduced into tobacco plants; the transcriptional activity was confirmed by real-time PCR, and the rhPL levels reached 1% of the total soluble protein (TSP) content in plants cultivated in the greenhouse. In vitro bioassays using the rat insulinoma (INS-1) cell line showed that recombinant protein was able to induce cell proliferation and activate the JAK-2/STAT-5 signal transduction pathway, demonstrating that plant cells can produce the biologically active hPL protein. To further characterize the plant expression system for hPL production, we analyzed the stability of the protein during the life cycle of tobacco plants as well as the transmission of the transgenic trait to the progeny. The recombinant protein was stably accumulated in young leaves, reaching the maximum level in the first month (6.51 µg/g of fresh weight), but showing a decreasing trend of 26% from the initial sampling time until the end of plant's life cycle. The progeny of the selected pNEKhPL2 plant showed in vitro expression levels of up to 1.1% of TSP. Our results therefore indicate that transgenic plants are a suitable expression system for hPL production.

Oral immunization using tuber extracts from transgenic potato plants expressing rabbit hemorrhagic disease virus capsid protein.

Rabbit hemorrhagic disease, which is caused by a calicivirus, is a lethal infection of adult animals that is characterized by acute liver damage and disseminated intravascular coagulation. In this study, we report the production of the major structural protein VP60 of rabbit hemorrhagic disease virus in transgenic tubers of potato plants and its use as an oral immunogen in rabbits.