Design of a fungal cellulose-binding domain for PCR amplification and expression in E. coli

A new fungal cellulose binding domain (CBD) from Stachybotris sp. has been cloned. Multiple sequence alignment of the CBD from 34 fungi shows highest sequence identity at the ends of the domains. The two primers from these regions were amplified by PCR giving a 120-bp product. Two of these, from Trichoderma sp. and Stachybotris sp. were subsequently cloned, sequenced and confirmed to be of the CBD family. The CBD from Stachybotris sp. was expressed in E. coli fused to g3p of the M13 phage and with a c-myc tag. The secreted fusion protein adsorbed on acid-swollen cellulose thereby confirming its functionality.     

Newly synthesized dopamine ester derivatives and assessment of their antioxidant,antimicrobial and hemolytic activities

Preparation of dopamine derivatives was carried out as a response to the increasing demand for new lipophilized antioxidants in food, cosmetic and pharmaceutical industries. A large series of dopamine esters (DA-C₃ to DA-C_18:1) with increasing lipophilicity was synthesized using lipase from Candida antarctica (Novozyme 435) as a biocatalyst. The highest conversion yield (52.75%) was reached when caprylic acid (DA-C₈) was used as acyl donor. Synthesized compounds were purified and evaluated for their antioxidant activity using the DPPH and the ABTS tests. Results showed that esterification had little effect on radical-scavenging capacity. However, long chain fatty acid esters displayed higher protective effect of oil against oxidation at 70 °C as compared to the parent dopamine or to the BHT. The hemolytic activity of dopamine esters was studied. Middle chain length derivatives (DA-C₈ and DA-C₁₂) of dopamine and oleic acid derivative (DA-C_18:1) showed the highest hemolytic activity against human erythrocyte. The antimicrobial activities of dopamine esters were also evaluated using well diffusion and minimal inhibition concentration methods. Among all the tested compounds, DA-C₈ and DA-C₁₂ exhibited the highest antibacterial activities. These results open up potential applications by using dopamine derivatives as antioxidants and antimicrobial compounds in cosmetic, food and pharmaceutical industries.     

Ectopic Expression of StERF94 Transcription Factor in Potato Plants Improved Resistance to Fusarium solani Infection

ERF proteins (ethylene-responsive factors), which belong to the AP2/ERF superfamily, play essential roles in plant development, growth, and response to abiotic and biotic constraints. In a previous study, we cloned a cDNA encoding the StERF94 factor from potato plants and the phylogenetic analyses showed that it belongs to group IX of the ERF family. Genes of this group are known to be involved in plant response to biotic stress. The StERF94 cDNA was overexpressed in transgenic potato plants and the resulting transgenic plants showed a high tolerance to salinity. In this study, we investigated the response of StERF94 transgenic plants to biotic stress by evaluating their resistance to Fusarium solani infection. A significant enhanced resistance to the fungus was noticed in the transgenic plants which displayed limited malondialdehyde and H₂O₂ production and increasing antioxidant enzyme activities. Our findings also revealed that overexpression of StERF94 in potato enhanced expression of relevant defense genes like those encoding PR proteins (pathogenesis related) which led to a protection against disease propagation and reduction of fungus development in plant tissues.

     

World Health Organization Global Estimates and Regional Comparisons of the Burden of Foodborne Disease in 2010

Illness and death from diseases caused by contaminated food are a constant threat to public health and a significant impediment to socio-economic development worldwide. To measure the global and regional burden of foodborne disease (FBD), the World Health Organization (WHO) established the Foodborne Disease Burden Epidemiology Reference Group (FERG), which here reports their first estimates of the incidence, mortality, and disease burden due to 31 foodborne hazards. We find that the global burden of FBD is comparable to those of the major infectious diseases, HIV/AIDS, malaria and tuberculosis. The most frequent causes of foodborne illness were diarrheal disease agents, particularly norovirus and Campylobacter spp. Diarrheal disease agents, especially non-typhoidal Salmonella enterica, were also responsible for the majority of deaths due to FBD. Other major causes of FBD deaths were Salmonella Typhi, Taenia solium and hepatitis A virus. The global burden of FBD caused by the 31 hazards in 2010 was 33 million Disability Adjusted Life Years (DALYs); children under five years old bore 40% of this burden. The 14 subregions, defined on the basis of child and adult mortality, had considerably different burdens of FBD, with the greatest falling on the subregions in Africa, followed by the subregions in South-East Asia and the Eastern Mediterranean D subregion. Some hazards, such as non-typhoidal S. enterica, were important causes of FBD in all regions of the world, whereas others, such as certain parasitic helminths, were highly localised. Thus, the burden of FBD is borne particularly by children under five years old–although they represent only 9% of the global population–and people living in low-income regions of the world. These estimates are conservative, i.e., underestimates rather than overestimates; further studies are needed to address the data gaps and limitations of the study. Nevertheless, all stakeholders can contribute to improvements in food safety throughout the food chain by incorporating these estimates into policy development at national and international levels.

Summary Points

• Thirty-one foodborne hazards caused 600 (95% uncertainty interval [UI] 420–960) million foodborne illnesses and 420,000 (95% UI 310,000–600,000) deaths in 2010.
• The global burden of FBD caused by the 31 hazards studied was 33 (95% UI 25–46) million DALYs in 2010.
• The most frequent causes of foodborne illness were diarrheal disease agents; particularly norovirus and Campylobacter spp.
• Foodborne diarrheal disease agents, particularly non-typhoidal Salmonella enterica, caused 230,000 (95% UI 160,000–320,000) deaths
• Other major causes of FBD deaths were Salmonella Typhi, Taenia solium, hepatitis A virus and aflatoxin.
• 40% of the FBD burden was among children under 5 years old.
• The African (AFR), South-East Asian (SEAR) and Eastern Mediterranean (EMR) D subregions had the highest FBD burden.
• Diarrheal disease agents were the leading cause of FBD burden in most subregions, and non-typhoidal Salmonella enterica caused an important burden in all subregions, particularly in the subregions in Africa.
• Other main causes of diarrheal FBD burden were enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli and Vibrio cholerae in low-income subregions, and Campylobacter spp. in high-income subregions.
• The burden of aflatoxin was high in the AFR D, Western Pacific (WPR) B and SEAR B subregions, whereas dioxins caused the highest burden in SEAR D, EMR D and European (EUR) A and C subregions.
• In the South-East Asian subregions, there was a considerable burden of Salmonella Typhi; the burden of Opisthorchis spp. was concentrated in the SEAR B region, where the seafoodborne trematodes Paragonimus spp. and Clonorchis sinensis were also important.
• In Central and South American (AMR B and AMR D) subregions, T. solium and Toxoplasma gondii contributed significantly to the FBD burden.
• These estimates should inform policy development at national and international levels to improve food safety throughout the food chain.

     

Gene cloning and molecular characterization of the Talaromyces thermophilus lipase catalyzed efficient hydrolysis and synthesis of esters

A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083 bp encoding a protein of 269 Aa with an estimated molecular mass of 30 kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides.     

Expression of a functional recombinant C‐type lectin‐like protein lebecetin in the human embryonic kidney cells

Lebecetin is an anticoagulant C‐type lectin‐like protein that was previously isolated from Macrovipera lebetina venom and described to consist of two subunits (alpha and beta). It was reported to potently prevent platelet aggregation by binding to glycoprotein Ib and to exhibit a broad spectrum of inhibitory activities on various integrin‐mediated functions of tumor cells, including adhesion, proliferation, and cell migration. This study aimed to investigate the structure‐function of lebecetin. Accordingly, the cDNA of each subunit was cloned and separately or jointly expressed in the human embryonic kidney cells using two vectors with different selectable tags. The immunofluorescence analysis of transfected cells revealed significant expression levels and co‐localization of the two lebecetin subunits. The recombinant proteins were efficiently secreted and purified using metal‐chelating affinity chromatography. We found that the Lebecetin alpha and beta subunits were produced as a mixture of homodimers and heterodimers and that the heterodimerization represents a prerequisite for functioning. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Cellobiose dehydrogenase influences the production of S. microspora β-glucosidase

BglG, a Stachybotrys microspora β-glucosidase produced in the presence of glucose and cellobiose, was used individually as sole carbon source. The time course synthesis of BglG showed two aspects: (1) an exponential curve, observed in glucose Mandels medium, and (2) a cloche curve, observed in cellobiose containing cultures. A decrease was observed in bglG production at the 6th, 8th and 10th days during mycelium growth in cellobiose Mandels medium, which allowed for the assumption that the anabolism of a bglG inhibitor factor was produced with cellobiose but not with glucose. Cellobiose dehydrogenases (CDH) activity was, on another hand detected in cellobiose grown cultures but not in glucose containing ones. The aliquots, withdrawn at the time course of bglG production in the presence of cellobiose, gave rise to an inhibitory effect on bglG activity. This result was obtained with and without the heat treatment (5 min at 100°C) of the aliquots, which supported the non-proteinaceous nature of the inhibitor factor. Furthermore, sugar chromatographic analyses revealed the appearance of a secondary metabolite in the cellobiose Mandels medium and indicated that the factor behind the bglG activity cloche curve was a δ-gluconolactone. Seeing that the latter follows a strong inhibitory effect on bglG activity, it is speculated that the decrease in bglG activity during the time course of bglG synthesis in cellobiose Mandels medium is assigned to the release of δ-gluconolactone. This paper presents and validates an explanatory model for this hypothesis.