The NF-κB factor Relish is essential for the epithelial defenses protecting against δ-endotoxin dependent effects of Bacillus thuringiensis israelensis infection in the Drosophila model.

Bacillus thuringiensis israelensis is largely regarded as the most selective, safe and ecofriendly biopesticide used for the control of insect vectors of human diseases. Bti enthomopathogenicity relies on the Cry and Cyt δ-endotoxins, produced as crystalline inclusions during sporulation. Insecticidal selectivity of Bti is mainly ascribed to the binding of the Cry toxins to receptors in the gut of target insects. However, the contribution of epithelial defenses in limiting Bti side effects in non-target species remains largely unexplored. Here, taking advantage of the genetically tractable Drosophila melanogaster model and its amenability for deciphering highly conserved innate immune defenses, we unravel a central role of the NF-κB factor Relish in the protection against the effects of ingested Bti spores in a non-susceptible host. Intriguingly, our data indicate that the Bti-induced Relish response is independent of its canonical activation downstream of peptidoglycan sensing and does not involve its longstanding role in the regulation of antimicrobial peptides encoding genes. In contrast, our data highlight a novel enterocyte specific function of Relish that is essential for preventing general septicemia following Bti oral infections strictly when producing δ-endotoxins. Altogether, our data provide novel insights into Bti-hosts interactions of prominent interest for the optimization and sustainability of insects' biocontrol strategies.

Biochemical limitations of Bacillus thuringiensis based biopesticides production in a wheat bran culture medium.

Bacillus thuringiensis, a gram-positive sporulating bacteria found in the environment, produces, during its sporulation phase, crystals responsible for its insecticidal activity, constituted of an assembly of pore-forming δ-endotoxins. This has led to its use as a biopesticide, an eco-friendly alternative to harmful chemical pesticides. To minimize production cost, one endemic Bacillus thuringiensis sv. kurstaki (Btk) strain Lip, isolated from Lebanese soil, was cultivated in a wheat bran (WB) based medium (IPM-4-Citrus project EC n° 734921). With the aim of studying the biochemical limitations of Btk biopesticide production in a wheat bran based medium, the WB was sieved into different granulometries, heat treated, inoculated with Btk Lip at flask scale, then filtered and separated into an insoluble and a permeate fractions. Several biochemical analyses, ie. bio performances, starch, elemental composition, total nitrogen and ashes, were then conducted on both fractions before and after culture. On a morphological level, two populations were distinguished, the fine starch granules and the coarse lignocellulosic particles. The biochemical analyses showed that both the raw and sieved WB have a similar proteins content (0.115 g/gdm WB), water content (0.116 g/gdm WB) and elemental composition (carbon: 45%, oxygen: 37%, nitrogen: 3%, hydrogen: 6%, ashes: 5%). The starch content was 17%, 14% and 34% and the fermentable fraction was estimated to 32.1%, 36.1% and 51.1% respectively for classes 2, 3 and 4. Both the elemental composition and Kjeldahl analyses showed that the nitrogen is the limiting nutrient of the culture.

Expression of the methylcytosine dioxygenase ten-eleven translocation-2 and connexin 43 in inflammatory bowel disease and colorectal cancer.

BACKGROUND: Inflammatory bowel disease (IBD) constitutes a substantial risk factor for colorectal cancer. Connexin 43 (Cx43) is a protein that forms gap junction (GJ) complexes involved in intercellular communication, and its expression is altered under pathological conditions, such as IBD and cancer. Recent studies have implicated epigenetic processes modulating DNA methylation in the pathogenesis of diverse inflammatory and malignant diseases. The ten-eleven translocation-2 (TET-2) enzyme catalyzes the demethylation, hence, regulating the activity of various cancer-promoting and tumor-suppressor genes. AIM: To investigate Cx43 and TET-2 expression levels and presence of 5-hydroxymethylcytosine (5-hmC) marks under inflammatory conditions both in vitro and in vivo. METHODS: TET-2 expression was evaluated in parental HT-29 cells and in HT-29 cells expressing low or high levels of Cx43, a putative tumor-suppressor gene whose expression varies in IBD and colorectal cancer, and which has been implicated in the inflammatory process and in tumor onset. The dextran sulfate sodium-induced colitis model was reproduced in BALB/c mice to evaluate the expression of TET-2 and Cx43 under inflammatory conditions in vivo. In addition, archived colon tissue sections from normal, IBD (ulcerative colitis), and sporadic colon adenocarcinoma patients were obtained and evaluated for the expression of TET-2 and Cx43. Expression levels were reported at the transcriptional level by quantitative real-time polymerase chain reaction, and at the translational level by Western blotting and immunofluorescence. RESULTS: Under inflammatory conditions, Cx43 and TET-2 expression levels increased compared to non-inflammatory conditions. TET-2 upregulation was more pronounced in Cx43-deficient cells. Moreover, colon tissue sections from normal, ulcerative colitis, and sporadic colon adenocarcinoma patients corroborated that Cx43 expression increased in IBD and decreased in adenocarcinoma, compared to tissues from non-IBD subjects. However, TET-2 expression and 5-hmC mark levels decreased in samples from patients with ulcerative colitis or cancer. Cx43 and TET-2 expression levels were also investigated in an experimental colitis mouse model. Interestingly, mice exposed to carbenoxolone (CBX), a GJ inhibitor, had upregulated TET-2 levels. Collectively, these results show that TET-2 levels and activity increased under inflammatory conditions, in cells downregulating gap junctional protein Cx43, and in colon tissues from mice exposed to CBX. CONCLUSION: These results suggest that TET-2 expression levels, as well as Cx43 expression levels, are modulated in models of intestinal inflammation. We hypothesize that TET-2 may demethylate genes involved in inflammation and tumorigenesis, such as Cx43, potentially contributing to intestinal inflammation and associated carcinogenesis.

Suggested policy and legislation reforms to reduce deleterious effect of pesticides in Lebanon.

Countrywide pesticide management activities are resource draining, even for developed countries, which sometimes fall short in achieving the optimum protection against pesticides deleterious effects on humans and environment. Additionally, in Lebanon, basic flaws exist at different levels of pesticide management cycle. In this study, through an extensive review of relevant literature regarding the pesticides impact on humans and environment in Lebanon and adopted policies in existing legislation, several gaps have been identified. Accordingly, recommendations to reduce pesticide risk through a combination of reforms at the policy level and its tools, particularly legislation, are proposed. In our opinion, the starting point is to adopt a minimum list of lower risk pesticides supported by a combination of: "prescriptions" based on a comprehensive registration and an effective implementation systems, a suitable IPM/ICM government-supported credit system, traceability systems of agricultural commodities and pesticides containers, Pesticide stock management system to reduce the quantity of obsolete pesticides, and containers recycling system. For a global sustainability of pesticides risk reduction, a binding global intervention fostered by the UN, based on human rights for safe food, is called upon to ban hazardous pesticides-except those of WHO class IV- trafficking in developing countries scoring low in an international official assessment of their pesticides lifecycle management. At the same time, global funds should support pesticides alternatives and the enhancement of the developing countries capacities for pesticides lifecycle management, which is a part of a larger global matrix in risk reduction.

A simple method for the separation of Bacillus thuringiensis spores and crystals.

A simple new method, for separating Bacillus thuringiensis crystals from spores and cell debris, is described. The developed purification method uses hexane and low speed centrifugation and does not require any expensive material or reagents.

Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera: Pyralidae) larvae.

The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC50 of Lip and HD1 were 33.27 and 128.61 µg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.

Differentiation between Bacillus thuringiensis strains by gyrB PCR-Sau3AI fingerprinting.

gyrB DNA fragments of seven Bacillus thuringiensis local collection family representatives were amplified by PCR and sequenced. Several differences in their corresponding sequences were evidenced. Both in silico and in vitro restriction maps of gyrB sequences and fragments respectively confirmed that EcoRI and Sau3AI could be used to differentiate between B. thuringiensis strains. However, the phylogeny analysis showed that only the gyrB PCR-Sau3AI allows a strains classification that correlates very well with that obtained on the basis of the sequences analysis. Thus, these finds show that gyrB PCR- Sau3AI digestion could be considered as an efficient, rapid, and easy method to make a distinction, not only between strains belonging to the Bacillus cereus group, but also between those belonging to B. thuringiensis.