The lectin concanavalin-A signals MT1-MMP catalytic independent induction of COX-2 through an IKKγ/NF-κB-dependent pathway

The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) which expression is increased in brain cancer. Given that MT1-MMP correlated to high expression of cyclooxygenase (COX)-2 in gliomas with increasing histological grade, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP’s catalytic function. ConA- and MT1-MMP-mediated intracellular signaling of COX-2 was also confirmed in wild-type and in Nuclear Factor-kappaB (NF-κB) p65−/− mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-κB1 (p50)−/− and in I kappaB kinase (IKK) γ−/− mutant MEF cells. Collectively, our results highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells, and prompts for the design of new therapeutic strategies that target cell surface carbohydrate structures and MT1-MMP-mediated signaling. Concise summary Concanavalin-A (ConA) mimics biological lectin/carbohydrate interactions that regulate the proinflammatory phenotype of cancer cells through yet undefined signaling. Here we highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of cyclooxygenase-2, and that could be responsible for the therapy resistance phenotype of glioblastoma cells

BioWF: A naturally‐fused, di‐domain biocatalyst from biotin biosynthesis displays an unexpectedly broad substrate scope

The carbon backbone of biotin is constructed from the C(7) di‐acid pimelate, which is converted to an acyl‐CoA thioester by an ATP‐dependent, pimeloyl‐CoA synthetase (PCAS, encoded by BioW). The acyl‐thioester is condensed with ʟ‐alanine in a decarboxylative, Claisen‐like reaction to form an aminoketone (8‐amino‐7‐oxononanoic acid, AON). This step is catalysed by the pyridoxal 5’‐phosphate (PLP)‐dependent enzyme (AON synthase, AONS, encoded by BioF). Distinct versions of Bacillus subtilis BioW (BsBioW) and E. coli BioF (EcBioF) display strict substrate specificity. In contrast, a BioW‐BioF fusion from Corynebacterium amycolatum (CaBioWF) accepts a wider range of mono‐ and di‐fatty acids. Analysis of the active site of the BsBioW : pimeloyl‐adenylate complex suggested a key role for a Phe (F192) residue in the CaBioW domain; a F192Y mutant restored the substrate specificity to pimelate. This surprising substrate flexibility also extends to the CaBioF domain, which accepts ʟ‐alanine, ʟ‐serine and glycine. Structural models of the CaBioWF fusion provide insight into how both domains interact with each other and suggest the presence of an intra‐domain tunnel. The CaBioWF fusion catalyses conversion of various fatty acids and amino acids to a range of AON derivatives. Such unexpected, natural broad substrate scope suggests that the CaBioWF fusion is a versatile biocatalyst that can be used to prepare a number of aminoketone analogues

Spontaneous post-implantation embryo resolution: A new concept in embryo loss

Objective: To determine and compare in an ART population the chances of complete abortion rate, and of spontaneous post-implantation embryo resolution (SPIER) (partial re-absorption of 1 or more gestational sacs) in clinical pregnancies according to the original number of gestational sacs observed. Design: A prospective, on going study in a University-based private practice and in a public hospital facility ART units. Materials and Methods: We studied the outcome of 620 pregnancies originated from ART. There were 414 singletons (67%), 132 twins (21%), 48 triplets (8%), 19 quadruplets (3%) and 7 quintuplets (1%). At the first ultrasound observation of the clinical pregnancies (20-30 days, post-conception) we detected 414 single gestational scans (44%), 264 double (28%), 144 triple (10%) 76 quadruple (8%) and 35 quintuple sacs (4%). Results: A) complete pregnancy loss was observed in 22% of all pregnancies (n:160) with the following distribution: Singleton (29%), twin (10%), triplet (8%), quadruplet pregnancies (10%), and quintuplets (14%) respectively. The pregnancy loss decreased proportional to the magnitude of the multiple gestation, being statistically significantly higher in the group of patients with singletons (p<0.05). B) SPIER was found to be overall of 29% (n:293), with the following distribution; 29% in singletons, 25% in twins 24% in triplets, 45% in quadruplet pregnancies, and 54% in quintuplets, respectively. The chance of losing a gestational sac does not differ between patients that originally had a singleton, twins or triplet pregnancies. However, it was statistically greater in those that began the gestation as a quadruplet. (p<0.005). Conclusions: These results show that the frequency of complete pregnancy loss and the partial-resolution of gestational sacs (SPIER) do not have a parallel correlation in singleton and multiple pregnancies (up to quadruplets). While the complete pregnancy wastage is increased in singleton vs. multiples, the SPIER is augmented only in high order multiple gestations. This information should be used for 1) counseling of patients about the prognosis and outcome of their pregnancies from ART once clinically confirmed, and 2) to assist in making decisions about the multifetal pregnancy reductions

Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli

Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines

Adaptive duplication and functional diversification of Protein kinase R contribute to the uniqueness of bat-virus interactions

Abstract Several bat species act as asymptomatic reservoirs for many viruses that are instead highly pathogenic in other mammals. Here, we have characterized the functional diversification of the Protein kinase R (PKR), a major antiviral innate defense system. Our data indicate that PKR has evolved under positive selection and has undergone repeated genomic duplications in bats, in contrast to all studied mammals that possess a single copy of the gene. Functional testing of the relationship between PKR and poxvirus antagonists revealed how an evolutionary conflict with ancient pathogenic poxviruses has shaped a specific bat host-virus interface. More importantly, we determined that duplicated PKRs of the Myotis species have undergone functional diversification allowing them to collectively escape from and enhance control of DNA and RNA viruses. These findings suggest that viral-driven adaptations in PKR contribute to modern virus-bat interactions and may account for bat specific immunity