Study of the role played by NfsA,NfsB nitroreductase and NemA flavin reductase from <Emphasis Type="Italic">Escherichia coli</Emphasis> in the conversion of ethyl 2-(2′-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties

Benzohydroxamic acids, such as 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), exhibit interesting herbicidal, fungicidal and bactericidal properties. Recently, the chemical synthesis of D-DIBOA has been simplified to only two steps. In a previous paper, we demonstrated that the second step could be replaced by a biotransformation using Escherichia coli to reduce the nitro group of the precursor, ethyl 2-(2′-nitrophenoxy)acetate and obtain D-DIBOA. The NfsA and NfsB nitroreductases and the NemA xenobiotic reductase of E. coli have the capacity to reduce one or two nitro groups from a wide variety of nitroaromatic compounds, which are similar to the precursor. By this reason, we hypothesised that these three enzymes could be involved in this biotransformation. We have analysed the biotransformation yield (BY) of mutant strains in which one, two or three of these genes were knocked out, showing that only in the double nfsA/nfsB and in the triple nfsA/nfsB/nemA mutants, the BY was 0%. These results suggested that NfsA and NfsB are responsible for the biotransformation in the tested conditions. To confirm this, the nfsA and nfsB open reading frames were cloned into the pBAD expression vector and transformed into the nfsA and nfsB single mutants, respectively. In both cases, the biotransformation capacity of the strains was recovered (6.09 ± 0.06% as in the wild-type strain) and incremented considerably when NfsA and NfsB were overexpressed (40.33% ± 9.42% and 59.68% ± 2.0% respectively).     

Metabolic engineering and protein directed evolution increase the yield of L-phenylalanine synthesized from glucose in Escherichia coli

L-phenylalanine (L-Phe) is an aromatic amino acid with diverse commercial applications. Technologies for industrial microbial synthesis of L-Phe using glucose as a starting raw material currently achieve a relatively low conversion yield (Y(Phe/Glc)). The purpose of this work was to study the effect of PTS (phosphotransferase transport system) inactivation and overexpression of different versions of feedback inhibition resistant chorismate mutase-prephenate dehydratase (CM-PDT) on the yield (Y(Phe/Glc)) and productivity of L-Phe synthesized from glucose. The E. coli JM101 strain and its mutant derivative PB12 (PTS(-)Glc(+) phenotype) were used as hosts. PB12 has an inactive PTS, but is capable of transporting and phosphorylating glucose by using an alternative system constituted by galactose permease (GalP) and glucokinase activities (Glk). JM101 and PB12 were transformed with three plasmids, harboring genes that encode for a feedback inhibition resistant DAHP synthase (aroG(fbr)), transketolase (tktA) and either a truncated CM-PDT (pheA(fbr)) or its derived evolved genes (pheA(ev1) or pheA(ev2)). Resting-cells experiments with these engineered strains showed that JM101 and PB12 strains expressing either pheA(ev1) or pheA(ev2) genes produced l-Phe from glucose with Y(Phe/Glc) of 0.21 and 0.33 g/g, corresponding to 38 and 60% of the maximum theoretical yield (0.55 g/g), respectively. In addition, in both engineered strains the reached q(Phe) high levels of 40 mg/g-dcw.h. The metabolic engineering strategy followed in this work, including a strain with an inactive PTS, resulted in a positive impact over the Y(Phe/Glc), enhancing it nearly 57% compared with its PTS(+) counterpart. This is the first report wherein PTS inactivation was a successful strategy to improve the Y(Phe/Glc).     

Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.     

Learning Contrast-Invariant Cancellation of Redundant Signals in Neural Systems

Cancellation of redundant information is a highly desirable feature of sensory systems, since it would potentially lead to a more efficient detection of novel information. However, biologically plausible mechanisms responsible for such selective cancellation, and especially those robust to realistic variations in the intensity of the redundant signals, are mostly unknown. In this work, we study, via in vivo experimental recordings and computational models, the behavior of a cerebellar-like circuit in the weakly electric fish which is known to perform cancellation of redundant stimuli. We experimentally observe contrast invariance in the cancellation of spatially and temporally redundant stimuli in such a system. Our model, which incorporates heterogeneously-delayed feedback, bursting dynamics and burst-induced STDP, is in agreement with our in vivo observations. In addition, the model gives insight on the activity of granule cells and parallel fibers involved in the feedback pathway, and provides a strong prediction on the parallel fiber potentiation time scale. Finally, our model predicts the existence of an optimal learning contrast around 15% contrast levels, which are commonly experienced by interacting fish.     

Erythropoietin responses and physical characterization of erythroid progenitor cells in Rauscher virus infected BALB/c mice.

Infection of BALB/c mice with Rauscher leukemia virus (RLV) gives rise to pronounced erythrocytopoiesis manifesting in splenomegaly and is associated with progressive development of anemia. In the spleen erythroid colony forming units (CFU-E) increase exponentially up to 800-fold that of normal levels by the third week of infection. In vitro these CFU-E are dependent on erythropoietin for colony formation, their erythropoietin requirements being higher than that of CFU-E from normal mice. Numbers of CFU-E in spleen and degree of splenomegaly in anemic RLV infected mice were also shown to be modified by red blood cell transfusion, but progression of the disease was not stopped. Erythroid burst forming units (BFU-E) were also responsive to erythropoietin. However, a small proportion of cells also formed BFU-E colonies at concentrations which did not support growth of normal marrow BFU-E. When compared to normal, CFU-E found in RLV-infected spleen have similar velocity sedimentation rates. However, buoyant density separation of leukemic spleen cells indicated that CFU-E were more homogeneous (modal density 1.0695 g/cm3) than CFU-E from normal spleen. Analysis of physical properties of CFU-E and the nonhemoglobinized erythroblast-like cells, which accumulate in the spleen showed that they differed mainly in their distribution of cell diameter. Our findings show that erythroid progenitor cells in RLV infected mice are responsive to erythropoietin in vitro. Also in vivo erythropoiesis appears to be under control of erythropoietin but other factors which lead to progression of RLV disease apparently exist. Most proerythroblast-like cells, which are characteristic of this disease, apparently lack the potential to form colonies and may be more mature than CFU-E.     

Centrosome migration into the Drosophila oocyte is independent of BicD and egl, and of the organisation of the microtubule cytoskeleton

During early Drosophila oogenesis, one cell from a cyst of 16 germ cells is selected to become the oocyte, and accumulates oocyte-specific proteins and the centrosomes from the other 15 cells. Here we show that the microtubule cytoskeleton and the centrosomes follow the same stepwise restriction to one cell as other oocyte markers. Surprisingly, the centrosomes still localise to one cell after colcemid treatment, and in BicD and egl mutants, which abolish the localisation of all other oocyte markers and the polarisation of the microtubule cytoskeleton. In contrast, the centrosomes fail to migrate in cysts mutant for Dynein heavy chain 64C, which disrupts the fusome. Thus, centrosome migration is independent of the organisation of the microtubule cytoskeleton, and seems to depend instead on the polarity of the fusome.     

Genome-wide association study identifies single nucleotide polymorphism in DYRK1A associated with replication of HIV-1 in monocyte-derived macrophages

Background

HIV-1 infected macrophages play an important role in rendering resting T cells permissive for infection, in spreading HIV-1 to T cells, and in the pathogenesis of AIDS dementia. During highly active anti-retroviral treatment (HAART), macrophages keep producing virus because tissue penetration of antiretrovirals is suboptimal and the efficacy of some is reduced. Thus, to cure HIV-1 infection with antiretrovirals we will also need to efficiently inhibit viral replication in macrophages. The majority of the current drugs block the action of viral enzymes, whereas there is an abundance of yet unidentified host factors that could be targeted. We here present results from a genome-wide association study identifying novel genetic polymorphisms that affect in vitro HIV-1 replication in macrophages.

Methodology/Principal Findings

Monocyte-derived macrophages from 393 blood donors were infected with HIV-1 and viral replication was determined using Gag p24 antigen levels. Genomic DNA from individuals with macrophages that had relatively low (n = 96) or high (n = 96) p24 production was used for SNP genotyping with the Illumina 610 Quad beadchip. A total of 494,656 SNPs that passed quality control were tested for association with HIV-1 replication in macrophages, using linear regression. We found a strong association between in vitro HIV-1 replication in monocyte-derived macrophages and SNP rs12483205 in DYRK1A (p = 2.16×10⁻⁵). While the association was not genome-wide significant (p<1×10⁻⁷), we could replicate this association using monocyte-derived macrophages from an independent group of 31 individuals (p = 0.0034). Combined analysis of the initial and replication cohort increased the strength of the association (p = 4.84×10⁻⁶). In addition, we found this SNP to be associated with HIV-1 disease progression in vivo in two independent cohort studies (p = 0.035 and p = 0.0048).

Conclusions/Significance

These findings suggest that the kinase DYRK1A is involved in the replication of HIV-1, in vitro in macrophages as well as in vivo.