Exploiting adaptive laboratory evolution of Streptomyces clavuligerus for antibiotic discovery and overproduction

Adaptation is normally viewed as the enemy of the antibiotic discovery and development process because adaptation among pathogens to antibiotic exposure leads to resistance. We present a method here that, in contrast, exploits the power of adaptation among antibiotic producers to accelerate the discovery of antibiotics. A competition-based adaptive laboratory evolution scheme is presented whereby an antibiotic-producing microorganism is competed against a target pathogen and serially passed over time until the producer evolves the ability to synthesize a chemical entity that inhibits growth of the pathogen. When multiple Streptomyces clavuligerus replicates were adaptively evolved against methicillin-resistant Staphylococcus aureus N315 in this manner, a strain emerged that acquired the ability to constitutively produce holomycin. In contrast, no holomycin could be detected from the unevolved wild-type strain. Moreover, genome re-sequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid, and acquired several single nucleotide polymorphisms in genes that have been shown to affect secondary metabolite biosynthesis. These results demonstrate that competition-based adaptive laboratory evolution can constitute a platform to create mutants that overproduce known antibiotics and possibly to discover new compounds as well.     

Structure–function analysis of hepatitis C virus envelope glycoproteins E1 and E2

Hepatitis C virus (HCV) is the leading cause of chronic liver disease in humans. The envelope proteins of HCV are potential candidates for vaccine development. The absence of three-dimensional (3D) structures for the functional domain of HCV envelope proteins [E1.E2] monomer complex has hindered overall understanding of the virus infection, and also structure-based drug design initiatives. In this study, we report a 3D model containing both E1 and E2 proteins of HCV using the recently published structure of the core domain of HCV E2 and the functional part of E1, and investigate immunogenic implications of the model. HCV [E1.E2] molecule is modeled by using aa205–319 of E1 to aa421–716 of E2. Published experimental data were used to further refine the [E1.E2] model. Based on the model, we predict 77 exposed residues and several antigenic sites within the [E1.E2] that could serve as vaccine epitopes. This study identifies eight peptides which have antigenic propensity and have two or more sequentially exposed amino acids and 12 singular sites are under negative selection pressure that can serve as vaccine or therapeutic targets. Our special interest is ₂₈₅FLVGQLFTFSPRRHW₂₉₉ which has five negatively selected sites (L286, V287, G288, T292, and G303) with three of them sequential and four amino acids exposed (F285, L286, T292, and R296). This peptide in the E1 protein maps to dengue envelope vaccine target identified previously by our group. Our model provides for the first time an overall view of both the HCV envelope proteins thereby allowing researchers explore structure-based drug design approaches.     

A study on dosimetric properties of electronic portal imaging device and its use as a quality assurance tool in Volumetric Modulated Arc Therapy

Aim

In this study, the dosimetric properties of the electronic portal imaging device were examined and the quality assurance testing of Volumetric Modulated Arc Therapy was performed.

Background

RapidArc involves the variable dose rate, leaf speed and the gantry rotation. The imager was studied for the effects like dose, dose rate, field size, leaf speed and sag during gantry rotation.

Materials and methods

A Varian RapidArc machine equipped with 120 multileaf collimator and amorphous silicon detector was used for the study. The characteristics that are variable in RapidArc treatment were studied for the portal imager. The accuracy of a dynamic multileaf collimator position at different gantry angles and during gantry rotation was examined using the picket fence test. The control of the dose rate and gantry speed was verified using a test field irradiating seven strips of the same dose with different dose rate and gantry speeds. The control over leaf speed during arc was verified by irradiating four strips of different leaf speeds with the same dose in each strip. To verify the results, the RapidArc test procedure was compared with the X-Omat film and verified for a period of 6 weeks using EPID.

Results

The effect of gantry rotation on leaf accuracy was minimal. The dose in segments showed good agreement with mean deviation of 0.8% for dose rate control and 1.09% for leaf speed control over different gantry speeds.

Conclusion

The results provided a precise control of gantry speed, dose rate and leaf speeds during RapidArc delivery and were consistent over 6 weeks.     

Comment on causality and pathway search in microarray time series experiment

Contact: nagarajanradhakrish{at}uams.edu Associate Editor: Martin Bishop     

Reductive glutaraldehydation of amine groups for identification of protein N-termini

In the present work, reductive alkylation of proteins and peptides with glutaraldehyde (reductive glutaraldehydation) is reported. The reaction is highly efficient and forms piperidine at the N-terminus as well as the side chain of lysine residues. The complete modification of protein amines was achieved by reductive glutaraldehydation in solution or in the gel in less than 15 min. The glutaraldehyde-modified peptides display an enhanced intensity in mass spectra and show higher retention time in reversed phase chromatography in comparison to unmodified peptides. Fragmentation of glutaraldehyde-modified proteins and peptides generates a1 fragment ions with enhanced intensity in MS/MS spectra. Thus, a method based on reductive glutaraldehydation and LC-MS/MS analysis has been developed to determine the N-terminal residue of proteins with free N-termini.     

Protective effect of oleanolic acid and ursolic acid against lipid peroxidation.

In a search for plant products against cancer, the protective effect of two plant products, ursolic acid isolated from Ocimum sanctum and oleanolic acid from Eugenia jumbolana against free radical induced damage was studied. Three different standard systems viz., ascorbic acid, carbon tetrachloride, ADP/Iron were used to induce lipid peroxidation in isolated rat liver microsomes in vitro. Both oleanolic acid and ursolic acid offered remarkable protection of 90% and 60% respectively. Both the compounds did not induce lipid peroxidation by themselves that improved the therapeutic application.     

Juglans regia L. protects against UVB induced apoptosis in human epidermal keratinocytes

The present study was aimed to investigate the photoprotective effect of the male flower of J. regia L. (MEJR) against ultraviolet-B induced apoptosis in human skin cells. Human skin epidermal keratinocytes were pretreated with the MEJR (80 µg/ml, has been selected after MTT assay), prior to 30 min UVB-irradiation at a dose of 20 mJ/cm². Mitochondrial membrane potential was evaluated using Rhodamine-123 staining; the % apoptosis by Hoechst staining and acridine orange staining; DNA damage was measured by comet assay. The levels of p53, Bax, Bcl-xL, Bcl-2, Cytochrome c, Caspase-9 and Caspase-3 expression in HaCaT cells were analyzed by western blotting and RT-PCR. Pretreatment with MEJR 80 µg/ml prior to UVB-irradiation significantly prevents apoptotic characteristics, DNA damage and loss of mitochondrial membrane potential. Thus, MEJR protects UVB-mediated human skin cells, by modulating the expression of apoptotic markers and UVB-induced DNA damage in HaCaT cells.     

Unmasking Anticooperative DNA-binding interactions of vaccinia DNA topoisomerase I

Vaccinia DNA topoisomerase (vTopo) catalyzes highly specific nucleophilic substitution at a single phosphodiester linkage in the pentapyrimidine recognition sequence 5'-(C/T)+5C4+C3+T+2T+1p \N-1 using an active-site tyrosine nucleophile, thereby expelling a 5' hydroxyl leaving group of the DNA. Here, we report the energetic effects of subtle modifications to the major-groove hydrogen-bond donor and acceptor groups of the 3'-GGGAA-5' consensus sequence of the nonscissile strand in the context of duplexes in which the scissile strand length was progressively shortened. We find that the major-groove substitutions become energetically more damaging as the scissile strand is shortened from 32 to 24 and 18 nucleotides, indicating that enzyme interactions with the duplex region present in the 32-mer but not the 24- or 18-mer weaken specific interactions with the DNA major groove. Regardless of strand length, the destabilizing effects of the major-groove substitutions increase as the reaction proceeds from the Michaelis complex to the transition state for DNA cleavage and, finally, to the phosphotyrosine-DNA covalent complex. These length-dependent anticooperative interactions involving the DNA major groove and duplex regions 3' to the cleavage site indicate that the major-groove binding energy is fully realized late during the reaction for full-length substrates but that smaller more flexible duplex substrates feel these interactions earlier along the reaction coordinate. Such anticooperative binding interactions may play a role in strand exchange and supercoil unwinding activities of the enzyme.     

Sensory representation abnormalities that parallel focal hand dystonia in a primate model

In our hypothesis of focal dystonia, attended repetitive behaviors generate aberrant sensory representations. Those aberrant representations interfere with motor control. Abnormal motor control strengthens sensory abnormalities. The positive feedback loop reinforces the dystonic condition. Previous studies of primates with focal hand dystonia have demonstrated multi-digit or hairy-glabrous responses at single sites in area 3b, receptive fields that average ten times larger than normal, and high receptive field overlap as a function of horizontal distance. In this study, we strengthen and elaborate these findings. One animal was implanted with an array of microelectrodes that spanned the border between the face and digits. After the animal developed hand dystonia, responses in the initial hand representation increasingly responded to low threshold stimulation of the face in a columnar substitution. The hand-face border that is normally sharp became patchy and smeared over 1 mm of cortex within 6 weeks. Two more trained animals developed a focal hand dystonia variable in severity across the hand. Receptive field size, presence of multi-digit or hairy-glabrous receptive fields, and columnar overlap covaried with the animal's ability to use specific digits. A fourth animal performed the same behaviors without developing dystonia. Many of its physiological measures were similar to the dystonic animals, but receptive field overlap functions were minimally abnormal, and no sites shared response properties that are normally segregated such as hairy-glabrous combined fields, or multi-digit fields. Thalamic mapping demonstrated proportionate levels of abnormality in thalamic representations as were found in cortical representations.