A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli

<p>Abstract</p> <p>Background</p> <p>The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc.</p> <p>Results</p> <p>We describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP) as the reporter gene and is constructed in such a way that the <it>E. coli </it>cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts.</p> <p>Conclusions</p> <p>This is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein.</p

EXPedite: A System for Encoded XML Processing

As XML becomes an increasingly popular format for information exchange, the efficient processing of broadcast XML data on a constrained device (for example, a cell phone or a PDA) becomes a critical task. In this paper we present the EXPedite system: a new model of data processing in an information exchange environment, which migrates the power of the data-sending server to receivers for efficient processing. It consists of a simple and general encoding scheme for servers, and streaming query processing algorithms on encoded XML stream for data receivers with constrained computing abilities. Experiments show the impressive performance of EXPedite

Efficient Update of Indexes for Dynamically Changing Web Documents

The original publication is available at www.springerlink.comRecent work on incremental crawling has enabled the indexed document collection of a search engine to be more synchronized with the changing World Wide Web. However, this synchronized collection is not immediately searchable, because the keyword index is rebuilt from scratch less frequently than the collection can be refreshed. An inverted index is usually used to index documents crawled from the web. Complete index rebuild at high frequency is expensive. Previous work on incremental inverted index updates have been restricted to adding and removing documents. Updating the inverted index for previously indexed documents that have changed has not been addressed. In this paper, we propose an efficient method to update the inverted index for previously indexed documents whose contents have changed. Our method uses the idea of landmarks together with the diff algorithm to significantly reduce the number of postings in the inverted index that need to be updated. Our experiments verify that our landmark-diff method results in significant savings in the number of update operations on the inverted index

Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection

<p>Abstract</p> <p>Background</p> <p>Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy.</p> <p>Results</p> <p>We developed a recombinant lysis-deficient <it>Staphylococcus aureus </it>phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in <it>S. aureus </it>strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (<it>cat</it>) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the <it>in vivo </it>efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic <it>S. aureus </it>at a dose that results in 80% mortality (LD₈₀). Treatment with the endolysin-deficient phage rescued mice from the fatal <it>S. aureus </it>infection.</p> <p>Conclusions</p> <p>A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant <it>S. aureus </it>without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage effectively rescued mice from fatal <it>S. aureus </it>infection. To our knowledge this is the first report of a lysis-deficient staphylococcal phage.</p

Asn12 and Asn278: Critical Residues for In Vitro Biological Activity of Reteplase

Reteplase (rPA) is a thrombolytic agent used for the treatment of acute myocardial infarction. We studied the expression of rPA and its selected asparagine mutants after integration into the Pichia genome. Though methanol induction of the native and the rPA mutants showed similar expression levels (~200–250 mg/L), the mutants displayed significant loss of protease activity. Strikingly, the clot lysis activities of these mutants were considerably different. While mutation of Asn12 (N12P) of the Kringle 2 domain showed delayed clot lysis activity (t1/2 = 38 min) compared to the native rPA (t1/2 = 33 min), a faster rate of clot lysis (t1/2 = 27 min) was observed when the Asn278 (N278S) of the serine protease domain was mutated. Interestingly, the slowest clot lysis activity (t1/2 = 49 min) demonstrated by the double mutant (N12P, N278S) suggests the dominant role of Asn12 in regulating the fibrinolytic activity of rPA. The results presented in this paper indicate that the fibrinolytic and the proteolytic activities of rPA are independent of each other

Enhanced fluorescent properties of an OmpT site deleted mutant of Green Fluorescent Protein

<p>Abstract</p> <p>Background</p> <p>The green fluorescent protein has revolutionized many areas of cell biology and biotechnology since it is widely used in determining gene expression and for localization of protein expression. Expression of recombinant GFP in <it>E. coli </it>K12 host from pBAD24M-GFP construct upon arabinose induction was significantly lower than that seen in <it>E. coli </it>B cells with higher expression at 30°C as compared to 37°C in <it>E. coli </it>K12 hosts. Since OmpT levels are higher at 37°C than at 30°C, it prompted us to modify the OmpT proteolytic sites of GFP and examine such an effect on GFP expression and fluorescence. Upon modification of one of the two putative OmpT cleavage sites of GFP, we observed several folds enhanced fluorescence of GFP as compared to unmodified GFPuv (Wild Type-WT). The western blot studies of the WT and the SDM II GFP mutant using anti-GFP antibody showed prominent degradation of GFP with negligible degradation in case of SDM II GFP mutant while no such degradation of GFP was seen for both the clones when expressed in BL21 cells. The SDM II GFP mutant also showed enhanced GFP fluorescence in other <it>E. coli </it>K12 OmpT hosts like <it>E. coli </it>JM109 and LE 392 in comparison to WT GFPuv. Inclusion of an OmpT inhibitor, like zinc with WT GFP lysate expressed from an <it>E. coli </it>K12 host was found to reduce degradation of GFP fluorescence by two fold.</p> <p>Results</p> <p>We describe the construction of two GFP variants with modified putative OmpT proteolytic sites by site directed mutagenesis (SDM). Such modified genes upon arabinose induction exhibited varied degrees of GFP fluorescence. While the mutation of K79G/R80A (SDM I) resulted in dramatic loss of fluorescence activity, the modification of K214A/R215A (SDM II) resulted in four fold enhanced fluorescence of GFP.</p> <p>Conclusions</p> <p>This is the first report on effect of OmpT protease site modification on GFP fluorescence. The wild type and the GFP variants showed similar growth profile in bioreactor studies with similar amounts of recombinant GFP expressed in the soluble fraction of the cell. Our observations on higher levels of fluorescence of SDM II GFP mutant over native GFPuv in an OmpT⁺host like DH5α, JM109 and LE392 at 37°C reiterates the role played by host OmpT in determining differences in fluorescent property of the expressed GFP. Both the WT GFP and the SDM II GFP plasmids in <it>E. coli </it>BL21 cells showed similar expression levels and similar GFP fluorescent activity at 37°C. This result substantiates our hypothesis that OmpT protease could be a possible factor responsible for reducing the expression of GFP at 37°C for WT GFP clone in K12 hosts like DH5α, JM109, LE 392 since the levels of GFP expression of SDM II clone in such cells at 37°C is higher than that seen with WT GFP clone at the same temperature.</p

A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME) from Phage K and its development into a potent antistaphylococcal protein

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>is a major cause of nosocomial and community-acquired infections. However, the rapid emergence of antibiotic resistance limits the choice of therapeutic options for treating infections caused by this organism. Muralytic enzymes from bacteriophages have recently gained attention for their potential as antibacterial agents against antibiotic-resistant gram-positive organisms. Phage K is a polyvalent virulent phage of the <it>Myoviridae </it>family that is active against many <it>Staphylococcus </it>species.</p> <p>Results</p> <p>We identified a phage K gene, designated <it>orf</it>56, as encoding the phage tail-associated muralytic enzyme (TAME). The gene product (ORF56) contains a C-terminal domain corresponding to cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), which demonstrated muralytic activity on a staphylococcal cell wall substrate and was lethal to <it>S. aureus </it>cells. We constructed N-terminal truncated forms of ORF56 and arrived at a 16-kDa protein (Lys16) that retained antistaphylococcal activity. We then generated a chimeric gene construct encoding Lys16 and a staphylococcal cell wall-binding SH3b domain. This chimeric protein (P128) showed potent antistaphylococcal activity on global clinical isolates of <it>S. aureus </it>including methicillin-resistant strains. In addition, P128 was effective in decolonizing rat nares of <it>S. aureus </it>USA300 in an experimental model.</p> <p>Conclusions</p> <p>We identified a phage K gene that encodes a protein associated with the phage tail structure. The muralytic activity of the phage K TAME was localized to the C-terminal CHAP domain. This potent antistaphylococcal TAME was combined with an efficient <it>Staphylococcus</it>-specific cell-wall targeting domain SH3b, resulting in the chimeric protein P128. This protein shows bactericidal activity against globally prevalent antibiotic resistant clinical isolates of <it>S. aureus </it>and against the genus <it>Staphylococcus </it>in general. <it>In vivo</it>, P128 was efficacious against methicillin-resistant <it>S. aureus </it>in a rat nasal colonization model.</p

A new derivation of the generating function for the major index

We present a new proof of the well-known combinatorial result [nk]q = [Sigma]w qMaj(w) where w is a permutation of 0k1n-k, by showing a bijection between the set of partitions of an integer m that fit in a k x n - k rectangle and the set consisting of all permutations w of 0k1n-k having Maj(w) = m.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28617/1/0000429.pd

In vitro anticancer activity of ethanolic extract of Stoechospermum marginatum against HT-29 human colon adenocarcinoma cells

169-175Colorectal cancer is a one of the leading causes of death globally and its clinical management of cancer involves chemotherapy. Increase in the development of resistance to the drugs used in the cancer treatment and serious side effects associated with chemotherapeutic drugs are the major limitations in cancer therapy. Hence, there exists a huge need to develop safer natural therapeutic products for cancer therapy. In this study, ethanolic extract of Stoechospermum marginatum was evaluated for its anticancer activity. The cytotoxicity of S. marginatum extract was evaluated on HT-29 cells by MTT assay. Trypan blue cell viability was also carried out to evaluate cytotoxicity and antiproliferative effect. The apoptosis-inducing potential of the extract was analyzed by acridine orange and ethidium bromide dual staining method, mitochondrial membrane potential assay and FITC Annexin V-Propidium iodide staining method. The ethanolic extract of S. marginatum showed significant dose-dependent cytotoxicity in HT-29 cells Treatment with S. marginatum extract increased number of apoptotic cells in HT-29 cells and caused damage to mitochondrial membrane potential. The findings of the present study confirmed in vitro anticancer activity of ethanolic extract S. marginatu