Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release

Background

In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles.

Results

Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 10³ to 10⁶ PFU/ml, could also be reliably detected.

Conclusions

This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics.     

Reward-related behavioral paradigms for addiction research in the mouse: performance of common inbred strains

The mouse has emerged as a uniquely valuable species for studying the molecular and genetic basis of complex behaviors and modeling neuropsychiatric disease states. While valid and reliable preclinical assays for reward-related behaviors are critical to understanding addiction-related processes, and various behavioral procedures have been developed and characterized in rats and primates, there have been relatively few studies using operant-based addiction-relevant behavioral paradigms in the mouse. Here we describe the performance of the C57BL/6J inbred mouse strain on three major reward-related paradigms, and replicate the same procedures in two other commonly used inbred strains (DBA/2J, BALB/cJ). We examined Pavlovian-instrumental transfer (PIT) by measuring the ability of an auditory cue associated with food reward to promote an instrumental (lever press) response. In a separate experiment, we assessed the acquisition and extinction of a simple stimulus-reward instrumental behavior on a touch screen based task. Reinstatement of this behavior was then examined following either continuous exposure to cues (conditioned reinforcers, CRs) associated with reward, brief reward and CR exposure, or brief reward exposure followed by continuous CR exposure. The third paradigm examined sensitivity of an instrumental (lever press) response to devaluation of food reward (a probe for outcome insensitive, habitual behavior) by repeated pairing with malaise. Results showed that C57BL/6J mice displayed robust PIT, as well as clear extinction and reinstatement, but were insensitive to reinforcer devaluation. DBA/2J mice showed good PIT and (rewarded) reinstatement, but were slow to extinguish and did not show reinforcer devaluation or significant CR-reinstatement. BALB/cJ mice also displayed good PIT, extinction and reinstatement, and retained instrumental responding following devaluation, but, unlike the other strains, demonstrated reduced Pavlovian approach behavior (food magazine head entries). Overall, these assays provide robust paradigms for future studies using the mouse to elucidate the neural, molecular and genetic factors underpinning reward-related behaviors relevant to addiction research.     

Anaerospora hongkongensis gen. nov. sp. nov., a novel genus and species with ribosomal DNA operon heterogeneity isolated from an intravenous drug abuser with pseudobacteremia

A bacterium was isolated from the blood culture of an intravenous drug abuser with pseudobacteremia. The cells were strictly anaerobic, straight or slightly curved, sporulating, Gram-negative rods. It grew on sheep blood agar as non-hemolytic, pinpoint colonies after 48 hr of incubation at 37 C in an anaerobic environment. It was motile but did not produce catalase or cytochrome oxidase. 16S ribosomal DNA (rDNA) sequencing revealed three different copies of 16S rDNA sequences. More than 90% of the differences among them were due to differences in the lengths of the sequences. Phylogenetically, the bacterium is clustered with Dendrosporobacter, Sporomusa, and Propionispora, the other three genera of anaerobic, sporulating, Gram-negative rods. There were 8.6-11.1% differences between the 16S rDNA sequences of the bacterium and that of D. quercicolus, 4.7-15.1% differences between the 16S rDNA sequences of it and those of S. acidovorans, S. aerivorans, S. malonica, S. ovata, S. paucivorans, S. silvacetica, S. spaeroides, and S. termitida, and 7.6-13.1% differences between the 16S rDNA sequences of it and those of P. hippei and P. vibrioides. The G+C content of the bacterium (mean +/- SD) was 46.8 +/- 3.2%. For these reasons, a new genus and species, Anaerospora hongkongensis gen. nov. sp. nov., is proposed, for which HKU15T is the type strain.     

Molecular and biochemical characterisation of a serine acetyltransferase of onion, Allium cepa (L.)

We have previously cloned a cDNA, designated SAT1, corresponding to a gene coding for a serine acetyltransferase (SAT) from onion (Allium cepa L.). The SAT1 locus was mapped to chromosome 7 of onion using a single-stranded conformation polymorphism (SSCP) in the 3' UTR of the gene. Northern analysis has demonstrated that expression of the SAT1 gene is induced in leaf tissue in response to low S-supply. Phylogenetic analysis has placed SAT1 in a strongly supported group (100% bootstrap) that comprises sequences that have been characterised biochemically, including Allium tuberosum, Spinacea oleracea, Glycine max, Citrullus vulgaris, and SAT5 (AT5g56760) of Arabidopsis thaliana. This group can be divided further with the SAT1 of A. cepa sequence grouping strongly with the A. tuberosum sequence. Translation of SAT1 from onion generates a protein of 289 amino acids with a calculated molecular mass of 30,573 Da and pI of 6.52. The conserved G277 and H282 residues that have been identified as critical for L-cysteine inhibition are observed at G272 and H277. SAT1 has been cloned into the pGEX plasmid, expressed in E. coli and SAT activity of the recombinant enzyme has been measured as acetyl-CoA hydrolysis detected at 232 nm. A Km of 0.72 mM was determined for l-serine as substrate, a Km of 92 microM was calculated with acetyl-CoA as substrate, and an inhibition curve for L-cysteine generated an IC50 value of 3.1 microM. Antibodies raised against the recombinant SAT1 protein recognised a protein of ca. 33 kDa in whole leaf onion extracts. These properties of the SAT1 enzyme from onion are compared with other SAT enzymes characterised from closely related species.     

Fish oil: what the prescriber needs to know

There is a general belief among doctors, in part grounded in experience, that patients with arthritis need nonsteroidal anti-inflammatory drugs (NSAIDs). Implicit in this view is that these patients require the symptomatic relief provided by inhibiting synthesis of nociceptive prostaglandin E₂, a downstream product of the enzyme cyclo-oxygenase (COX), which is inhibited by NSAIDs. However, the concept of 'safe' NSAIDs has collapsed following a multiplicity of observations establishing increased risk for cardiovascular events associated with NSAID use, especially but not uniquely with the new COX-2-selective NSAIDs. This mandates greater parsimony in the use of these agents. Fish oils contain a natural inhibitor of COX, reduce reliance on NSAIDs, and reduce cardiovascular risk through multiple mechanisms. Fish oil thus warrants consideration as a component of therapy for arthritis, especially rheumatoid arthritis, in which its symptomatic benefits are well established. A major barrier to the therapeutic use of fish oil in inflammatory diseases is ignorance of its mechanism, range of beneficial effects, safety profile, availability of suitable products, effective dose, latency of effects and instructions for administration. This review provides an evidence-based resource for doctors and patients who may choose to prescribe or take fish oil.     

Antigene property of PNA conjugated to the nuclear localization signal peptide

Peptide nucleic acid (PNA) is a DNA mimic with antigene properties. To enhance its capacity to enter in the cell and internalize in the nucleus, PNA has been conjugated to the nuclear localization signal (NLS) peptide, PKKKRKV PNA-NLS conjugates form stable hybrids with complementary DNA strands and poorly tolerate mismatched base pairing. Employed against cancer-associated genes, PNA-NLS exhibited a potent and specific antigene activity, suggesting exciting therapeutic approaches to cancer.     

Do nine-primaried passerines have nine or ten primary feathers? The evolution of a concept

The number of primary feathers in a birds wing has been used as a systematic character since the first half of the nineteenth century. During the years, though, the definition of which feathers to count as a primary has changed and today the species historically denoted as having only nine primaries are instead said to have, for example, nine functional primaries. In this study, I investigated the borderline between nine-primaried and ten-primaried birds to search for a proper definition of the term nine-primaried. A total of 161 specimens of 104 bird species, mainly passerines, were examined. All species examined had ten primaries although the nine-primaried species had primary ten more or less concealed under primary covert nine. The number of primary coverts has decreased over time, with ten primary coverts as the ancestral state within Passeriformes and nine primary coverts among most oscine species. In conclusion, a proper definition of nine-primaried might be with primary ten concealed by primary covert nine. This definition includes all taxa historically denoted nine-primaried, i.e. systematically it is a definition of a paraphyletic group. The term nine-primaried is thus too inclusive to be of more than very limited systemtic value and, consequently, the New World nine-primaried oscines group might gain from a new denotation.Electronic Supplementary Material Supplementary material is available for this article at