Use of Mycobacterium smegmatis Deficient in ADP-Ribosyltransferase as Surrogate for Mycobacterium tuberculosis in Drug Testing and Mutation Analysis

Rifampicin (Rif) is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr) in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs). Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase) causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr). The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC) for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil) strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G). Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.     

An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.     

SBOL Visual: A Graphical Language for Genetic Designs

Synthetic Biology Open Language (SBOL) Visual is a graphical standard for genetic engineering. It consists of symbols representing DNA subsequences, including regulatory elements and DNA assembly features. These symbols can be used to draw illustrations for communication and instruction, and as image assets for computer-aided design. SBOL Visual is a community standard, freely available for personal, academic, and commercial use (Creative Commons CC0 license). We provide prototypical symbol images that have been used in scientific publications and software tools. We encourage users to use and modify them freely, and to join the SBOL Visual community: http://www.sbolstandard.org/visual.     

Genetics of resistance to Cry1C toxin from Bacillus thuringiensis in Spodoptera litura (Fab.)

The present study was undertaken to determine the genetics of Cry1C resistance in Spodoptera litura. Selection of S. litura (Fab.) with Cry1C was done for eight generations to develop resistance. Reciprocal crosses between resistant and susceptible populations were made to understand the population genetics of Cry1C resistance in S. litura. Generation wise selection with Cry1C was evaluated for resistance development in S. litura. The LC₅₀ of Cry1C was 0.14 µg/cm² for the first selected generation and it increased to 23.98 µg/cm² after eight selected generations, which is a 285.47-fold increase in resistance compared with the susceptible strain. The estimated realized heritability (h²) after eight generations of selection with Cry1C insecticidal protein was 0.44. The number of generations required for the tenfold increase in LC₅₀ (1/R) was estimated to be 3.33. Response to Cry1C selection in S. litura was 0.30, the estimated selection differential was 0.69 and the pheonotypic standard deviation (dP) was 0.24. Reciprocal crosses between Cry1C resistant and susceptible strain of S. litura showed autosomal resistance.     

Phenothiazine-mediated rescue of cognition in tau transgenic mice requires neuroprotection and reduced soluble tau burden

Background

It has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection.

Results

Here, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember?, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced.

Conclusions

Neuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.

     

The dynamic nature of contact networks in infectious disease epidemiology

Although contact network models have yielded important insights into infectious disease transmission and control throughout the last decade, researchers have just begun to explore the dynamic nature of contact patterns and their epidemiological significance. Most network models have assumed that contacts are static through time. Developing more realistic models of the social interactions that underlie the spread of infectious diseases thus remains an important challenge for both data gatherers and modelers. In this article, we review some recent data-driven and process-driven approaches that capture the dynamics of human contact, and discuss future challenges for the field.     

Evidence that the C‐terminal domain (CtD) autoinhibits neural repression by Drosophila E(spl)M8

Analysis of the retinal defects of a CK2 phosphomimetic variant of E(spl)M8 (M8S¹⁵⁹D) and the truncated protein M8* encoded by the E(spl)D allele, suggest that the nonphosphorylated CtD “autoinhibits” repression. We have investigated this model by testing for inhibition (in “trans”) by the CtD fragment in its nonphosphorylated (M8‐CtD) and phosphomimetic (M8SD‐CtD) states. In N⁺ flies, ectopic M8‐CtD compromises lateral inhibition, i.e., elicits supernumerary bristles as with loss of N signaling. This antimorphic activity of M8‐CtD strongly rescues the reduced eye and/or bristle loss phenotypes that are elicited by ectopic M8SD or wild type M8. Additionally, the severely reduced eye of N^spl/Y; E(spl)D/+ flies is also rescued by M8‐CtD. Rescue is specific to the time and place, the morphogenetic furrow, where “founding” R8 photoreceptors are specified. In contrast, the phosphomimetic M8SD‐CtD that is predicted to be deficient for autoinhibition, exhibits significantly attenuated or negligible activity. These studies provide evidence that autoinhibition by the CtD regulates M8 activity in a phosphorylation‐dependent manner. genesis 48:44–55, 2010. © 2009 Wiley‐Liss, Inc.