Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline

A number of different processes that impact on telomere length dynamics have been identified but factors that affect the turnover of repeats located proximally within the telomeric DNA are poorly defined. We have identified a particular repeat type (CTAGGG) that is associated with an extraordinarily high mutation rate (20% per gamete) in the male germline. The mutation rate is affected by the length and sequence homogeneity of the (CTAGGG)_n array. This level of instability was not seen with other sequence-variant repeats, including the TCAGGG repeat type that has the same composition. Telomeres carrying a (CTAGGG)_n array are also highly unstable in somatic cells with the mutation process resulting in small gains or losses of repeats that also occasionally result in the deletion of the whole (CTAGGG)_n array. These sequences are prone to quadruplex formation in vitro but adopt a different topology from (TTAGGG)_n (see accompanying article). Interestingly, short (CTAGGG)₂ oligonucleotides induce a DNA damage response (γH2AX foci) as efficiently as (TTAGGG)₂ oligos in normal fibroblast cells, suggesting they recruit POT1 from the telomere. Moreover, in vitro assays show that (CTAGGG)_n repeats bind POT1 more efficiently than (TTAGGG)_n or (TCAGGG)_n. We estimate that 7% of human telomeres contain (CTAGGG)_n repeats and when present, they create additional problems that probably arise during telomere replication.     

Novozym 435 for production of biodiesel from unrefined palm oil: Comparison of methanolysis methods

Biodiesel (BD) is commonly produced from refined vegetable oils by alkali-catalyzed methanolysis. Unrefined vegetable oils are economically attractive but not suitable for alkali catalysis because of their high content of free fatty acids (FFAs). Novozym 435 (immobilized Candida antarctica lipase B), which accepts both FFA and oil as substrates, was, therefore, employed to convert unrefined palm oil to BD. Three different methanolysis methods, namely, t-butanol mediated system (method-1), LiCl solution based controlled release system for methanol (method-2) and solvent-free system with three successive additions of methanol (method-3), were compared. The optimal methanol to oil molar ratios in the method-1, -2 and -3 are 6:1, 3:1 and 3:1, respectively. BD yield at an optimal methanol concentration reaches 91–92% after 10, 20 and 24 h in the method-1, -2 and -3, respectively. BD yield remains the same over five repeated cycles in the method-1, while it drops to 68 and 71% by the fifth cycle in the method-2 and -3, respectively. The results show that the method-1 is the most effective for production of BD from a low cost feedstock like unrefined palm oil.     

Mating duration and egg production of the predaceous mite Neoseiulus californicus (Acari: Phytoseiidae) vary with temperature

Effects of constant temperature on mating duration and total fecundity of Neoseiulus californicus females mated once were investigated at 18 °C, 25 °C, 30 °C, and 35 °C with a photoperiod of 16L:8D. Adult mites grown and maintained at 25 °C mated for 315.3 min on average and produced 46.1 eggs per female. These values varied significantly by temperature: 553.6 and 13.9 (18 °C), 261.2 and 26.6 (30 °C), and 253.6 and 23.9 (35 °C), respectively. Duration of copulation was negatively correlated with temperature. However, total egg production peaked at 25 °C and decreased at lower and higher temperatures. Reduced sperm transfer and/or survival rate of sperm in the female body may account for decreased egg production when temperatures are not optimal.     

Tsukamurella tyrosinosolvens - An unusual case report of bacteremic pneumonia after lung transplantation

Intravesical administration of Bacillus Calmette-Guérin is used as a treatment method in superficial bladder cancer. While it is generally well tolerated, serious side effects may develop. Granulomatous hepatitis cases have been previously reported; however, only one case with tuberculous peritonitis exists in the current literature. We hereby present two cases, one of which is the second tubercular peritonitis case following Bacillus Calmette-Guérin treatment to be reported, and the other a case with granulomatous hepatitis. Complete cure was achieved in both cases with specific therapy. In the patient who developed peritonitis, intravesical Bacillus Calmette-Guérin therapy was recommenced after antituberculosis treatment, and completed without further complications.     

Production of cell culture (MDCK) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process

The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg‐based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin‐Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25–30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers ≥8.6 log₁₀ FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn‐around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production. Biotechnol. Bioeng. 2010;106: 906–917. © 2010 Wiley Periodicals, Inc.     

Germination and inactivation of Bacillus subtilis spores induced by moderate hydrostatic pressure

In this study, we investigated the mechanisms of spore inactivation by high pressure at moderate temperatures to optimize the sterilization efficiency of high‐pressure treatments. Bacillus subtilis spores were first subjected to different pressure treatments ranging from 90 to 550 MPa at 40°C, with holding times from 10 min to 4 h. These treatments alone caused slight inactivation, which was related to the pressure‐induced germination of the spores. After these pressures treatments, the sensitivity of these processed spores to heat (80°C/10 min) or to high pressure (350 MPa/40°C/10 min) was tested to determine the pressure‐induced germination rate and the advancement of the spores in the germination process. The subsequent heat or pressure treatments were applied immediately after decompression from the first pressure treatment or after a holding time at atmospheric pressure. As already known, the spore germination is more efficient at low pressure level than at high pressure level. Our results show that this low germination efficiency at high pressure seemed not to be related either to a lower induction or a difference in the induction mechanisms but rather to an inhibition of enzyme activities which are involved in germination process. In fact, high pressure was necessary and very efficient in inducing spore germination. However, it seemed to slow the enzymatic digestion of the cortex, which is required for germinated spores to be inactivated by pressure. Although these results indicate that high‐pressure treatments are more efficient when the two treatments are combined, a small spore population still remained dormant and was not inactivated with any holding time or pressure level. Biotechnol. Bioeng. 2010;107: 876–883. © 2010 Wiley Periodicals, Inc.     

MrGrid: A Portable Grid Based Molecular Replacement Pipeline

Background

The crystallographic determination of protein structures can be computationally demanding and for difficult cases can benefit from user-friendly interfaces to high-performance computing resources. Molecular replacement (MR) is a popular protein crystallographic technique that exploits the structural similarity between proteins that share some sequence similarity. But the need to trial permutations of search models, space group symmetries and other parameters makes MR time- and labour-intensive. However, MR calculations are embarrassingly parallel and thus ideally suited to distributed computing. In order to address this problem we have developed MrGrid, web-based software that allows multiple MR calculations to be executed across a grid of networked computers, allowing high-throughput MR.

Methodology/Principal Findings

MrGrid is a portable web based application written in Java/JSP and Ruby, and taking advantage of Apple Xgrid technology. Designed to interface with a user defined Xgrid resource the package manages the distribution of multiple MR runs to the available nodes on the Xgrid. We evaluated MrGrid using 10 different protein test cases on a network of 13 computers, and achieved an average speed up factor of 5.69.

Conclusions

MrGrid enables the user to retrieve and manage the results of tens to hundreds of MR calculations quickly and via a single web interface, as well as broadening the range of strategies that can be attempted. This high-throughput approach allows parameter sweeps to be performed in parallel, improving the chances of MR success.     

The Sudden Dominance of bla CTX–M Harbouring Plasmids in Shigella spp. Circulating in Southern Vietnam

Background

Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs) has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges.

Methodology

We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla _CTX–M encoding plasmid.

Principal Findings

We show that two different bla _CTX-M genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla _CTX-M gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356) carried the bla _CTX-M-24 gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids.

Significance

The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.     

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord¹ have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons². This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate^3,4 and the chemoattractive activities of Netrin1⁵ and Sonic Hedgehog (Shh)⁶ in the floor plate in the spinal cord.Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels⁷. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag1⁸. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons^3,4 as well the necessity for this signal to direct the commissural trajectory⁹.Download video file.^{(108M, mp4)}