Substrate-dependent production and some properties of a thermostable, α-galactosidase from Rhodothermus marinus

-Galactosidase activity in Rhodothermus marinus is dependent on the composition of the growth media. A maximum of 46 U g^–1 cell dry weight was obtained using minimal medium with galactooligo- or polysaccharides as single carbon source. An enzyme hydrolysing both high and low molecular weight galacto-saccharides was partly purified from the cell fractions. The molecular weight was 200 kDa (native) and 50 kDa (monomer). It was optimally active at 85°C, with a half-life of 2 h at 75°C, and had a broad pH range (4–8).     

Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase

Unveiling the determinants for transferase and hydrolase activity in glycoside hydrolases would allow using their vast diversity for creating novel transglycosylases, thereby unlocking an extensive toolbox for carbohydrate chemists. Three different amino acid substitutions at position 220 of a GH1 β-glucosidase from Thermotoga neapolitana caused an increase of the ratio of transglycosylation to hydrolysis (r _s/r _h) from 0.33 to 1.45–2.71. Further increase in r _s/r _h was achieved by modulation of pH of the reaction medium. The wild-type enzyme had a pH optimum for both hydrolysis and transglycosylation around 6 and reduced activity at higher pH. Interestingly, the mutants had constant transglycosylation activity over a broad pH range (5–10), while the hydrolytic activity was largely eliminated at pH 10. The results demonstrate that a combination of protein engineering and medium engineering can be used to eliminate the hydrolytic activity without affecting the transglycosylation activity of a glycoside hydrolase. The underlying factors for this success are pursued, and perturbations of the catalytic acid/base in combination with flexibility are shown to be important factors.

     

Decreased Th1-type inflammatory cytokine expression in the skin is associated with persisting symptoms after treatment of erythema migrans

Background

Despite the good prognosis of erythema migrans (EM), some patients have persisting symptoms of various character and duration post-treatment. Several factors may affect the clinical outcome of EM, e.g. the early interaction between Borrelia (B.) burgdorferi and the host immune response, the B. burgdorferi genotype, antibiotic treatment as well as other clinical circumstances. Our study was designed to determine whether early cytokine expression in the skin and in peripheral blood in patients with EM is associated with the clinical outcome.

Methods

A prospective follow-up study of 109 patients with EM was conducted at the Åland Islands, Finland. Symptoms were evaluated at 3, 6, 12 and 24 months post-treatment. Skin biopsies from the EM and healthy skin were immunohistochemically analysed for expression of interleukin (IL)-4, IL-10, IL-12p70 and interferon (IFN)-γ, as well as for B. burgdorferi DNA. Blood samples were analysed for B. burgdorferi antibodies, allergic predisposition and levels of systemic cytokines.

Findings

None of the patients developed late manifestations of Lyme borreliosis. However, at the 6-month follow-up, 7 of 88 patients reported persisting symptoms of diverse character. Compared to asymptomatic patients, these 7 patients showed decreased expression of the Th1-associated cytokine IFN-γ in the EM biopsies (p = 0.003). B. afzelii DNA was found in 48%, B. garinii in 15% and B. burgdorferi sensu stricto in 1% of the EM biopsies, and species distribution was the same in patients with and without post-treatment symptoms. The two groups did not differ regarding baseline patient characteristics, B. burgdorferi antibodies, allergic predisposition or systemic cytokine levels.

Conclusion

Patients with persisting symptoms following an EM show a decreased Th1-type inflammatory response in infected skin early during the infection, which might reflect a dysregulation of the early immune response. This finding supports the importance of an early, local Th1-type response for optimal resolution of LB.     

Alternate method for endocardial pacemaker lead implantation: A hybrid mini-thoracotomy approach

Although the conventional methods for endo-cardial pacemaker lead implantation via subclavian or cephalic or axillary vein routes is common, but sometimes due to anatomical variations it is not feasible to access these veins Emergence of newer techniques are useful for lead implantation. This case report focuses on a hybrid approach of combined mini-thoracotomy for endocardial pacemaker lead implantation. This fluoroscopy guided minimal thoracotomy approach with endocardial MRI compatible lead placement had the benefits of simple procedural, minimal hospital stay, low early complication rates and economically viable to the patient.     

Regulating germline editing in assisted reproductive technology: An EU cross-disciplinary perspective

Potential applications of genome editing in assisted reproductive technology (ART) raise a vast array of strong opinions, emotional reactions and divergent perceptions. Acknowledging the need for caution and respecting such reactions, we observe that at least some are based on either a misunderstanding of the science or misconceptions about the content and flexibility of the existing legal frameworks. Combining medical, legal and ethical expertise, we present and discuss regulatory responses at the national, European and international levels. The discussion has an EU starting point and is meant as a contribution to the general international regulatory debate. Overall, this paper concludes that gene editing technologies should not be regulated autonomously. Rather, potential uses should be regulated under general, existing frameworks and where applicable by reference to sufficiently equivalent technologies and techniques already subject to specific regulation. To be clear, we do not argue for the hasty introduction of gene editing as a reproductive treatment option in the immediate future. We call for caution with regard to overreaching moratoria and prohibitions that will also affect basic research. We recommend flexible regulations that allow for further responsible research into the potential development of the technology. We call for an open and inclusive debate and argue that scientific communication should claim a more prominent role to counter the danger of widespread misinformation. A high level of transparency and accuracy should guide scientific communication while simultaneously global-scale responsibility and governance should be fostered by promoting cross-disciplinary thinking and multi-level stakeholder involvement in legal and regulatory processes.     

Postharvest growth dynamic of <Emphasis Type="Italic">Schoenoplectus californicus</Emphasis> along fluvio-estuarine and flooding gradients

The emergent wetland plant Schoenoplectus californicus is used as fiber in several American countries, but the effects of harvesting on this species have not previously been studied. We analyzed the biomass production, stem density and morphometry of this species along fluvio-estuarine and flooding gradients and evaluated the effects of harvesting on growth and recovery capacity in the Santa Lucía River (Uruguay), comparing river sections and surface elevations. Differences in biomass, length and stem density were associated with the dynamics of the hydrological regime. The mean biomass and length growth rates were 3.0 ± 2.8 g day^?1 and 0.8 ± 0.5 cm day^?1, respectively. The analysis of the postharvest growth dynamic showed variation among the stems, suggesting the existence of mechanisms of plant compensation for the harvest effect. Six months after the harvest, S. californicus had recovered, e.g., in stem length and density, while the biomass showed a slower recovery. Our results suggest that the recovery capacity of this species depends on the population structure before the harvest and on favorable conditions during the recovery period. Based on our results, we recommend strategies for sustainable harvest management.     

Metals in motor neuron diseases

Degenerative processes within the nervous system are common features in disease entities such as dementia of Alzheimer type (DAT), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease with unknown etiology; widespread muscle wasting and respiratory failure lead to death within a few years. Denervation can be detected with electromyography and axonal deterioration monitored by motor unit number estimates. Several suggestions about the cause of ALS have emerged but no solid theory has yet precipitated. Lead or mercury exposure has been suggested. Exposure data alone cannot support this connection. Alterations in metal kinetics may underlie the deterioration of motor function observed in patients with ALS. In this review the role of metals in motor neuron disease is discussed. Both classic studies on exposure and recent understanding of metal binding proteins are considered. Aspects of peak exposure and excretion are merged toward an understanding of metal dynamics in ALS. An overview of chemical and electrophysiological investigations is given in the context of neurodegeneration.     

Susceptibility of mouse minute virus to inactivation by heat in two cell culture media types

Viral contaminations of biopharmaceutical manufacturing cell culture facilities are a significant threat and one for which having a risk mitigation strategy is highly desirable. High temperature, short time (HTST) mammalian cell media treatment may potentially safeguard manufacturing facilities from such contaminations. HTST is thought to inactivate virions by denaturing proteins of the viral capsid, and there is evidence that HTST provides ample virucidal efficacy against nonenveloped or naked viruses such as mouse minute virus (MMV), a parvovirus. The aim of the studies presented herein was to further delineate the susceptibility of MMV, known to have contaminated mammalian cell manufacturing facilities, to heat by exposing virus‐spiked cell culture media to a broad range of temperatures and for various times of exposure. The results of these studies show that HTST is capable of inactivating MMV by three orders of magnitude or more. Thus, we believe that HTST is a useful technology for the purposes of providing a barrier to adventitious contamination of mammalian cell culture processes in the biopharmaceutical industry. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009