Conditional inactivation of the Cacna1a gene in transgenic mice

Ca(v)2.1 (P/Q-type) voltage-gated calcium channels play an important role in neurotransmitter release at many brain synapses and at the neuromuscular junction. Mutations in the CACNA1A gene, encoding the pore forming alpha(1) subunit of Ca(v)2.1 channels, are associated with a wide spectrum of neurological disorders. Here we generated mice with a conditional, floxed, Cacna1a allele without any overt phenotype. Deletion of the floxed Cacna1a allele resulted in ataxia, dystonia, and lethality during the fourth week, a severe phenotype similar to conventional Ca(v)2.1 knockout mice. Although neurotransmitter release at the neuromuscular junction was not affected in the conditional mice, homozygous deletion of the floxed allele caused an ablation of Ca(v)2.1 channel-mediated neurotransmission that was accompanied by a compensatory upregulation of Ca(v)2.3 (R-type) channels at this synapse. Pharmacological inhibition of Ca(v)2.1 channels is possible, but the contributing cell-types and time windows relevant to the different Ca(v)2.1-related neurological disorders can only be reliably determined using Cacna1a conditional mice.     

Temporal bone bank: complying with European union directives on human tissue and cells

Background

Availability of allograft tympano-ossicular systems (ATOS) provides unique reconstructive capabilities, allowing more radical removal of middle ear pathology. To provide ATOS, the University of Antwerp Temporal Bone Bank (UATB) was established in 1988. ATOS use was stopped in many countries because of safety issues concerning human tissue transplantation. Our objective was to maintain an ATOS tissue bank complying with European Union (EU) directives on human tissues and cells.

Methods

The guidelines of the Belgian Superior Health Council, including EU directive requirements, were rigorously applied to UATB infrastructure, workflow protocols and activity. Workflow protocols were updated and an internal audit was performed to check and improve consistency with established quality systems and changing legislations. The Belgian Federal Agency of Medicines and Health Products performed an inspection to examine compliance with national legislatives and EU directives on human tissues and cells. A sample of important procedures was meticulously examined in its workflow setting next to assessment of the infrastructure and personnel.

Results

Results are reported on infrastructure, personnel, administrative workflow, procurement, preparation, processing, distribution, internal audit and inspection by the competent authority. Donors procured: 2006, 93 (45.1%); 2007, 64 (20.6%); 2008, 56 (13.1%); 2009, 79 (6.9%). The UATB was approved by the Minister of Health without critical or important shortcomings. The Ministry accords registration each time for 2?years.

Conclusions

An ATOS tissue bank complying with EU regulations on human allografts is feasible and critical to assure that the patient receives tissue, which is safe, individually checked and prepared in a suitable environment.     

Community shifts in a seeded 3-chlorobenzoate degrading membrane biofilm reactor: indications for involvement of in situ horizontal transfer of the clc-element from inoculum to contaminant bacteria

Pseudomonas putida BN210, carrying the self- transferable clc-element encoding degradation of 3-chlorobenzoate on the chromosome, was used as inoculum in different membrane biofilm reactors treating 3-chlorobenzoate-contaminated model wastewater. Analysis of the bacterial population in the effluent and in the biofilm showed the loss of BN210 beyond detection from the reactors and the appearance of several novel 3-chlorobenzoate mineralizing bacteria mainly belonging to the beta-proteobacteria. In contrast, in non-inoculated reactors, no 3-chlorobenzoate degradation was observed and no 3-chlorobenzoate degraders could be recovered. Southern blots hybridization of genomic DNA using clc-element-specific probes and FIGE analysis indicated the presence of the complete clc-element in one or more copies in the isolates. Moreover, the isolates could transfer the clc genes to Ralstonia metallidurans recipients. Two representative reactor isolates, Ralstonia sp. strains KP3 and KP9 demonstrated a higher growth rate on 3-chlorobenzoate than strain BN210 in batch cultures. When BN210, KP3 and KP9 were simultaneously inoculated in a membrane reactor supplied with 3-chlorobenzoate, strain KP3 outcompeted the two other strains and remained the major 3-chlorobenzoate degrading population in the reactor. Our data suggest that in situ horizontal transfer of the clc-element from the inoculum to contaminant bacteria in the reactors was involved in the establishment of novel 3-chlorobenzoate degrading populations that were more competitive under the defined reactor conditions than the inoculum strain.     

HDAC6 at the Intersection of Neuroprotection and Neurodegeneration

Histone deacetylase 6 (HDAC6) catalyzes multiple reactions. We summarize the current knowledge on HDAC6, its targets and functions. Among others, HDAC6 recognizes damaged proteins and assures that these proteins are destroyed by autophagy. On the other hand, HDAC6 also modifies the tracks used by the clearance mechanism so that axonal transport becomes less efficient. We hypothesize that a disturbance in the equilibrium between the different functions of HDAC6 could play an important role in neurodegeneration.     

Liquid medium additions to established tissue cultures to improve elongation and rooting in vivo

In order to save manual labour in plant tissue propagation, we tried adding liquid media to established, exhausted cultures, instead of transplanting the tissues to a fresh medium. This has been investigated for the elongation and for root induction stages with Cordyline, Philodendron, Magnolia and Spathiphyllum. The salt concentration and the application time are important factors, determining the success of this method. Charcoal added to the liquid stage IIIa medium eliminated the residual effects of BA applied during stage II.     

Nature and distribution of large sequence polymorphisms in Saccharomyces cerevisiae

To obtain a better understanding of the genome-wide distribution and the nature of large sequence polymorphisms (LSPs) in Saccharomyces cerevisiae, we hybridized genomic DNA of 88 haploid or homozygous diploid S. cerevisiae strains of diverse geographic origins and source substrates onto high-density tiling arrays. On the basis of loss of hybridization, we identified 384 LSPs larger than 500 bp that were located in 188 non-overlapping regions of the genome. Validation by polymerase chain reaction-amplification and/or DNA sequencing revealed that 39 LSPs were due to deletions, whereas 74 LSPs involved sequences diverged far enough from the S288c reference genome sequence as to prevent hybridization to the microarray features. The LSP locations were biased toward the subtelomeric regions of chromosomes, where high genetic variation in genes involved in transport or fermentation is thought to facilitate rapid adaptation of S. cerevisiae to new environments. The diverged LSP sequences appear to have different allelic ancestries and were in many cases identified as Saccharomyces paradoxus introgressions.     

HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1-induced Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system. Mutations in the 27-kDa small heat-shock protein gene (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). We developed and characterized transgenic mice expressing two different HSPB1 mutations (S135F and P182L) in neurons only. These mice showed all features of CMT or distal HMN dependent on the mutation. Expression of mutant HSPB1 decreased acetylated α-tubulin abundance and induced severe axonal transport deficits. An increase of α-tubulin acetylation induced by pharmacological inhibition of histone deacetylase 6 (HDAC6) corrected the axonal transport defects caused by HSPB1 mutations and rescued the CMT phenotype of symptomatic mutant HSPB1 mice. Our findings demonstrate the pathogenic role of α-tubulin deacetylation in mutant HSPB1-induced neuropathies and offer perspectives for using HDAC6 inhibitors as a therapeutic strategy for hereditary axonopathies.     

Endoscopic procurement of tympano-ossicular allografts: alternative to the transcranial or retroauricular technique

A tympano-ossicular tissue bank complying with European Union regulations on human allografts is feasible and critical to assure that the patient receives tissue which is safe, individually checked and prepared in a suitable environment. The transcranial procurement technique has become the standard approach to procure tympano-ossicular allografts since the 1970s because it can provide en bloc allografts. Over the last 10–20 years, en bloc allografts have been abandoned and only the malleus (hammer) is left attached to the tympanic membrane. This modification enables introduction of the transmeatal procurement technique. Transmeatal procurement using readily available nasal 0° and 30° endoscopes is a feasible alternative which avoids contact with the dura mater and is not esthetically invasive to the donor. It involves a more time-consuming procurement but avoids the need for preparation of the temporal bone plug and is therefore generally more time-efficient.     

Rapamycin increases survival in ALS mice lacking mature lymphocytes

Background

Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease. Disease pathophysiology is complex and not yet fully understood, but is proposed to include the accumulation of misfolded proteins, as aggregates are present in spinal cords from ALS patients and in ALS model organisms. Increasing autophagy is hypothesized to be protective in ALS as it removes these aggregates. Rapamycin is frequently used to increase autophagy, but is also a potent immune suppressor. To properly assess the role of rapamycin-induced autophagy, the immune suppressive role of rapamycin should be negated.

Findings

Autophagy is increased in the spinal cord of ALS mice. Dietary supplementation of rapamycin increases autophagy, but does not increase the survival of mutant SOD1 mice. To measure the effect of rapamycin in ALS independent of immunosuppression, we tested the effect of rapamycin in ALS mice deficient of mature lymphocytes. Our results show that rapamycin moderately increases the survival of these ALS mice deficient of mature lymphocytes.

Conclusions

Rapamycin could suppress protective immune responses while enhancing protective autophagy reactions during the ALS disease process. While these opposing effects can cancel each other out, the use of immunodeficient mice allows segregation of effects. Our results indicate that maximal therapeutic benefit may be achieved through the use of compounds that enhance autophagy without causing immune suppression.