The effects of prolonged deep freezing on the biomechanical properties of osteochondral allografts

Musculo-skeletal allografts sterilized and deep frozen are among the most common human tissue to be preserved and utilized in modern medicine. The effects of a long deep freezing period on cortical bone has already been evaluated and found to be insignificant. However, there are no reports about the influences of a protracted deep freezing period on osteochondral allografts. One hundred osteochondral cylinders were taken from a fresh specimen and humeral heads of 1 year, 2 years, 3 years and 4 year old bones. Twenty chips from each period, with a minimum of 3 chips per humeral head. Each was mechanically tested by 3 point compression. The fresh osteochondral allografts were significantly mechanically better than the deep frozen osteochondral allografts. There was no statistical significant time dependent difference between the deep frozen groups in relation to the freezing period. Therefore, we conclude that, from the mechanical point of view deep freezing of osteochondral allografts over a period of 4 years, is safe without further deterioration of the biomechanical properties of the osteochondral allografts.     

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.     

The first potassium channel toxin from the venom of the Iranian scorpion Odonthobuthus doriae

The very first member of K(+) channels toxins from the venom of the Iranian scorpion Odonthobuthus doriae (OdK1) was purified, sequenced and characterized physiologically. OdK1 has 29 amino acids, six conserved cysteines and a pI value of 4.95. Based on multiple sequence alignments, OdK1 was classified as alpha-KTx 8.5. The pharmacological effects of OdK1 were studied on six different cloned K(+) channels (vertebrate Kv1.1-Kv1.5 and Shaker IR) expressed in Xenopus laevis oocytes. Interestingly, OdK1 selectively inhibited the currents through Kv1.2 channels with an IC50 value of 183+/-3 nM but did not affect any of the other channels.     

Size-selective predation by all-male prawns: implications for sustainable biocontrol of snail invasions

All-male populations of the freshwater prawn Macrobrachium rosenbergii were recently produced by a novel temporal RNA interference (RNAi)-based biotechnology for aquaculture purposes. This biotechnology opens the way to the wide use of all-male prawn populations as sustainable biocontrol agents against invading populations of freshwater snails, for which there is currently no environmentally friendly solution. Among the most damaging of the invasive freshwater snail species are the apple snails (Pomacea spp.), which inflict major damage on natural ecosystems and rice fields. The proposed use of all-male prawn populations as environmentally friendly biocontrol agents against invasive freshwater snails has several advantages: efficient predation by the prawns over a wide range of freshwater snails, the ready availability of the prawns, and the monosex non-reproductive nature of the biocontrol agents. Since the aquatic predators are strongly size selective, we quantified the predation rate as a function of body size of both predator and prey (M. rosenbergii and P. caniculata). Medium-sized and large prawns (~10–30 g) efficiently preyed small and medium-sized snails (up to 15 mm), while small prawns (up to 4 g) immediately and completely eradicated snail hatchlings. Medium-sized prawns (~22 g) exterminated a significant fraction of snail biomass within 24 h (up to 58% of their body mass) after being introduced into a tank of snails. A typical ‘climbing-to-the surface’ anti-predator behavior of the snails was recorded. The potential of all-male prawns as efficient biocontrol agents over hatchling and adult apple snails as part of an integrated pest management program is discussed. Our experiments set the stage for evaluating the ecological and economic implications of this generic solution for a wide variety of habitats.     

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.     

Leishmania species: Detection and identification by nested PCR assay from skin samples of rodent reservoirs

Many rodent species act as reservoir hosts of zoonotic cutaneous leishmaniasis in endemic areas. In the present study a simple and reliable assay based on nested PCR was developed for the detection and identification of Leishmania parasites from rodent skin samples. We designed Leishmania-specific primers that successfully amplified ITS regions of Leishmania major, Leishmania gerbilli and Leishmania turanica using nested PCR. Out of 95 field collected Rhombomys opimus, 21 were positive by microscopic examination and 48 by nested PCR. The percentage of gerbils infected with L. major, L. gerbilli and L. turanica was 3.2%, 1.1% and 27.4%, respectively. In 15.8% of the rodents, we found mixed natural infections by L. major and L. turanica, 1.1% by L. major and L. gerbilli, and 2.1% by the three species. We concluded that this method is simple and reliable for detecting and identifying Leishmania species circulating in rodent populations.     

Bcl-2 proteins link programmed cell death with growth and morphogenetic adaptations in the fungal plant pathogen Colletotrichum gloeosporioides

Proteins belonging to the Bcl-2 family regulate apoptosis in mammals by controlling mitochondria efflux of cytochrome c and other apoptosis-related proteins. Homologues of human Bcl-2 proteins are found in different metazoan organisms where they play a similar role, while they seem to be absent in plants and fungi. Nonetheless, Bcl-2 protein members can induce or prevent yeast cell death, suggesting that enough functional conservation exists between apoptotic machineries of mammals and fungi. Here we show that induction or prevention of apoptosis by Bcl-2 proteins in the fungal plant pathogen Colletotrichum gloeosporioides is tightly linked with growth and morphogenetic adaptation that occur throughout the entire fungal life cycle. Isolates expressing the pro-apoptotic Bax protein underwent cell death with apoptotic characteristics, and showed alterations in conidial germination that are associated with pathogenic and non-pathogenic life styles. Isolates expressing the anti-apoptotic Bcl-2 protein had prolonged longevity, were protected from Bax-induced cell death, and exhibited enhanced stress resistance. These isolates also had enhanced mycelium and conidia production, and were hyper virulent to host plants. Our findings show that apoptotic-associated machinery regulates morphogenetic switches, which are critical for proper responses and adaptation fungi to different environments.