Effect of a pedicle-screw-based motion preservation system on lumbar spine biomechanics: A probabilistic finite element study with subsequent sensitivity analysis

Pedicle-screw-based motion preservation systems are often used to support a slightly degenerated disc. Such implants are intended to reduce intradiscal pressure and facet joints forces, while having a minimal effect on the motion patterns.In a probabilistic finite element study with subsequent sensitivity analysis, the effects of 10 input parameters, such as elastic modulus and diameter of the elastic rod, distraction of the segment, level of bridged segments, etc. on the output parameters intervertebral rotations, intradiscal pressures, and facet joint forces were determined. A validated finite element model of the lumbar spine was employed. Probabilistic studies were performed for seven loading cases: upright standing, flexion, extension, left and right lateral bending and left and right axial rotation.The simulations show that intervertebral rotation angles, intradiscal pressures and facet joint forces are in most cases reduced by a motion preservation system. The influence on intradiscal pressure is small, except in extension. For many input parameter combinations, the values for intervertebral rotations and facet joint forces are very low, which indicates that the implant is too stiff in these cases. The output parameters are affected most by the following input parameters: loading case, elastic modulus and diameter of the elastic rod, distraction of the segment, and angular rigidity of the connection between screws and rod.The designated functions of a motion preservation system can best be achieved when the longitudinal rod has a low stiffness, and when the connection between rod and pedicle screws is rigid.     

Synthesis, Copper(II) Complexation, (64)Cu-Labeling, and Bioconjugation of a New Bis(2-pyridylmethyl) Derivative of 1,4,7-Triazacyclononane

A new ligand derivative of 1,4,7-triazacyclononane (TACN), 2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic acid ( 6), has been synthesized and its complexation behavior toward Cu2+ ions investigated. The ligand 6 has been characterized by spectroscopic methods, and a molecular structure of a corresponding Cu(II) complex has been elucidated by single-crystal X-ray analysis. The suitability of 6 for conjugation to peptide substrates has been shown by amide coupling of 6 to the stabilized derivative of bombesin (BN), beta Ala-beta Ala-[Cha13, Nle14]BN(7-14), to give the conjugate 8. The free ligand 6 and the bioconjugate 8 were labeled with 64Cu2+, and the resulting complexes, 64Cu subset6 and 64Cu subset8 , were found to be stable in the presence of a large excess of a competing ligand (cyclam) or copper-seeking superoxide dismutase (SOD), as well as in rat plasma. Biodistribution studies of 64Cu subset8 in Wistar rats showed a high activity uptake into the pancreas (5.76 +/- 0.25 SUV, 5 min p.i.; 3.93 +/- 0.25 SUV, 1 h p.i.), which is the organ with high levels of gastrin-releasing peptide receptor (GRPR). This receptor is overexpressed in a large number of breast and prostate carcinomas. The novel 64Cu subset6 complex had a dominating influence on the nonspecific activity biodistribution of its BN conjugate, since the distribution data of 64Cu subset6 are similar to those of 64Cu subset8 . The 64Cu complexes exhibited a low activity accumulation in the liver tissue and an extensive renal clearance, which was distinctively different to the biodistribution of 64CuCl 2, suggesting that 64Cu subset6 does not undergo significant demetalation, but rather exhibits high in vivo stability.     

Detailed analysis concerning the biodistribution and metabolism of human calcitonin-derived cell-penetrating peptides

The interest in using small peptides for therapeutic and diagnostic in vivo applications is based on several advantageous features such as good penetration into tissues and rapid clearance from the body. Because of their size, they can easily be synthesized chemically. The recently discovered cell-penetrating peptides (CPP) and among them CPP derived from the native peptide hormone human calcitonin (hCT) could meet these requirements. Therefore, they are nowadays widely used as delivery vectors for a variety of bioactive molecules. However, the knowledge about the distribution and metabolism of CPP in vivo is very limited. Hence, evaluation of the pharmacological features of any promising peptide is a crucial challenge in its development process. Herein, we studied the in vivo radiopharmacology of (68)Ga radiolabeled DOTA-modified, hCT-derived CPP in rats using small animal PET. Furthermore, the arterial blood at different time points and urine were analyzed for radio-metabolites. It was shown that d-amino acid modifications of the sequence hCT(9-32) resulted in an increased in vivo stability and lower retention in the kidney cortex of this peptide.     

Distinct mechanisms of apoptosis-induced compensatory proliferation in proliferating and differentiating tissues in the Drosophila eye

In multicellular organisms, apoptotic cells induce compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. Here we show that a second mechanism of apoptosis-induced compensatory proliferation exists. This mechanism is dependent on effector caspases which trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Furthermore, whereas Dpp and Wg signaling is preferentially employed in apoptotic proliferating tissues, Hh signaling is activated in differentiating eye tissues. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell-cycle reentry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent nonapoptotic function.     

Recombinant expression of Garlic virus C (GARV-C) capsid protein in insect cells and its potential for the production of specific antibodies

Garlic cultivars in Brazil are infected by a complex of viruses and for some virus species, such as the allexivirus, purification of the virions is sometimes cumbersume. To overcome this problem, recombinant expression of viral proteins in heterologous systems is an alternative method for producing antibodies. The capsid gene from Garlic virus C (GarV-C), an Allexivirus, was inserted into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) generating the recombinant virus vSynGarV-C. The recombinant protein expression was confirmed by SDS-PAGE and western-blot of extracts from recombinant virus infected insect cells, where a protein band of approximately 32 KDa was observed only in extracts from recombinant infected cells. This protein corresponded to the predicted size of the capsid protein of the GarV-C. A rabbit polyclonal antibody was raised against this protein, shown to be specific for the GarV-C protein in western-blot and dot-Elisa, however with a low titer.     

Characterization of the methanogenic Archaea within two-phase biogas reactor systems operated with plant biomass

The two-phase leach-bed system is a biogas reactor system optimized for the utilization of energy crop silages at maximized loading rates under maintenance of an optimal microbial activity. In this study, a characterization of the methanogenic microbial community within this reactor system was conducted for the first time. Accordingly, effluent samples from the anaerobic filter and the silage digesting leach-bed reactors of both a laboratory-scale two-phase biogas reactor system and a scaled-up commercial on-farm pilot plant were investigated. In total, five Archaea-specific 16S rDNA libraries were constructed and analyzed by amplified rDNA restriction analysis (ARDRA), with subsequent phylogenetic analysis of nucleotide sequences for individual ARDRA patterns. A quantification of major methanogenic Archaea groups was conducted by real-time PCR. A total of 663 clones were analyzed and 45 operational taxonomic units (OTUs) related to methanogenic Archaea were detected. These OTUs were related to the orders Methanosarcinales, Methanomicrobiales and Methanobacteriales, as well as the hitherto uncultured CA-11 and ARC-I groups, and most of them occurred throughout all the compartments of both two-phase biogas reactors. The proportion of acetotrophic to hydrogenotrophic methanogens differed between the laboratory and the pilot scale system. A total of 56% of the clones from the 16S rDNA library derived from the laboratory biogas system were assigned to presumably acetotrophic members of Methanosarcinales. In contrast, these OTUs were less abundant in the 16S rDNA library derived from samples of the pilot plant. Therein, the most dominant OTUs were Methanoculleus-related OTUs, which presumably indicated the predominant presence of hydrogenotrophic methanogens. These findings were confirmed by group-specific quantitative real-time PCR assays. The results indicated that the fraction of acetotrophic and hydrogenotrophic methanogens within a biogas reactor caused certain variations, which may reflect varying substrate utilization during methanogenesis.     

The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tissue growth non-autonomously

Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.     

Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.     

Archaea diversity within a commercial biogas plant utilizing herbal biomass determined by 16S rDNA and mcrA analysis

Aims: The Archaea diversity was evaluated in an agricultural biogas plant supplied with cattle liquid manure and maize silage under mesophilic conditions. Methods and Results: Two different genes (16S rRNA; methyl‐coenzyme‐M‐reductase, MCR) targeted by three different PCR primer sets were selected and used for the construction of three clone libraries comprising between 104 and 118 clones. The clone libraries were analysed by restriction fragment polymorphism (RFLP). Between 11 and 31 operational taxonomic units (OTUs) were detected and assigned to orders Methanomicrobiales, Methanosarcinales and Methanobacteriales. Over 70% of all Archaea OTUs belong to the order Methanomicrobiales which mostly include hydrogenotrophic methanogens. Acetotrophic methanogens were detected in minor rates. Similar relative values were obtained by a quantitative real‐time PCR analysis. Conclusions: The results implied that in this biogas plant the most of the methane formation resulted from the conversion of H₂ and CO₂. Significance and Impact of the Study: This study reports, for the first time, a molecular analysis of the archaeal community in this type of agricultural biogas plants. Therein the hydrogenotrophic methanogenesis seems to be the major pathway of methane formation. These results are in contrast with the common thesis that in biogas fermentations the primary substrate for methanogenesis is acetate.