Molecular insights into the axon guidance molecules Sidestep and Beaten path.

The transmembrane protein Sidestep (Side) functions as a substrate-bound attractant for motor axons in Drosophila. Outgrowing motor axons recognize Side via Beaten path Ia (Beat) and migrate along Side-expressing tissues. Here, we report a structure-function analysis of these guidance molecules using a variety of mutant lines and transgenic constructs. Investigation of Side mutants shows that the exchange of a single amino acid (L241H) in the second immunoglobulin domain disturbs Side function and subcellular localization. Overexpression of Side and Beat deletion constructs in S2 cells and muscles demonstrate that the first Ig domains of both proteins are necessary for their interaction. Furthermore, subcellular distributions of several Beat constructs identify functional domains and suggest a potential posttranslational processing step in ER compartments. In fact, fusing full-length Beat at both the N- and C-terminus with GFP and mCherry, respectively, shows that the N-terminal domain is transported to the plasma membrane and exposed on the cell surface, while the C-terminal domain accumulated in the nucleus. Taken together, these results give insights into the interaction of Side and Beat and imply that Beat might be subject to proteolytic cleavage during maturation.

Rearrangements in the musculature correlate with jumping behaviour in legless Mediterranean fruit fly larvae Ceratitis capitata (Tephritidae).

Larvae of holometabolic insects evolved different crawling strategies depending on the presence or absence of larval legs or life style. A rather unusual mode of locomotion has independently evolved in legless larvae of several dipteran species. Maggots of the Mediterranean fruit fly Ceratitis capitata developed an effective jumping mechanism to increase locomotion speed or to deter predators during the search for suitable pupation sites. Here, we use high-speed videography to visualize even the fastest movements during jump preparation and take-off. Quantification of kinetic and biometric parameters reveal that maggots jump up to 15-fold of their body length from a standing position and gain speed with 27 times the acceleration of gravity. Videos at high spatial resolution show the mechanism of latch formation and release in unprecedented detail. Mouth hooks insert in the caudal segment and raise a cuticular fold that serves as a handle to pressurize the body prior to launch. Since locomotion behaviour should be intrinsically linked to neuromuscular systems, we dissected third instar larvae and determined the precise pattern of abdominal muscles fibres. Compared to non-jumping dipteran larvae, such as Drosophila melanogaster, the overall arrangement is highly similar, but a few muscle fibres show characteristic re-arrangements in orientation and strength that are consistent with a role in bending and jumping. These results suggest that body wall muscles show adaptations to jumping behaviour in Ceratitis larvae, and possibly also in other species with different jumping techniques.

Paradox of sonic hedgehog (SHH) transcriptional regulation: Alternative transcription initiation overrides the effect of downstream promoter DNA methylation.

Recently, DNA methylation has been suggested as a potential mechanism involved in the transcriptional regulation of SHH gene expression in cancer. However, detailed analyses on the underlying transcriptional mechanisms of SHH expression have not been presented so far and were therefore the focus of this study. We found that the genomic region of SHH contains two different transcriptional start sites and four CpG islands spread from the 5' promoter region to the 3' end of the SHH gene. Based on this CpG island topology we analyzed the influence of DNA methylation within the promoter region as well as in exon 2 and exon 3 on SHH mRNA expression in a large set (n = 14) of benign and malignant human cell lines, and further elucidated the functionality of the two identified SHH transcription initiation sites. Methylation-specific PCR (MSP) clearly showed that SHH is expressed independently of DNA methylation within exon 2 and exon 3 of its genomic region, while methylation of the promoter region is able to abrogate SHH expression. Most interesting, we found activation of the upstream SHH promoter in several breast cancer cell lines when the downstream SHH promoter is methylated. These observations lead us to propose a transcriptional model for the SHH gene, in which combined mechanisms of DNA methylation and alternative promoter usage coordinate the transcriptional activity of this important developmental gene.

Primary stability of anterior lumbar stabilization: interdependence of implant type and endplate retention or removal.

This is a comparative in vitro biomechanical study of the primary stability of an anterior lumbar interbody stabilization. The objective was to compare the stability of a interbody stabilizing titanium cage with and without the retention of the bordering vertebral endplates, as well as to compare the titanium cage with a tricalcium phosphate block when the endplates are removed. An adequate blood supply is critical for interbody fusion, which suggests surgical treatment of the bordering endplates. On the other hand, primary stability is improved by the retention of the endplates. Furthermore, bone substitute materials are finding more frequent use due to complications associated with autologous bone grafts. Ten bovine lumbar spine motion segments (average age 6 months) were investigated. Pure bending loadings as well as eccentric axial compression loadings were applied. A titanium cage and tricalcium phosphate block, were tested in conjunction with an anterior augmentation (MACS). Range of motion, neutral zone (NZ) and bending stiffness were measured under pure bending to 10 Nm, and bending stiffness under axial loads of up to 1,500 N. Range of motion of both implants in flexion-extension was significantly smaller than physiologic (cage without endplates 4.3 degrees , cage with 2.8 degrees , block without 3.4 degrees , and physiologic 6.6 degrees , all p<0.001). The cage with endplates and the block without endplates were both significantly stiffer than physiologic in all directions except left lateral bending. The block without endplates and the cage with endplates were both stiffer than the cage without endplates. The results suggest that the use of the bone substitute block provides better stability than the cage when the endplates are removed.

Comparative biomechanical testing of anterior and posterior stabilization procedures.

STUDY DESIGN: This is a comparative in vitro biomechanical study in a calf lumbar spine model. OBJECTIVES: The objective was to compare the primary stability of an anterior instrumentation, an intercorporal cage in combination with an anterior instrumentation, and a posterior instrumentation for monosegmental spondylodesis. SUMMARY OF BACKGROUND DATA: Spondylodesis can be achieved through a posterior lumbar fusion, posterior lumbar intercorporal fusion, or an anterior lumbar intercorporal fusion. The posterior lumbar fusion is the gold standard, although the anterior approach offers some potential advantages to the transpedicular posterior techniques. METHODS: Stability testing was performed on 30 calf lumbar spine motion segments in a physiologic state (n = 30), with either an isolated anterior (MACS) or posterior instrumentation (SOCON), and with an anterior instrumentation augmented with an intercorporal cage (MACS-Cage, n = 10, respectively). Range of motion, neutral zone, and bending stiffness were measured under pure bending to 10 Nm, and bending stiffness under axial loads of up to 1500 N. RESULTS: The isolated posterior instrumentation was found to be more stable than the isolated or augmented anterior instrumentation in flexion/extension, although no significant differences were observed in lateral bending or axial rotation. The results of this biomechanical study suggest that an augmented anterior instrumentation provides similar stability for bony fusion as does the golden standard posterior instrumentation, with the exception of flexion/extension. CONCLUSION: An augmented anterior instrumentation may provide similar stability for bony fusion as does the posterior instrumentation.