Craniofacial shortening by contraction osteogenesis: an experimental model

Application of gradual external forces to correct craniofacial deformities challenges many procedures in conventional craniomaxillofacial surgery. Distraction osteogenesis is replacing traditional osteotomies for correction of patients with craniomaxillofacial deficiencies. However, the reverse concept, contraction osteogenesis, has yet to be established for patients with craniomaxillofacial excesses. The purpose of this investigation is to demonstrate the contraction osteogenesis phenomenon applied in a controlled animal model during the craniofacial growth period. Twenty-six 26-day-old rabbits were assigned to one of four groups: 0, control; 1, pin control (pin insertion); 2, no contraction (pins and contraction device application, without active contraction); and 3, contraction (pin insertion, contraction device application, and active contraction). An external fixator was placed across the incisive-maxillary suture, and the effects after 4.5 weeks of contraction at a rate of 0.5 mm twice a week were compared with control groups. The results were assessed by craniometric and cephalometric measurements and by histologic examination. Gross alterations were evident in the contraction group, characterized by midface anteroposterior shortening, maxillary regression, snout deviation, and anterior crossbite. Histologic examination of the contraction group demonstrated a significant increase in osteoblastic activity. Contraction osteogenesis is a new treatment concept in craniofacial development and may offer therapeutic opportunities for shortening skeletal structures without the need of osteotomies, thus taking advantage of the potential of craniofacial growth and remodeling.     

Seasonal Pattern of Tomato Mosaic Tobamovirus Infection and Concentration in Red Spruce Seedlings

Tomato mosaic tobamovirus (ToMV) infects red spruce (Picea rubens) and causes significant changes in its growth and physiology. The mechanism of infection and the pattern of virus concentration in seedling roots and needles were investigated. One-year-old red spruce seedlings were obtained from the nursery in April and June 1995 and August 1996 and tested for ToMV using enzyme-linked immunosorbent assay (ELISA). Virus-free seedlings were divided into three treatments: control, root inoculated, and needle inoculated. Two control, five root-inoculated, and five needle-inoculated seedlings were sampled destructively at biweekly intervals for 3 months and then tested for ToMV by ELISA. ToMV was transmitted to seedlings by root but not by needle inoculation. The virus was detected in 67 to 100% of roots but in less than 7% of needles of root-inoculated seedlings. The percent infection of root-inoculated seedlings differed significantly between the April and June and between the April and August inoculation periods. Virus concentration in infected seedling roots increased initially, peaked within 4 weeks postinoculation, and steadily declined thereafter. Significant differences in ToMV concentrations in roots also were detected among inoculation periods and sampling dates. Early spring may represent the optimal time for infection of seedlings, as well as for assaying roots for ToMV.     

Monitoring of Brain Spiking Activity and Autoantibodies to N-Terminus Domain of GluR1 Subunit of AMPA Receptors in Blood Serum of Rats with Cobalt-Induced Epilepsy

Abstract: We have monitored EEG spontaneous spiking activity and analyzed serum from rats with cobalt-induced epilepsy for the presence of autoreactive antibodies to α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) glutamate receptor subunits. The presence and the level of autoantibodies were assessed using immunoblot and ELISA with synthetic peptide specific to the N-terminus domain of the GluR1 subunit of the AMPA receptor. Rats with cobalt-induced epilepsy exhibited strong GluR1 immunoreactivity at the end of the first week after surgery compared with vehicle-treated rats. We showed that GluR1 autoantibodies in blood serum of rats with cobalt-induced epilepsy preceded the spiking activity maximum in the EEG. Levels of autoantibodies to GluR1 detected in blood of these rats remained elevated when EEG spiking activity was significantly reduced and seizures disappeared. The EEG monitoring of spiking activity showed a correlation with accumulation of GluR1 autoantibodies in blood serum of rats with cobalt-induced epilepsy.     

Calpain-3 Impairs Cell Proliferation and Stimulates Oxidative Stress-Mediated Cell Death in Melanoma Cells

Calpain-3 is an intracellular cysteine protease, belonging to Calpain superfamily and predominantly expressed in skeletal muscle. In human melanoma cell lines and biopsies, we previously identified two novel splicing variants (hMp78 and hMp84) of Calpain-3 gene (CAPN3), which have a significant lower expression in vertical growth phase melanomas and, even lower, in metastases, compared to benign nevi. In the present study, in order to investigate the pathophysiological role played by the longer Calpain-3 variant, hMp84, in melanoma cells, we over-expressed it in A375 and HT-144 cells. In A375 cells, the enforced expression of hMp84 induces p53 stabilization, and modulates the expression of a few p53- and oxidative stress-related genes. Consistently, hMp84 increases the intracellular production of ROS (Reactive Oxygen Species), which lead to oxidative modification of phospholipids (formation of F₂-isoprostanes) and DNA damage. Such events culminate in an adverse cell fate, as indicated by the decrease of cell proliferation and by cell death. To a different extent, either the antioxidant N-acetyl-cysteine or the p53 inhibitor, Pifithrin-α, recover cell viability and decrease ROS formation. Similarly to A375 cells, hMp84 over-expression causes inhibition of cell proliferation, cell death, and increase of both ROS levels and F₂-isoprostanes also in HT-144 cells. However, in these cells no p53 accumulation occurs. In both cell lines, no significant change of cell proliferation and cell damage is observed in cells over-expressing the mutant hMp84^C42S devoid of its enzymatic activity, suggesting that the catalytic activity of hMp84 is required for its detrimental effects. Since a more aggressive phenotype is expected to benefit from down-regulation of mechanisms impairing cell growth and survival, we envisage that Calpain-3 down-regulation can be regarded as a novel mechanism contributing to melanoma progression.     

Thrombospondin‐1 is part of a Slug‐independent motility and metastatic program in cutaneous melanoma,in association with VEGFR‐1 and FGF‐2

Differently from most transformed cells, cutaneous melanoma expresses the pleiotropic factor thrombospondin‐1 (TSP‐1). Herein, we show that TSP‐1 (RNA and protein), undetectable in four cultures of melanocytes and a RGP melanoma, was variously present in 13 cell lines from advanced melanomas or metastases. Moreover, microarray analysis of 55 human lesions showed higher TSP‐1 expression in primary melanomas and metastases than in common and dysplastic nevi. In a functional enrichment analysis, the expression of TSP‐1 correlated with motility‐related genes. Accordingly, TSP‐1 production was associated with melanoma cell motility in vitro and lung colonization potential in vivo. VEGF/VEGFR‐1 and FGF‐2, involved in melanoma progression, regulated TSP‐1 production. These factors were coexpressed with TSP‐1 and correlated negatively with Slug (SNAI2), a cell migration master gene implicated in melanoma metastasis. We conclude that TSP‐1 cooperates with FGF‐2 and VEGF/VEGFR‐1 in determining melanoma invasion and metastasis, as part of a Slug‐independent motility program.     

Hospital Admission of Cancer Patients: Avoidable Practice or Necessary Care?

Background

Cancer patients are frequently admitted to hospital due to acute conditions or refractory symptoms. This occurs through the emergency departments and requires medical oncologists to take an active role. The use of acute-care hospital increases in the last months of life.

Patients and methods

We aimed to describe the admissions to a medical oncology inpatient service within a 16-month period with respect to patients and tumor characteristics, and the outcome of the hospital stay.

Results

672 admissions of 454 patients were analysed. The majority of admissions were urgent (74.1%), and were due to uncontrolled symptoms (79.6%). Among the chief complaints, dyspnoea occurred in 15.7%, pain in 15.2%, and neurological symptoms in 14.5%. The majority of the hospitalizations resulted in discharge to home (60.6%); in 26.5% the patient died and in 11.0% was transferred to a hospice. Admissions due to symptoms correlated with a longer hospital stay and a higher incidence of in-hospital death.

Conclusion

We suggest that hospital use is not necessarily a sign of inappropriately aggressive care: inpatient care is probably an unavoidable step in the cancer trajectory. Optimization of inpatient supportive procedures should be a specific task of modern medical oncology.     

Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility,Gating Residues and Multiple Binding Pockets

The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.     

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.