Surgical Treatment of Multilevel Degenerative Cervical Myelopathy: Open-Door Laminoplasty and Fixation via Unilateral Approach. A Feasibility Study.

OBJECTIVE: We compared open-door laminoplasty via a unilateral approach and additional unilateral lateral mass screw fixation (uLP) with laminectomy and bilateral lateral mass screw fixation (LC) in the surgical treatment of multilevel degenerative cervical myelopathy (mDCM). METHODS: A retrospective cohort analysis of 46 prospectively enrolled patients (23 uLP and 23 LC). The minimum follow-up was 1 year. Neck and arm pains were evaluated with visual analog scales and disability with the Neck Disability Index (NDI). Myelopathy was rated with the modified Japanese Orthopaedic Association (mJOA) score. Cervical sagittal parameters were measured on plain and functional X-ray films with a specific software. The statistical significance was set at p < 0.05. Fusion was defined as <2 degrees of intersegmental motion on flexion/extension radiographs. RESULTS: The two groups were similar in age and comorbidities. The mean operation time and the mean hospital stay were shorter in the uLP group (p = 0.015). The intraoperative blood loss did not exceed 200 mL in both groups. At follow-up, the groups showed comparable clinical outcome data. The sagittal profile did not deteriorate in either group. Fusion rates were 67% in the uLP group and 92% in the LC group. No infections occurred in either group. In the LC group, one patient developed a transient C5 palsy. Revision surgery was required for a malpositioned screw (LC) and for one implant failure (uLP). CONCLUSION: Laminoplasty and unilateral fixation via a unilateral approach achieved comparable clinical and radiologic results with laminectomy and bilateral fixation, despite a lower fusion rate. However, the surgical traumatization was less.

Biochemical Analyses of Human Iron-Sulfur Protein Biogenesis and of Related Diseases.

Maturation of Fe/S proteins in mammals is an intricate process mediated by two assembly systems located in the mitochondrial and cytosolic-nuclear compartments. Malfunction particularly of the mitochondrial system gives rise to severe neurological, metabolic, or hematological disorders, often with fatal outcome. In this chapter, we describe approaches for the differential biochemical investigation of cellular Fe/S protein maturation in mitochondria, cytosol, and nucleus. The analyses may also facilitate the identification of the affected Fe/S protein assembly step in diseased state. As Fe/S cluster insertion into target apoproteins is a frequent determinant of protein stability, examination of protein steady-state levels in biological samples frequently permits reliable first clues about the maturation process. In some specific cases, this approach allows the assessment of enzymatic or regulatory functions of Fe/S proteins, including the formation of lipoate cofactor by mitochondrial lipoic acid synthase or the posttranscriptional regulation of transferrin receptor and ferritin expression by the cytosolic iron regulatory proteins. More direct Fe/S protein maturation assays like enzymatic analyses may further validate the observed maturation defects. Here, we present a simple protocol for the determination of dihydropyrimidine dehydrogenase enzyme activity by thin-layer chromatography. In order to directly monitor Fe/S cluster insertion into target apoproteins, we have developed a 55Fe radiolabeling technique tracing the in vivo Fe/S cofactor formation in mammalian tissue culture. The combination of the presented techniques represents a comprehensive strategy to assess the multiple facets of Fe/S protein assembly for both mechanistic analyses and for the elucidation of specific defects in Fe/S diseases.

pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs.

The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-ß-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins.IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced cellular proteins. The IFN-induced proteins of the IFITM family block IAV entry into target cells and can restrict viral spread and pathogenicity. Here we show for the first time that the sensitivity of IAVs to the IFN-induced antiviral state and IFITM2 and IFITM3 proteins depends on the pH value at which the viral HA undergoes a conformational transition and mediates membrane fusion. Our data imply that the high pH optimum of membrane fusion typical of zoonotic IAVs of gallinaceous poultry, such as H5N1 and H7N9, may contribute to their enhanced virulence in humans.