Soft Landing technique as a possible prevention strategy for proximal junctional failure following adult spinal deformity surgery

BackgroundThis cross sectional study describes a "Soft Landing" strategy utilizing hooks for minimizing proximal junctional kyphosis (PJK) and proximal junctional failure (PJF). The technique creates a gradual transition from a rigid segmental construct to unilateral hooks at the upper instrumented level and preservation of the soft tissue attachments on the contralateral side of the hooks. Authors devise a novel classification system for better grading of PJK severity.MethodsThirty-nine consecutive adult spinal deformity (ASD) patients at a single institution received the "Soft Landing" technique. The proximal junctional angle was measured preoperatively and at last follow-up using standing 36-inch spinal radiographs. Changes in proximal junctional angle and rates of PJK and PJF were measured and used to create a novel classification system for evaluating and categorizing ASD patients postoperatively.ResultsThe mean age of the cohort was 61.4 years, and 90% of patients were women. Average follow up was 2.2 years. The mean change in proximal junctional angle was 8° (SD 7.4°) with the majority of patients (53%) experiencing less than 10° and only 1 patients with proximal junctional angle over 20°. Four patients (10%) needed additional surgery for proximal extension of the uppermost instrumented vertebra (UIV) secondary to PJF.ConclusionsSoft Landing technique is a possibly effective treatment strategy to prevent PJK and PJF following ASD that requires further evaluation. The described classification system provides management framework for better grading of PJK. The "Soft Landing" technique warrants further comparison to other techniques currently used to prevent both PJK and failure

<i>In Vivo</i> Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency

<div><p>Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in <i>SBDS</i> are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15^Ink4b and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to be p53-dependent. Our findings therefore point to cell/tissue-specific responses to p53-activation that include distinction between apoptosis and senescence pathways, in the context of translation disruption.</p></div

SDS brain is apoptotic.

<p>Histochemistry of transverse rhombencephalon (E11.5; <b>A</b>) and telencephalon (E14.5; <b>B</b>) brain sections indicate hypocellularity and neuronal cell death (green in TUNEL panels) in post-mitotic regions of <i>Sbds</i>^{<i>R126T/R126T</i>} mice with earlier onset in the <i>Sbds</i>^{<i>R126T/–</i>}mice (compare TUNEL panels in <b>A</b> and <b>B</b>). Bromodeoxyuridine labeling (brown in BrdU panels) highlighted reduced proliferation of neural progenitors. V, lateral ventricle; VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars represent 25 μm.</p

Tgfβ and p53 response in the SDS pancreas.

<p><b>A,</b> Steady state protein levels paralleled observed transcript changes with decreased Myc and increased p21^Cip and Tgfβ expression, along with changes in Smad2 and Smad3 phosphorylation status in mutants. Representative immunoblots of lysates from four littermate pairs at 3 weeks of age are shown. Associated densitometry is shown in right graphs, with Myc, Tgfβ, and p21 relative to Gapdh expression, and phosphorylated-Smad2 and Smad3 relative to total Smad2 and Smad3, respectively. Horizontal lines in scatter plots indicate mean values. <b>B,</b> Representative immunoblot indicates stabilization of p53 protein in the SDS pancreas at 3 weeks of age with associated densitometry (expression relative to calnexin) below. Horizontal lines indicate mean values. <b>C,</b> Immunohistochemistry indicated p53 stabilization as early as 15 days of age (sections shown are of littermates). Yellow arrows highlight examples of positive nuclei. p53 staining was observed specifically in nuclei of acinar cells of the SDS pancreas model (islets denoted with pale yellow dashed outlines). Scale bars represent 50 μm.</p

Senescence-associated markers in the SDS pancreas.

<p><b>A,</b> Senescence-associated β-galactosidase activity (SA-bgal, bright blue) was detected in acini of the SDS pancreas at 30 days of age (N = 5). <i>Sbds</i>^{<i>P–/+</i>} and <i>Sbds</i>^{<i>P–/R126T</i>} are shorthand for <i>Sbds</i>^<i>CKO/+</i>; <i>Ptf1a</i>^<i>Cre/+</i> and <i>Sbds</i>^{<i>CKO/R126T</i>}; <i>Ptf1a</i>^<i>Cre/+</i>, respectively [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005288#pgen.1005288.ref051" target="_blank">51</a>]. Scale bars represent 100 μm. <b>B,</b> 84 cellular-senescence associated genes were assayed using the SABiosciences Cellular Senescence RT² Profiler PCR Array (QIAGEN) with total RNA isolated from pancreata of mice at 15 and 25 days of age. Table lists transcripts that showed statistically significant changes relative to control genes at at least one of the two assayed time points (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005288#pgen.1005288.s010" target="_blank">S2 Table</a>). Fold change: <i>Sbds</i>^{<i>P-/R126T</i>}/<i>Sbds</i>^<i>P-/+</i>. Criteria for significance (as per supplier’s instructions): ≥3 fold difference with a <i>P</i>-value of <0.05, N = 3 at each time point. <b>C</b> and <b>D,</b> Quantitative transcript analysis. In <b>C</b>, fold change: <i>Sbds</i>^{<i>P-/R126T</i>}/<i>Sbds</i>^<i>P-/+</i>; N = 4 at each time point, except at E18.5 where N = 3. Criteria for significance: ≥2 fold change, <i>P</i><0.05. E18.5 pancreas expression is relative to Tbp; P8-P25 expression is relative to Gapdh. In <b>D</b>, fold change: <i>Sbds</i>^{<i>R126T/R126T</i>}/<i>Sbds</i>^{<i>R126T/+</i>}; N = 4. Criteria for significance: ≥2 fold change, <i>P</i><0.05. Brain and liver expression is relative to Actb; bone expression is relative to Tbp. All <i>P</i>-values calculated using unpaired, two-tailed T-tests. Red indicates down-regulation, blue indicates up-regulation. Abbreviations in <b>B</b>: <i>Akt1</i>: <i>Thymoma viral proto-oncogene 1; Cd44</i>: <i>CD44 antigen; Cdkn1a</i>: <i>Cyclin-dependent kinase inhibitor 1A; Ckdn2b</i>: <i>Cyclin-dependent kinase inhibitor 2B; Col1a1</i>: <i>Collagen</i>, <i>type I</i>, <i>alpha 1; Creg1</i>: <i>Cellular repressor of E1A-stimulated genes 1; Egr1</i>: <i>Early growth response 1; Ets1</i>: <i>E26 avian leukemia oncogene 1</i>, <i>5’ domain; Fn1</i>: <i>Fibronectin 1; Hras1</i>: <i>Harvey rat sarcoma virus oncogene 1; Ifng</i>: <i>Interferon gamma; Igfbp5</i>: <i>Insulin-like growth factor binding protein 5; Igfbp7</i>: <i>Insulin-like growth factor binding protein 7; Interferon regulatory factor 3; Irf5</i>: <i>Interferon regulatory factor 5; Irf7</i>: <i>Interferon regulatory factor 7; Myc</i>: <i>Myelocytomatosis oncogene; Nfkb1</i>: <i>Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1</i>, <i>p105; Sparc</i>: <i>Secreted acidic cysteine rich glycoprotein (osteonectin; Tbx3</i>: <i>T-box-3; Tgfb1</i>: <i>Transforming growth factor</i>, <i>beta 1; Tgfb1i1</i>: <i>Transforming growth factor beta 1 induced transcript 1</i>.</p

Sbds mutants display ribosomopathy and SDS phenotypes.

<p><b>A,</b> Embryos with biallelic mutations in <i>Sbds</i> have decreased mass compared with littermate controls, **<i>P</i><4X10^-6. <i>Sbds</i>^{<i>R126T/–</i>}embryos are smaller than <i>Sbds</i>^{<i>R126T/R126T</i>} embryos, *<i>P</i> = 1.9X10^-4 (Wilcoxon Rank Sum Test; Kruskal-Wallis <i>P</i> = 3.0X10^-8). Error bars represent ±SEM. Scale bar represents 5 mm (upper panel). <b>B,</b> Decreased granulocytes (dark purple, H&E, E18.5) in liver (cell cluster examples are indicated with yellow arrowheads) and bone marrow (black arrowheads) with loss of <i>Sbds</i>; N = 3 (<i>Sbds</i>^{<i>R126T/R126T</i>}) and 4 (<i>Sbds</i>^{<i>R126T/–</i>}). Scale bars represent 100 μm. <b>C,</b> Decreased bone ossification was observed in transverse metacarpal sections of mutants (corresponding regions of littermate controls that maintain red Safranin O staining in mutants are highlighted with magenta arrowheads, E18.5). Scale bars represent 100 μm.</p

Atrophic SDS pancreas phenotype is p53-dependent.

<p><b>A,</b> Improved mass of SDS pancreas with loss of p53 (<i>Sbds</i>^{<i>P–/R126T</i>}; <i>Trp53</i>^{<i>–/–</i>}; 30 days of age); *<i>P</i> = 0.0025, **<i>P</i><4X10^-5, Wilcoxon Rank Sum Test (Kruskal-Wallis Test <i>P</i> = 3.2X10^-5). Inset numbers = N. Error bars represent ±SEM. <b>B,</b> Resolution of atrophy and hypocellularity as well as fat infiltration with loss of p53 in SDS pancreas at 60 days of age (H&E). However, multiple apoptotic acinar cells (TUNEL) per field of view and increased expression of dedifferentiation markers in acini (examples of Hes1 and Pdx1 positive cells, yellow arrowheads) highlighted dysplasia. I: islets. Scale bar represents 100 μm. <b>C,</b> Improvement in digestive enzyme expression and abrogation of senescence-related changes in Tgfβ/p15^Ink4b and Myc expression in double mutants at 25 days of age. Fold changes correspond to the comparison of <i>Sbds</i>^{<i>P–/R126T</i>};<i>Trp5</i>^<i>+/–</i>to <i>Sbds</i>^{<i>P–/+</i>};<i>Trp53</i>^<i>+/–</i>or <i>Sbds</i>^{<i>P–/R126T</i>};<i>Trp53</i>^{<i>–/–</i>}to <i>Sbds</i>^{<i>P–/+</i>};<i>Trp53</i>^{<i>–/–</i>}transcript levels. <i>P</i>-values calculated using unpaired, two-sided T-tests.</p

SDS pancreas senescence is downstream of p53-dependent changes in protein synthesis.

<p><b>A,</b> Increased nuclei per acinar area (*<i>P</i> = 0.029, Wilcoxon Rank Sum Test) and <b>B,</b> decreased mean acinus diameter (**<i>P</i> = 0.0073 and <i>NS</i> = not significant, <i>P</i> = 0.142; unpaired, two-sided T-test) in <i>Sbds</i>^{<i>P–/R126T</i>}; <i>Trp53</i>^{<i>–/–</i>}pancreas at 30 days of age. Error bars represent ±SEM; in boxplots, whiskers represent extreme values; circles, outliers. <b>C,</b> Coomassie and silver staining of pancreas lysate SDS-PAGE illustrated p53-dependent altered protein expression in the SDS pancreas (20 days of age, 6 μg total protein loaded). <b>D,</b> Immunoblotting confirmed that digestive enzymes were reduced in expression in the SDS pancreas, with expression of protease carboxypeptidase (Cpa1) increasing in a <i>Trp53</i>^{<i>–/–</i>}genetic background (3 weeks of age, 25 μg total protein loaded). Representative blots are shown. Associated densitometry, with expression relative to Gapdh, is shown in lower panels, <i>Sbds</i>^{<i>P–/+</i>} black, <i>Sbds</i>^{<i>P–/R126T</i>} grey, horizontal lines indicate mean values. <b>E,</b> Restoration of zymogen granules (example, white arrowhead) with loss of p53 was observed by one week of age (electron micrographs). Scale bar represents 5 μm. <b>F,</b> Representative polysome traces illustrate restoration of 80S peak in mutants to levels similar to those of controls with loss of p53 (20 days of age, 79 μg RNA loaded, N = 4 (<i>Trp53</i>^<i>+/–</i>) and 3(<i>Trp53</i>^{<i>–/–</i>})). P: polysomes.</p

Shwachman-Bodian Diamond syndrome is a multi-functional protein implicated in cellular stress responses

Shwachman-Diamond syndrome (SDS; OMIM 260400) results from loss-of-function mutations in the Shwachman-Bodian Diamond syndrome (SBDS) gene. It is a multi-system disorder with clinical features of exocrine pancreatic dysfunction, skeletal abnormalities, bone marrow failure and predisposition to leukemic transformation. Although the cellular functions of SBDS are still unclear, its yeast ortholog has been implicated in ribosome biogenesis. Using affinity capture and mass spectrometry, we have developed an SBDS-interactome and report SBDS binding partners with diverse molecular functions, notably components of the large ribosomal subunit and proteins involved in DNA metabolism. Reciprocal co-immunoprecipitation confirmed the interaction of SBDS with the large ribosomal subunit protein RPL4 and with DNA-PK and RPA70, two proteins with critical roles in DNA repair. Function for SBDS in response to cellular stresses was implicated by demonstrating that SBDS-depleted HEK293 cells are hypersensitive to multiple types of DNA damage as well as chemically induced endoplasmic reticulum stress. Furthermore, using multiple routes to impair translation and mimic the effect of SBDS-depletion, we show that SBDS-dependent hypersensitivity of HEK293 cells to UV irradiation can be distinguished from a role of SBDS in translation. These results indicate functions of SBDS beyond ribosome biogenesis and may provide insight into the poorly understood cancer predisposition of SDS patients