3D matrix‐embedding inhibits cycloheximide‐mediated sensitization to TNF‐alpha‐induced apoptosis of human endothelial cells

The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Others and we previously demonstrated endothelial biology being intertwined with biochemical and structural composition of the subendothelial basement membrane. We now demonstrate that a three‐dimensional growing environment significantly shields endothelial cells from cytokine‐induced apoptosis. Detailed analysis reveals differences in intracellular signaling pathways in naive endothelial cells and cytokine‐stimulated endothelial cells when cells are grown within a three‐dimensional collagen‐based matrix compared to cells grown on two‐dimensional tissue culture plates. Main findings are significantly reduced p53 expression and level of p38‐phosphorylation in three‐dimensional grown endothelial cells. Despite similar concentrations of focal adhesion kinase, three‐dimensional matrix‐embedded endothelial cells express significantly less tyrosine‐phosphorylated focal adhesion kinase. Pretreatment with antibodies against integrin α_vβ₃ partially reversed the protective effect of three‐dimensional matrix‐embedding on endothelial apoptosis. Our findings provide detailed insights into the mechanisms of endothelial apoptosis with respect to the spatial matrix environment. These results enhance our understanding of endothelial biology and may otherwise help in the design of tissue‐engineered materials. Furthermore, findings on focal adhesion kinase phosphorylation might enhance our understanding of clinical studies with tyrosine kinase inhibitors.     

Proteinase-activated receptor-2-mediated signaling and inhibition of DNA synthesis in human pancreatic cancer cells

Summary Conclusion Proteinase-activated receptor-2 (PAR-2)-mediated effects contribute to the intracellular signaling network in pancreatic tumor cells. A role of PAR-2 as negative regulator in human pancreatic tumor growth might be implied. Background Using the human pancreatic tumor cell line MIA PaCa-2, we evaluated cellular effects of trypsin and the PAR-2-activating peptide SLIGRL on [Ca²⁺]_i mobilization, Ins(1,4,5)P₃ level, and protein kinase (PKC) activation. Furthermore, PAR-2 involvement in the regulation of cell proliferation has been estimated by measurement of [³H]thymidine incorporation in MIA PaCa-2 cells. Results Trypsin and the PAR-2 synthetic peptide agonist SLIGRL induced [Ca²⁺]_i mobilization, transient increase in inositol (1,4,5) triphosphate level, and PKC translocation in MIA PaCa-2 cells. In addition, SLIGRL induced a decrease in DNA synthesis in MIA PaCa-2 cells.     

Robotic-assisted total mesorectal excision (TME) for rectal cancer results in a significantly higher quality of TME specimen compared to the laparoscopic approach—report of a single-center experience

Aim

Robotic surgery allows for a better visualization and more precise dissection especially in the narrow male pelvis and mid and lower third of the rectum. However, superiority to laparoscopic TME has yet to be proven. We therefore analyzed short-term outcomes of laparoscopic and robotic low anterior rectal resection for rectal cancer.

Patients and methods

From 2011 to 2016, 44 robotic (RTME) and 41 laparoscopic (LTME) low anterior rectal resection with total mesorectal excision were performed at a single institution. Specimen quality was assessed and reported by an independent pathologist following international guidelines.

Results

The groups did not differ significantly regarding gender, age, ASA stage, BMI, and distance of the lower tumor margin from the anal verge. More patients in the RTME group underwent preoperative chemoradiation (43.2 vs. 19.5%, p?=?0.019). The quality of the TME specimen was significantly better in the RTME group (complete/nearly complete/incomplete for RTME 97/0/3% and for LTME 78/17/5%, p?=?0.03). The conversion rate tended to be lower in the RTME group (7 vs. 17%, p?=?0.143). There was no difference in CRM positivity between the groups.

Conclusion

Robotic surgery is safe and can improve the quality of TME for rectal cancer compared to laparoscopy. Any effect on long-term survival remains to be established.

     

Disruption of EXOC6B in a patient with developmental delay,epilepsy, and a de novo balanced t(2;8) translocation

Most balanced chromosomal aberrations are not associated with a clinical phenotype, however, in some patients they may disrupt gene structure. With the development of various next-generation sequencing techniques, fast and specific analyses of the breakpoint regions of chromosomal rearrangements are possible. Here, we report on a 19-year-old woman with a de novo balanced translocation t(2;8)(p13.2;q22.1) and a severe clinical phenotype including intellectual disability, epilepsy, behavioral features resembling autism, and minor dysmorphic features. By next-generation sequencing, we defined the breakpoints and found disruption of the exocyst complex component 6B (EXOC6B) gene in intron 1 on chromosome 2p13.2 involving two Alu elements with a homology of 81%. No gene was found at the respective breakpoint on chromosome 8. Expression analysis of the EXOC6B in blood lymphocytes and buccal smear revealed reduced expression in the patient in comparison with the control. Our findings in combination with one recently published case and one other patient listed in DECIPHER v5.1 indicate EXOC6B as a gene relevant for intellectual development and electrophysiological stability.     

Early venous manifestation of Ehlers–Danlos syndrome Type IV through a novel mutation in COL3A1

Ehlers–Danlos syndrome (EDS) leads to abnormalities in the synthesis of collagen and complications involving arterial vessels. We describe here a mutation in the intron 14 of the COL3A1 gene leading to EDS Type IV (EDS IV) associated with venous manifestations only. The patient, an 18-year-old male, suffered from truncal varicosity of the long saphenous vein on both sides. Conventional stripping surgery of the left saphenous vein revealed an extremely vulnerable ectatic superficial femoral vein. An inserted vein graft occluded, and venous thrombectomy was unsuccessful. A conservative anticoagulant and compression therapy finally succeeded. This is the first report describing EDS IV due to a mutation in intron 14 of the COL3A1 gene leading to venous manifestations without affecting arterial vessels at clinical presentation. Our findings imply that molecular genetic analysis should be considered in patients with unusual clinical presentation and that conservative therapy should be applied until a suspected clinical diagnosis has been secured.     

Sex-specific Differences of the Infraacetabular Corridor: A Biomorphometric CT-based Analysis on a Database of 523 Pelves

BackgroundAn infraacetabular screw path facilitates the closure of a periacetabular fixation frame to increase the plate fixation strength in acetabular fractures up to 50%. Knowledge of the variance in corridor sizes and axes has substantial surgical relevance for safe screw placement.Questions/purposes(1) What proportion of healthy pelvis specimens have an infraacetabular corridor that is 5 mm or larger in diameter? (2) Does a universal corridor axis and specific screw entry point exist? (3) Are there sex-specific differences in the infraacetabular corridor size or axis and are these correlated with anthropometric parameters like age, body weight and height, or the acetabular diameter?MethodsA template pelvis with a mean shape from 523 segmented pelvis specimens was generated using a CT-based advanced image analyzing system. Each individual pelvis was registered to the template using a free-form registration algorithm. Feasible surface regions for the entry and exit points of the infraacetabular corridor were marked on the template and automatically mapped to the individual samples to perform a measurement of the maximum sizes and axes of the infraacetabular corridor on each specimen. A minimum corridor diameter of at least 5 mm was defined as a cutoff for placing a 3.5-mm cortical screw in clinical settings.ResultsIn 484 of 523 pelves (93%), an infraacetabular corridor with a diameter of at least 5 mm was found. Using the mean axis angulations (54.8° [95% confidence interval {CI}, 0.6] from anterocranial to posterocaudal in relation to the anterior pelvic plane and 1.5° [95% CI, 0.4] from anteromedial to posterolateral in relation to the sagittal midline plane), a sufficient osseous corridor was present in 64% of pelves. Allowing adjustment of the three-dimensional axis by another 5° included an additional 25% of pelves. All corridor parameters were different between females and males (corridor diameter, 6.9 [95% CI, 0.2] versus 7.7 [95% CI, 0.2] mm; p < 0.001; corridor length, 96.2 [95% CI, 0.7] versus 106.4 [95% CI, 0.6] mm; p < 0.001; anterior pelvic plane angle, 54.0° [95% CI, 0.9] versus 55.3° [95% CI, 0.8]; p < 0.01; sagittal midline plane angle, 4.3° [95% CI, 0.6] versus −0.3° [95% CI, 0.5]; p < 0.001).ConclusionThis study provided reference values for placement of a 3.5-mm cortical screw in the infraacetabular osseous corridor in 90% of female and 94% of male pelves. Based on the sex-related differences in corridor axes, the mean screw trajectory is approximately parallel to the sagittal midline plane in males but has to be tilted from medial to lateral in females. Considering the narrow corridor diameters, we suggest an individual preoperative CT scan analysis for fine adjustments in each patient.     

The Parkinson’s disease-associated gene ITPKB protects against α-synuclein aggregation by regulating ER-to-mitochondria calcium release

Inositol-1,4,5-triphosphate (IP₃) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP₃, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson’s disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by a variety of motor symptoms (including unbalanced gait, resting tremor, and bradykinesia) that are accompanied by psychosis and dementia at later stages of disease. The onset of motor symptoms is largely caused by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the corresponding depletion of dopamine innervation in the striatum. Although the cause of neuron loss is unknown, the hallmark pathological feature of PD is the presence of intraneuronal inclusions composed of misfolded and fibrillar α-synuclein (α-syn) in the neurites and soma, termed Lewy neurites and Lewy bodies, respectively (1). Protein-coding single-nucleotide polymorphisms (SNPs), duplications, and triplications in the gene encoding α-syn (SNCA) all cause early-onset, familial forms of PD and result in accelerated aggregation of α-syn protein into insoluble, fibrillar aggregates (1, 2). Recent evidence suggests that these aggregates can spread from cell to cell, leading to the propagation of pathology to neuroanatomically connected brain regions (3, 4). Therefore, therapeutic approaches that reduce the aggregation or spreading of pathological α-syn species represent potential disease-modifying therapies for PD.In addition to SNCA, mutations in several other genes have been identified that cause rare, familial forms of PD. These genes are primarily involved in the autophagic clearance of intracellular aggregates or damaged organelles, especially mitochondria (5–8). Genome-wide association studies (GWAS) have also identified common SNPs in several genes related to endolysosomal function, such as GBA and LRRK2, that are associated with increased risk of PD (8–11). GBA and LRRK2 mutations have been shown to affect lysosomal and mitochondrial phenotypes (12–14), which contribute to the accumulation of PD-like neuropathology in mouse models, primary neurons, and human iPSC-derived cells (15–18). These findings highlight the role of the lysosomal and mitochondria quality control pathways in PD and demonstrate that perturbations in these pathways are sufficient to increase α-syn aggregation. Despite this, the etiology of sporadic PD is not fully understood, and the specific genes and pathways that are tractable for therapeutic modulation remain elusive. Therefore, the discovery of new genes associated with sporadic PD may be critical for both understanding disease pathogenesis and identifying novel therapeutic approaches.Recently, Chang et al. conducted a GWAS analysis that identified 17 novel gene loci significantly associated with sporadic PD in a European population including more than 26,000 patients across three independent cohorts (19). The lead GWAS SNP in the novel 1q42 locus was rs4653767. This is an intronic SNP in the gene encoding inositol-1,4,5-triphosphate kinase B (ITPKB) and produces a thymine-to-cytosine nucleotide substitution that is protective against developing PD (odds ratio [OR] = 0.92, P = 2.4 × 10⁻¹⁰). A follow up meta-analysis study of 37,688 PD patients, which included the discovery cohort, and 18,618 proxy cases strengthened the GWAS finding at this locus (OR = 0.92, P = 1.4 × 10⁻¹⁵). Furthermore, this locus was still significant when the analysis was performed on only the new independent cases and proxy cases (OR = 0.92, P = 2.8 × 10⁻⁵) (20). The rs4653767-C allele is present in similar frequencies across populations (27% in non-Finnish European and 29% in East Asian populations) and was found to have the same direction of effect (OR = 0.87, P = 0.016) in a targeted replication study of the European PD loci in an East Asian cohort (21). ITPKB is also highly expressed in several brain regions related to PD, including the SNpc, striatum, and cerebral cortex (22).ITPKB is one of three ubiquitously expressed kinases known to phosphorylate inositol-1,4,5-triphosphate (IP₃), an intracellular messenger produced from phosphatidylinositol-4,5-bisphosphate by phospholipase C (23, 24). IP₃ binds to IP₃ receptors (IP₃Rs) in the endoplasmic reticulum (ER) to stimulate the release of calcium ions from the ER into the cytosol to mediate various downstream signaling pathways. IP₃ kinases (ITPKA, ITPKB, and ITPKC) add a fourth phosphate group to IP₃ producing inositol-1,3,4,5-tetrakisphosphate (IP₄), which has no activity on IP₃Rs. Thus, IP₃ kinases negatively regulate IP₃-mediated calcium release from the ER. While the role of this pathway in peripheral cell types under normal physiological conditions is well understood (25, 26), whether ITPKB is involved in the pathogenesis of PD is unknown. Here, we investigate whether the modulation of ITPKB expression or kinase activity impacts the accumulation of α-syn pathology in cellular models of PD.     

Use of cardiac magnetic resonance to assess viability

The accurate differentiation of viable and nonviable myocardium is crucial for therapy planning in patients with coronary artery disease and left ventricular dysfunction. Traditional techniques such as echocardiography, positron emission tomography, single photon emission computed tomography, and dobutamine echocardiography have established roles. Cardiac MRI (CMR) is a rapidly emerging new modality that is used at an increasing number of medical centers in Europe and the United States. This review describes the role of CMR for the assessment of myocardial viability in the setting of acute and chronic ischemic ventricular dysfunction.