Postmortem genetic testing should be recommended in sudden cardiac death cases due to thoracic aortic dissection

Background

Acute thoracic aortic dissections and ruptures, the main life-threatening complications of the corresponding aneurysms, are an important cause of sudden cardiac death. Despite the usefulness of the molecular diagnosis of these conditions in the clinical setting, the corresponding forensic field remains largely unexplored. The main goal of this study was to explore and validate a new massive parallel sequencing candidate gene​ assay as a diagnostic tool for acute thoracic aortic dissection autopsy cases.

Materials and methods

Massive parallel sequencing of 22 thoracic aortic disease candidate genes performed in 17 cases of thoracic aortic dissection using AmpliSeq and Ion Proton technologies. Genetic variants were filtered by location, type, and frequency at the Exome Aggregation Consortium and an internal database and further classified based on the American College of Medical Genetics and Genomics (ACMG) recommendations published in 2015. All prioritized results were confirmed by traditional sequencing.

Results

From the total of 10 potentially pathogenic genetic variants identified in 7 out of the 17 initial samples, 2 of them were further classified as pathogenic, 2 as likely pathogenic, 1 as possibly benign, and the remaining 5 as variants of uncertain significance, reaching a molecular autopsy yield of 23%, approximately.

Conclusions

This massive parallel sequencing candidate gene approach proved useful for the molecular autopsy of aortic dissection sudden cardiac death cases and should therefore be progressively incorporated into the forensic field, being especially beneficial for the anticipated diagnosis and risk stratification of any other family member at risk of developing the same condition.

     

The Simpson grading in meningioma surgery: does the tumor location influence the prognostic value?

Journal of Neuro-Oncology - In meningiomas, location-specific differences of the prognostic value of the Simpson classification are sparsely investigated but can influence strategy of surgery. We...     

Interleukins as new prognostic genetic biomarkers in non-small cell lung cancer

Background

Surgery is the standard treatment for early-stage NSCLC, and platinum-based chemotherapy remains as the treatment of choice for advanced-stage NSCLC patients with naïve EGFR status. However, overall 5-years relative survival rates are low. Interleukins (ILs) are crucial for processes associated with tumor development. In NSCLC, IL1B, IL6, IL12A, IL13 and IL16 gene polymorphisms may contribute to individual variation in terms of patient survival. The purpose of this study was to evaluate the association between IL gene polymorphisms and survival in NSCLC patients.

A pilot study of the association between cariogenic oral bacteria and preterm birth*

Objectives: We examined the associations between preterm birth and low birth weight and maternal caries history, maternal periodontal status, and salivary levels of mutans streptococci and Lactobacilli. Design: This study was a matched case–control study in women during their pregnancy or up to 8 weeks after delivery. Subjects and methods: Thirty‐four women delivering before 37 weeks gestation were recruited along with 73 term controls matched for age and race/ethnicity. Demographic and obstetric information was collected from questionnaires and medical records and oral examinations along with commercial salivary tests were completed within the study groups. Main outcome measures: The main outcome variables were the preterm birth and low birth weight status. The independent variables measured were the salivary levels of Lactobacilli and mutans streptococci and the caries and periodontal status of the subjects. Results: The odds ratio comparing low levels of bacteria in preterm mothers and controls was statistically significant for Lactobacilli (odds ratio (OR) = 3.45, 95% confidence interval (CI) = 1.27 to 10.00) and almost significant for mutans streptococci (OR = 2.63, 95% CI = 0.95 to 8.33). Clinical caries and periodontal disease measures did not differ significantly between groups. Conclusion: Within the limitation of our study, low levels of Lactobacilli in saliva were found to be associated with preterm birth.     

Folding and misfolding of potassium channel monomers during assembly and tetramerization

The dynamics and folding of potassium channel pore domain monomers are connected to the kinetics of tetramer assembly. In all-atom molecular dynamics simulations of Kv1.2 and KcsA channels, monomers adopt multiple nonnative conformations while the three helices remain folded. Consistent with this picture, NMR studies also find the monomers to be dynamic and structurally heterogeneous. However, a KcsA construct with a disulfide bridge engineered between the two transmembrane helices has an NMR spectrum with well-dispersed peaks, suggesting that the monomer can be locked into a native-like conformation that is similar to that observed in the folded tetramer. During tetramerization, fluoresence resonance energy transfer (FRET) data indicate that monomers rapidly oligomerize upon insertion into liposomes, likely forming a protein-dense region. Folding within this region occurs along separate fast and slow routes, with τ_fold ∼40 and 1,500 s, respectively. In contrast, constructs bearing the disulfide bond mainly fold via the faster pathway, suggesting that maintaining the transmembrane helices in their native orientation reduces misfolding. Interestingly, folding is concentration independent despite the tetrameric nature of the channel, indicating that the rate-limiting step is unimolecular and occurs after monomer association in the protein-dense region. We propose that the rapid formation of protein-dense regions may help with the assembly of multimeric membrane proteins by bringing together the nascent components prior to assembly. Finally, despite its name, the addition of KcsA’s C-terminal “tetramerization” domain does not hasten the kinetics of tetramerization.

Many homo- and hetero-tetrameric ion channel monomers contain two transmembrane (TM) helices connected by a re-entrant pore loop segment (p) and adopt a modular structure with a “TM-p-TM” topology (Fig. 1) (1). The ion permeation pathway is created by the juxtaposition of the p-loop of each monomer along the tetramer’s symmetry axis. The highly conserved p-loop contains a short 3.5-turn helix and the ion selectivity filter (1, 2). Mutations in this region are detrimental to potassium channel trafficking (3) and are associated with diseases such as weaver (4–6) and long-QT syndrome (3, 7, 8) (the QT interval is the time from the start of the Q wave to the end of the T wave).Open in a separate windowFig. 1.KcsA TM Domain [Protein Data Bank (PDB) ID 3EFF]. (A and B) Top, side views. (C) Monomer: TM helices TM1, TM2, pore helix, turret, and selectivity filter. (D) Surface residues (polar, basic, and acidic residues are colored green, red, and blue, respectively).The folding of helical membrane proteins is thought to proceed in a two-stage manner, with insertion of stable TM α-helices followed by lateral packing within the bilayer (9–12). A more intricate three-stage model has also been proposed, where insertion and lateral packing of α-helices is followed by folding of loops, ligand binding, and insertion of peripheral domains (10). The folding of multimeric membrane proteins also requires the assembly of independent subunits, “building blocks,” that may need to be at least partly folded before oligomerization. This raises the question: How folded are the ion channel’s subunits prior to tetramerization? In vivo, monomers are independently synthesized and inserted into the lipid bilayer from the ribosome–translocon complex. Then, for a K⁺ channel, four monomeric subunits associate to form a functional tetrameric channel (13). The tetramerization process may be assisted by cytosolic tetramerization domains that could help the monomers fold or assist in their association (13, 14). However, prior studies have also shown that the pore domain can form tetramers on its own (13, 15–18) (Fig. 1 A and B).Several biophysical properties of K⁺ channels are relevant to their dynamics. The ion-conducting pore of K⁺ channels, formed by the re-entrant segment located between the two hydrophobic TM helices (termed TM1 and TM2 in KcsA and S5 and S6 in Kv1.3), is lined with polar and charged residues (Fig. 1D) (19). If the helices retain the same structure and orientation as they do in the tetramer, the polar residues along with the non–hydrogen-bonded backbone atoms would be exposed to lipids in the channel’s monomeric form. As these interactions are energetically unfavorable, the dominant conformation of individual monomers prior to tetramerization is unclear. Previous thiol-labeling experiments have shown that for Kv1.3, the monomers maintain the helical structure of the re-entrant pore helix in a native-like orientation at the water–lipid interface (20, 21). Likewise, previous molecular dynamics (MD) simulations indicated that the monomers’ native conformation (i.e., the conformation observed in the assembled tetramer) was surprisingly stable on a timescale of ∼1 μs (21).In this paper, the dynamics of KcsA and Kv1.2 pore domain monomers are first investigated using MD and NMR. Then, we connect their dynamics to the kinetics of tetramerization using a gel-based refolding assay and fluoresence resonance energy transfer (FRET) on KcsA. The monomers are seen to associate in the bilayer prior to tetramerization, suggesting that the liposome provides a poor solvent environment for KcsA’s TM helices. A comparison between wild-type (WT) KcsA and an enhanced folding construct containing a disulfide bond linking TM1 and TM2 (A29C-A109C) indicates that the arrangement of the TM helices can influence the kinetics of folding and misfolding. Finally, the lack of concentration dependence in folding kinetics points to a late rate-limiting step occurring after monomer association.     

Inhibition of LPS toxicity by hepatic argininosuccinate synthase (ASS): novel roles for ASS in innate immune responses to bacterial infection

Lipopolysaccharide (LPS), a structural component of Gram-negative bacteria, is implicated in the pathogenesis of endotoxemia/sepsis and multi-organ injury, including liver damage. We have shown that argininosuccinate synthase (ASS), a hepatic enzyme of the urea cycle, accumulates in circulation within 1h after treatment with both LPS alone and hepatotoxic combination of LPS and D-Galactosamine. These findings indicate ASS as a sensitive biomarker of liver responses to bacterial endotoxin. Furthermore, we suggest that the ASS release represents a potential counteracting liver reaction to LPS, and demonstrates anti-LPS activity of recombinant ASS (rASS) in vitro and in rodent models of endotoxemia in vivo. rASS physically bound to LPS, as indicated by a gel shift assay, and suppressed Escherichia coli growth in cultures consistent with direct antimicrobial properties of ASS. rASS reduced LPS cytotoxicity, TNF-α production, and increased cell viability in cultured mouse macrophages, even when added one hour following LPS challenge. Intraperitoneal injection of rASS (5 mg/kg) after treatment with a high dose of LPS remarkably increased survival of rodents, with a concomitant decrease of sepsis markers TNF-α, C-reactive protein (CRP), and lactate dehydrogenase (LDH) levels in blood. These results suggest that the endogenous ASS constitutes a novel liver-derived component of the innate immune response to bacterial LPS, and that recombinant ASS could mitigate the lethal effects of bacterial endotoxins during sepsis.     

Detection of circulating tumor cells as a prognostic factor in patients undergoing radical surgery for non-small-cell lung carcinoma: comparison of the efficacy of the CellSearch Assay™ and the isolation by size of epithelial tumor cell method

Comparison of the efficacy of different enrichment methods for detection of circulating tumor cells (CTCs) before radical surgery is lacking in non‐small‐cell lung carcinoma (NSCLC) patients. Detection and enumeration of CTCs in 210 consecutive patients undergoing radical surgery for NSCLC were evaluated with the CellSearch Assay? (CS), using the CellSearch Epithelial Cell Kit, and by the isolation by size of epithelial tumor (ISET) method, using double immunolabeling with anti‐cytokeratin and anti‐vimentin antibodies. CTCs were detected in 144 of 210 (69%) patients using CS and/or ISET and in 104 of 210 (50%) and 82 of 210 (39%) patients using ISET and CS, respectively. Using ISET, 23 of 210 (11%) patients had vimentin‐positive cells with cytological criteria of malignancy. Disease‐free survival (DFS) was worse for patients with CTCs compared to patients without CTCs detected by CS alone (p < 0.0001; log rank = 30.59) or by ISET alone (p < 0.0001; log rank = 33.07). The presence of CTCs detected by both CS and ISET correlated even better with shorter DFS at a univariate (p < 0.0001; log rank = 42.15) and multivariate level (HR, 1.235; 95% CI, 1.056–1.482; p < 0.001). CS and ISET are complementary methods for detection of CTCs in preoperative radical surgery for NSCLC. CTC detection in resectable NSCLC patients using CS and/or ISET could be a prognostic biomarker of great interest and may open up new avenues into improved therapeutic strategies for lung carcinoma patients.