The impact of non-tumor-derived circulating nucleic acids implicates the prognosis of non-small cell lung cancer

Background

A high level of circulating DNA (cirDNA) in cancer patients has been correlated with poor outcomes. Studies have demonstrated the critical contributions of the tumor-derived cirDNA. In this report, we investigated the roles of the non-tumor-derived cirDNA (nt-cirDNA) in determining the prognosis of non-small cell lung cancer (NSCLC).

Materials and methods

Plasma samples from 58 advanced NSCLC patients and 52 controls were collected. The nt-cirDNA levels were assessed with qPCR assay to detect the unmethylation status of an epithelial-specific marker, the SHP-1 promoter 2 (unmethylated SHP1P2). Clinicopathological correlations were analyzed.

Results

There was a significant increase in the total amount of cirDNA in NSCLC patients compared with controls: 4.3 ng ml^?1 [0.82–49.8] and 2.0 ng ml^?1 [0.03–26.9], respectively (p < 0.01). An increased amount of the unmethylated SHP1P2 in advanced NSCLC was also detected: 3.4 ng ml^?1 [1.2–24.8] versus 2.0 ng ml^?1 [0.03–26.9] in the controls (p = 0.026). Survival analyses revealed that high levels of total cirDNA and unmethylated SHP1P2 were significantly associated with decreased survival. However, the total cirDNA had a better prognostic correlation than the unmethylated SHP1P2. Multivariate analysis identified total cirDNA (p = 0.004) and systemic treatment (p = 0.002) as independent prognostic parameters.

Conclusion

The level of total cirDNA in NSCLC is an important prognostic parameter that demonstrates the contributions from both tumor-derived sources and non-tumor-derived sources.     

Polyester-based polyurethanes derived from alcoholysis of polylactide as toughening agents for blends with shape-memory properties

A process for sizing down and functionalizing commercial polylactide (PLA) resin is developed by alcoholysis with 1,4-butanediol (BDO) and propylene glycol (PG) to medium-sized PLA-based diols, with lower cost than a bottom-up synthesis process. These are subsequently used as polyols in preparing polyurethanes (PU) by reacting with 1,6-diisocyanatohexane (HDI). The PLA-based PU has an excellent elongation at break of 487%. The products are suitable as toughening agents for brittle PLA resin due to their highly elastic properties and high compatibility with PLA. The PU products are blended with PLA resin at various compositions, and their physical and mechanical properties and shape recovery are examined. The tensile tests showed enhancements in elongation at break up to 160% with low modulus. The fracture morphology and FTIR results confirm that the blends show strong interfacial interaction and adhesion between the PLA-based PU disperse phase and the PLA matrix. The PLA/PU blends exhibit a high shape recovery efficiency, and their recovery mechanisms are identified. These flexible PLA/PU blends are promising for various applications where bio-compatibility/degradability and high ductility are required, especially as filaments for 3D bio-printing.

A process for sizing down and functionalizing polylactide (PLA) is developed by alcoholysis. These are used as polyols in preparing PLA-based polyurethanes for toughening of brittle PLA. The blends exhibit improved mechanical properties with a high shape recovery efficiency.     

HLa-class II (DRB & DQB1) in Thai sudden unexplained death syndrome (Thai SUDS) families (Lai-Tai families)

Thai Sudden Unexplained Death Syndrome (Thai SUDS), or Lai-Tai, is a major health problem among rural residents of northeastern Thailand. The cause has been identified as a genetic disease. SUDS, a disorder found in Southeast Asia, is characterized by an abnormal electrocardiogram with ST-segment elevation in leads V1-V3, identical to that seen in Brugada Syndrome (Brugada Sign, BS) and sudden death due to ventricular fibrillation and cardiac arrest (represents an arrhythmogenic marker that identifies high-risk for SUDS). SUDS victims have a sleeping disorder (narcolepsy). The HLA-DR locus is tightly associated with narcoleptic Japanese patients and HLA-DR2, DQ haplotypes were also found in Oriental narcoleptic patients. These circumstances prompted us to study the association between the disease and HLA Class II by HLA DNA typing using a PCR-SSO method, with five Thai SUDS families (18 BS-positive subjects as the cases, and 27 BS-negatives as the controls). We found that the HLA-DRB1 *12021 allele was significantly increased in BS-positive subjects (p = 0.02; OR = 4.5), the same as the HLA-DRB1*12021-DQB1 *0301/09 haplotype (p = 0.01; OR = 7.95). Our data suggests that the HLA-DRB1* 12021 allele associated with BS and the HLA-DRB1*12021-DQB1 *0301/09 is a haplotype susceptible to arrhythmogenic markers that can identify a high risk for SUDS.     

Pulmonary Arterial Hypertension in Previously Splenectomized Patients with β-Thalassemic Disorders

Our aim was to study the cause and describe the clinical features of pulmonary arterial hypertension (PHT) in splenectomized beta-thalassemia (beta-Thal) patients. Ten splenectomized beta-Thal patients with systolic pulmonary artery (PA) pressure >30 mm Hg were evaluated by echocardiography, right-heart catheterization, and pulmonary angiography. Five of these patients later underwent hemodynamic studies. Echocardiography and pulmonary angiography on the 10 patients showed normal values of left ventricular systolic function and no findings of acute or chronic pulmonary embolism. Hemodynamic evaluation showed very high PA pressures associated with markedly increased pulmonary vascular resistance indices (PVRIs). Hematological evaluation of the 10 patients showed marked anemia, markedly increased numbers of nucleated red blood cells (nRBCs), and serum ferritin. Mean platelet count, plasma beta2 thromboglobulin, and thrombin-antithrombin III complex levels were significantly increased. It was concluded that PHT can be found in splenectomized beta-Thal patients. Features associated with PHT were female sex, hemoglobin E/beta-Thal, status many years postsplenectomy, marked anemia, markedly increased nRBC count, thrombocytosis, and very high serum ferritin levels. PHT was not due to pulmonary emboli. Our findings suggested that severe PHT was due to increased PVRI from thrombotic pulmonary arteriopathy, likely from chronic low-grade hypercoagulability and platelet activation after splenectomy.     

Changes in heart rate variability during concentration meditation

This study aims at investigating changes in heart rate variability (HRV) measured during meditation. The statistical and spectral measures of HRV from the RR intervals were analyzed. Results indicate that meditation may have different effects on health depending on frequency of the resonant peak that each meditator can achieve. The possible effects may concern resetting baroreflex sensitivity, increasing the parasympathetic tone, and improving efficiency of gas exchange in the lung.     

Target Enrichment Metagenomics Reveals Human Pegivirus-1 in Pediatric Hematopoietic Stem Cell Transplantation Recipients

Human pegivirus-1 (HPgV-1) is a lymphotropic human virus, typically considered nonpathogenic, but its infection can sometimes cause persistent viremia both in immunocompetent and immunosuppressed individuals. In a viral discovery research program in hematopoietic stem cell transplant (HSCT) pediatric patients, HPgV-1 was detected in 3 out of 14 patients (21.4%) using a target enrichment next-generation sequencing method, and the presence of the viruses was confirmed by agent-specific qRT-PCR assays. For the first time in this patient cohort, complete genomes of HPgV-1 were acquired and characterized. Phylogenetic analyses indicated that two patients had HPgV-1 genotype 2 and one had HPgV-1 genotype 3. Intra-host genomic variations were described and discussed. Our results highlight the necessity to screen HSCT patients and blood and stem cell donors to reduce the potential risk of HPgV-1 transmission.     

Self-assembly of amphiphilic poly(styrene-b-acrylic acid) on magnetic latex particles and their application as a reusable scale inhibitor

The deposition of scale on membranes or container and pipe surfaces (clogging the system) is a costly issue in water treatment processes or water-cooling systems. To effectively cope with this issue, magnetic polymeric nanoparticles (MPNPs) have been developed and applied as promising scale inhibitors, due to their high surface-area-to-volume ratio, surface modifiability, and magnetic separation ability. Carboxylated MPNPs, having a monodisperse size distribution (236 ± 26 nm) with a high magnetic content of 70 wt% and superparamagnetic properties, were fabricated by using a 2-step process: (i) formation of clusters of hydrophobic magnetic nanoparticles stabilized by oleic acid (OA-MNPs), and (ii) self-assembly of the amphiphilic block copolymer of poly(styrene₂₇-b-acrylic acid₁₂₀) (PS₂₇-b-PAA₁₂₀) onto the cluster surfaces. With application of ultrasonication to 12.0 wt% OA-MNPs, a three-dimensional network was formed by particle–particle interactions, suppressing coalescence, and then creating stable magnetic clusters. The cluster surfaces were then adsorbed by amphiphilic PS₂₇-b-PAA₁₂₀via the attractive force between hydrophobic PS blocks. This moves longer hydrophilic PAA blocks containing carboxylic acid groups into the water phase. The formulated MPNPs acted as a nanosorbent for calcium ion (Ca²⁺) removal with a removal efficiency of 92%. The MPNPs can be effectively reused for up to 4 cycles. Based on the electrostatic interactions between the negatively-charged polymer and the hydrated Ca²⁺, the resulting precipitation leads to the prevention of calcium carbonate scale formation. Insights into this mechanism open up a new perspective for magnetic-material applications as effective antiscalants.

Carboxylated magnetic polymeric nanoparticles, having a high magnetic content, and superparamagnetic properties were prepared and applied as effective antiscalants.     

Talin determines the nanoscale architecture of focal adhesions

Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin–talin–actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.Cell adhesion to the ECM is a highly coordinated process that involves ECM-specific recognition by integrin transmembrane receptors, and their aggregation with numerous cytoplasmic proteins into dense supramolecular complexes called focal adhesions (FAs) (1). Actin stress fibers terminate at FAs where actomyosin contractility is transmitted to the ECM, generating traction (2–5). Mechanical tension impinging on each FA is implicated in key steps including the elongation, reinforcement, and maintenance of the FA structures (6). FA mechanotransduction is a major aspect of cellular microenvironment sensing with wide-ranging consequences in physiological and pathological processes (7–10). However, molecular-scale spatial parameters that specify FA nanoscale organization have been difficult to access experimentally. Nevertheless, these are essential to understand how mechanosensitivity arises within such complex molecular machines (11–15).Previously 3D superresolution fluorescence microscopy has unveiled the nanoscale organization of major FA components, whereby a core region of ∼30 nm interposes between the integrin and the actin cytoskeleton along the vertical (z) axis (16). The FA core consists of a membrane-proximal layer that contains signaling proteins such as FAK (focal adhesion kinase) and paxillin, an intermediate zone that contains force-transduction proteins such as talin and vinculin, and a stress fiber interfacial zone that contains actin-associated proteins such as VASP (vasodilator-stimulated protein) and α-actinin. Although such multilaminar architecture signifies a certain degree of compartmentalization within FAs that may serve to spatially constrain protein–protein interactions and dynamics, the structural connectivity, the molecular configuration and geometry of FA proteins, and the molecular basis of their higher-order organization remain unclear.Proteomic and interactome analysis of the integrin adhesome have uncovered several direct and multitier connections between integrins and actin (17–20). This suggests that multiple highly interconnected protein–protein interactions could collectively self-organize into FA structures; such redundancy could also account for the remarkable mechanical robustness of FAs after cellular disruption or perturbation (21). Alternatively, a specific FA component may play a dominant role in regulating FA architecture. Aspects of both scenarios may also act cooperatively or function at distinct stages of FA assembly and maturation. Superresolution microscopy of cells expressing fluorescent protein (FP)-tagged FA components has revealed that talin, a large cytoskeletal adaptor protein, adopts a highly polarized orientation in FAs (16), with the N terminus residing in the membrane-proximal layer and the C terminus elevated by z ∼30 nm to the FA/stress fiber interfacial zone. This led us to hypothesize that an array of integrin–talin–actin linkages may vertically span the FA core, serving a structural role in determining FA architecture (16).To test this hypothesis, we sought to perturb FAs by substituting endogenous talin with recombinant analogs having modified lengths. These were generated by retaining both the N-terminal FERM (band 4.1/Ezrin/Radixin/Moesin) and C-terminal THATCH (Talin/HIP1R/Sla2p Actin-tethering C-terminal Homology, or R13) domains but with selective deletion of the multiple helical bundles within the central region of talin. By using a siRNA-mediated knockdown/rescue approach, we found that such talin analogs were able to support FA formation, clustering of activated integrins, and linkages to the actin cytoskeleton. By mapping the z-position of the FPs tagged at either the N or the C termini, we show that talin and its analogs are linearly extended and oriented in FAs, with their lengths regulating FA nanoscale organization. Chimeric-talin analogs with a 30-nm spacer insertion are also able to support FA assembly, facilitating the precise determination of talin geometry in FAs. Our results indicate that talin is oriented at 15° relative to the plasma membrane, measuring ∼97 nm end to end. FA nanoscale architecture in vinculin-null mouse embryonic fibroblasts (MEFs) retained its stratified organization and talin polarization similar to that in other cell types, suggesting that vinculin is dispensable for the specification of FA architecture. Our measurements demonstrate how the integrin–talin–actin module serves as the primary, and surprisingly modular, structural and tension-bearing core of FAs and geometrically define how such complexes could integrate multiple cellular forces at adhesion sites.