Sessile droplet array for sensitive profiling of multiple extracellular vesicle immuno-subtypes.

The sensitive detection of the multiple immuno-subtypes of cancer-specific extracellular vesicles (EVs) has emerged as a promising method for multiclass cancer diagnosis; however, its limitations in sensitivity, accessibility, and multiple detection of EV subtypes have hindered its further implementation. Here, we present a platform for sensitive EV detection enabled by sessile droplet array (eSD) that exploits enhanced immuno-capture of EVs via evaporation-driven radial flows in a sessile droplet. Compared to a micro-well without internal flows, this platform demonstrates significantly enhanced EV capture and detection by detecting low levels of EVs with a detection limit of 384.7 EVs per microliter, which is undetectable in the micro-well. In addition, using a small sample consumption of ∼0.2 µL plasma per droplet, the platform detects EV immuno-subtypes against seven different antibodies in patient plasma samples of different cancer types (liver, colon, lung, breast and prostate cancers). Further, using the profiling data, the platform exhibits a sensitivity of 100% (95% confidence interval (CI): 83-100%) and a specificity of 100% (95% CI: 40-100%) for the diagnosis of cancer, and classified cancer types with an overall accuracy of 96% (95% CI: 86-100%) using a two-staged algorithm based on quadratic discriminant analysis technique for machine learning.

Human and Bacterial Toll-Interleukin Receptor Domains Exhibit Distinct Dynamic Features and Functions.

Toll-interleukin receptor (TIR) domains have emerged as critical players involved in innate immune signaling in humans but are also expressed as potential virulence factors within multiple pathogenic bacteria. However, there has been a shortage of structural studies aimed at elucidating atomic resolution details with respect to their interactions, potentially owing to their dynamic nature. Here, we used a combination of biophysical and biochemical studies to reveal the dynamic behavior and functional interactions of a panel of both bacterial TIR-containing proteins and mammalian receptor TIR domains. Regarding dynamics, all three bacterial TIR domains studied here exhibited an inherent exchange that led to severe resonance line-broadening, revealing their intrinsic dynamic nature on the intermediate NMR timescale. In contrast, the three mammalian TIR domains studied here exhibited a range in terms of their dynamic exchange that spans multiple timescales. Functionally, only the bacterial TIR domains were catalytic towards the cleavage of NAD+, despite the conservation of the catalytic nucleophile on human TIR domains. Our development of NMR-based catalytic assays allowed us to further identify differences in product formation for gram-positive versus gram-negative bacterial TIR domains. Differences in oligomeric interactions were also revealed, whereby bacterial TIR domains self-associated solely through their attached coil-coil domains, in contrast to the mammalian TIR domains that formed homodimers and heterodimers through reactive cysteines. Finally, we provide the first atomic-resolution studies of a bacterial coil-coil domain and provide the first atomic model of the TIR domain from a human anti-inflammatory IL-1R8 protein that undergoes a slow inherent exchange.

Mechanical Properties of Single Muscle Fibers: Understanding Poor Muscle Quality in Older Adults with Diabetes.

BACKGROUND: While aging causes muscle weakness, type 2 diabetes mellitus (T2DM) is also considered a high-risk factor for the induction of skeletal muscle weakness. Previous studies have reported increased collagen content in insulin-resistant skeletal muscles. Here, we studied the mechanical properties of aged skeletal muscle in subjects with T2DM to investigate whether aged skeletal muscles with T2DM induce higher passive tension due to the abundance of extracellular matrix (ECM) inside or outside of the muscle fibers. METHODS: Samples from the gluteus maximus muscles of older adults with diabetes (T2DM) and non-diabetic (non-DM) older adults who underwent elective orthopedic surgery were collected. Permeabilized single muscle fibers from these samples were used to identify their mechanical properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to quantify titin and fiber type distributions in these samples. RESULTS: We confirmed a significant predominance of type I fiber ratio in both T2DM and non-DM aged muscles. While the average cross-sectional area and maximal active tension of the single fibers were smaller in the T2DM group than those in the non-DM group, the difference was not statistically significant. T2DM subjects showed significantly greater passive tension and lower titin-/ECM-based passive tension ratios than those in non-DM subjects, which indicated that more ECM but less titin contributed to the total passive tension. CONCLUSION: Based on our findings, we concluded that T2DM may cause increased passive stiffness of single skeletal muscle fibers in older adults because of an excessive accumulation of ECM in and around single muscle fibers due to increased insulin resistance.

Effects of cisplatin on photosensitizer-mediated photodynamic therapy in breast tumor-bearing nude mice.

OBJECTIVE: This study aimed to evaluate the potential effects of cisplatin on photodynamic therapy (PDT) in breast cancer using a breast tumor-bearing mouse model. METHODS: In this study, breast tumor (experimental mammary tumour-6 cell)-bearing nude mice were used as experimental animals. Photolon® (photosensitizer, 2.5 mg/kg body weight [BW]) was injected intraperitoneally; after 2 hours, the tumors were irradiated (660 nm, 80 J/cm2) using a diode laser tool. Cisplatin (3 mg/kg BW) was injected intraperitoneally 1 hour before the Photolon® injection. RESULTS: Tumor volume increased over time in the control group and was not different from that in the cisplatin group. In the PDT group, the tumor volume increased on day 3, but not on day 7. In the cisplatin+PDT group, tumor volume increased on day 3 but decreased on day 7. There was no significant difference in the levels of thiobarbituric acid reactive substance (TBARS) in tumor tissues between the control and cisplatin groups. The levels of TBARS in the cisplatin+PDT group were higher (47%) than those in the PDT group. Analysis of tumor tissue transcriptomes showed that the expression of genes related to the inflammatory response including CL and XCL genes increased, while that of Fn1 decreased in the cisplatin+PDT group compared with the PDT group. CONCLUSION: These results suggest that cisplatin enhances the therapeutic effect of PDT in a breast tumor-bearing mouse model. However, further clinical studies involving patients with breast cancer is needed.

Nebulin stiffens the thin filament and augments cross-bridge interaction in skeletal muscle.

Nebulin is a giant sarcomeric protein that spans along the actin filament in skeletal muscle, from the Z-disk to near the thin filament pointed end. Mutations in nebulin cause muscle weakness in nemaline myopathy patients, suggesting that nebulin plays important roles in force generation, yet little is known about nebulin's influence on thin filament structure and function. Here, we used small-angle X-ray diffraction and compared intact muscle deficient in nebulin (using a conditional nebulin-knockout, Neb cKO) with control (Ctrl) muscle. When muscles were activated, the spacing of the actin subunit repeat (27 Å) increased in both genotypes; when converted to thin filament stiffness, the obtained value was 30 pN/nm in Ctrl muscle and 10 pN/nm in Neb cKO muscle; that is, the thin filament was approximately threefold stiffer when nebulin was present. In contrast, the thick filament stiffness was not different between the genotypes. A significantly shorter left-handed (59 Å) thin filament helical pitch was found in passive and contracting Neb cKO muscles, as well as impaired tropomyosin and troponin movement. Additionally, a reduced myosin mass transfer toward the thin filament in contracting Neb cKO muscle was found, suggesting reduced cross-bridge interaction. We conclude that nebulin is critically important for physiological force levels, as it greatly stiffens the skeletal muscle thin filament and contributes to thin filament activation and cross-bridge recruitment.

Thick-Filament Extensibility in Intact Skeletal Muscle.

Myofilament extensibility is a key structural parameter for interpreting myosin cross-bridge kinetics in striated muscle. Previous studies reported much higher thick-filament extensibility at low tension than the better-known and commonly used values at high tension, but in interpreting mechanical studies of muscle, a single value for thick-filament extensibility has usually been assumed. Here, we established the complete thick-filament force-extension curve from actively contracting, intact vertebrate skeletal muscle. To access a wide range of tetanic forces, the myosin inhibitor blebbistatin was used to induce low tetanic forces in addition to the higher tensions obtained from tetanic contractions of the untreated muscle. We show that the force/extensibility curve of the thick filament is nonlinear, so assuming a single value for thick-filament extensibility at all force levels is not justified. We also show that independent of whether tension is generated passively by sarcomere stretch or actively by cross-bridges, the thick-filament extensibility is nonlinear. Myosin head periodicity, however, only changes when active tension is generated under calcium-activated conditions. The nonlinear thick-filament force-extension curve in skeletal muscle, therefore, reflects a purely passive response to either titin-based force or actomyosin-based force, and it does not include a thick-filament activation mechanism. In contrast, the transition of myosin head periodicity to an active configuration appears to only occur in response to increased active force when calcium is present.

Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae.

Norcoclaurine synthase (NCS) catalyzes the enantioselective Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid (BIA) biosynthesis. NCS orthologs in available transcriptome databases were screened for variants that might improve the low yield of BIAs in engineered microorganisms. Databases for 21 BIA-producing species from four plant families yielded 33 assembled contigs with homology to characterized NCS genes. Predicted translation products generated from nine contigs consisted of two to five sequential repeats, each containing most of the sequence found in single-domain enzymes. Assembled contigs containing tandem domain repeats were detected only in members of the Papaveraceae family, including opium poppy (Papaver somniferum). Fourteen cDNAs were generated from 10 species, five of which encoded NCS orthologs with repeated domains. Functional analysis of corresponding recombinant proteins yielded six active NCS enzymes, including four containing either two, three or four repeated catalytic domains. Truncation of the first 25 N-terminal amino acids from the remaining polypeptides revealed two additional enzymes. Multiple catalytic domains correlated with a proportional increase in catalytic efficiency. Expression of NCS genes in Saccharomyces cereviseae also produced active enzymes. The metabolic conversion capacity of engineered yeast positively correlated with the number of repeated domains.

Physico-chemical properties and cytotoxic potential of Cordyceps sinensis metabolites.

This study was conducted to estimate the antioxidant activities, biochemical properties and biological activities of one of the entomopathogenic fungi, Cordyceps sinensis. Analysis of fungal metabolites indicated that the most abundant free sugar was glucose; the highest component of organic acids was citric acid from 10-day culture medium and the glutamate was the predominant amino acid observed from 3-day culture medium. Maximum total polyphenols and flavonoids were detected in the 15-day culture medium. For cytotoxicity test, three cancer cell lines, HepG2 (liver), MCF-7 (breast) and A549 (lung) were used. The IC50 values of the highest toxicity of HepG2 cell lines were observed from 10-day cultured medium, whereas the highest toxicity of MCF-7 and A549 was observed on 5-day cultured medium. This is the first study reporting on the strong antioxidant and cytotoxic potential of C. sinensis. Culture medium of C. sinensis may thus be used as an effective antioxidant and anticancer treatment of natural origin.

Calcium sensitivity and myofilament lattice structure in titin N2B KO mice.

The cellular basis of the Frank-Starling "Law of the Heart" is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information to altered calcium sensitivity has remained elusive. Here the effect of titin-based passive tension on the length-dependence of activation (LDA) was studied by measuring the tension-pCa relation in skinned mouse LV muscle at two sarcomere lengths (SLs). N2B KO myocardium, where the N2B spring element in titin is deleted and passive tension is elevated, was compared to WT myocardium. Myofilament lattice structure was studied with low-angle X-ray diffraction; the myofilament lattice spacing (d1,0) was measured as well as the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I1,1/I1,0) as an estimate of the degree of association of myosin heads with the thin filaments. Experiments were carried out in skinned muscle in which the lattice spacing was reduced with Dextran-T500. Experiments with and without lattice compression were also carried out following PKA phosphorylation of the skinned muscle. Under all conditions that were tested, LDA was significantly larger in N2B KO myocardium compared to WT myocardium, with the largest differences following PKA phosphorylation. A positive correlation between passive tension and LDA was found that persisted when the myofilament lattice was compressed with Dextran and that was enhanced following PKA phosphorylation. Low-angle X-ray diffraction revealed a shift in mass from thin filaments to thick filaments as sarcomere length was increased. Furthermore, a positive correlation was obtained between myofilament lattice spacing and passive tension and the change in I1,1/I1,0 and passive tension and these provide possible explanations for how titin-based passive tension might regulate calcium sensitivity.

Monochord sounds and progressive muscle relaxation reduce anxiety and improve relaxation during chemotherapy: a pilot EEG study.

BACKGROUND: Chemotherapy is the most distressing form of cancer treatment in oncology, but listening to music can be an adjuvant during chemotherapy. Monochord (MC) sounds are used in music therapy for the alleviation of pain, enhanced body perception, and relaxation. This study investigated the relaxation effect of MC sounds for patients during chemotherapy compared with progressive muscle relaxation (PMR), an established relaxation technique. METHODOLOGY/PRINCIPAL FINDINGS: Two randomized groups of patients were observed during chemotherapy. One group listened to recorded MC sounds (n=20) and the other group listened to recorded PMR (n=20). Each session was investigated pre and post using Spielberger's State Anxiety Inventory (SAI) and a questionnaire about the patient's physical and psychological states. Further, for the first and the last session, multivariate electroencephalogram (EEG) signals were recorded. Patients in both MC and PMR groups showed significant improvement in their physical and psychological states and in state anxiety. The EEG data showed that the MC and the PMR groups were associated with an increase of posterior theta (3.5-7.5 Hz) and a decrease of midfrontal beta-2 band (20-29.5 Hz) activity during the end phase of relaxation treatment. Further, the MC group was associated with decreased alpha band (8-12 Hz) activity in comparison with PMR group. CONCLUSIONS: This study shows that both listening to recorded MC sounds and practising PMR have a useful and comparable effect on gynaecologic oncological patients during chemotherapy, with partially overlapping but also notably divergent neural correlates. Future research should establish the systematic use of MC in oncological contexts.

Echocardiographic predictors of left ventricular function and clinical outcomes after successful mitral valve repair: conventional two-dimensional versus speckle-tracking parameters.

BACKGROUND: We aimed to compare conventional echocardiographic versus speckle tracking-derived parameters in predicting postoperative left ventricular (LV) dysfunction and clinical outcomes after successful mitral valve repair in patients with mitral regurgitation. METHODS: In 147 consecutive patients in sinus rhythm with severe MR, two-dimensional echocardiography and speckle-tracking imaging for global longitudinal, circumferential, and radial strains and strain rates were performed within 30 days before successful mitral valve repair. Echocardiography was repeated within 7 days in all patients, and more than 3 months after surgery in 112 patients. Clinical events were evaluated for 21±17 months. RESULTS: Multivariate linear regression analysis showed that preoperative LV systolic dimension (p=0.004) and volume (p=0.001) were independent determinants of immediate postoperative LV ejection fraction. Preoperative LV end-systolic dimension (p=0.004), LV ejection fraction (p=0.017), and circumferential strain (p=0.029) were independent predictors of late follow-up LV ejection fraction. By Cox regression analysis, preoperative end-systolic LV dimension (hazard ratio 1.26 for every 1 mm, 95% confidence interval 1.11 to 1.44, p<0.001) was the only predictor of hospital admission for heart failure. The best cutoff values of LV end-systolic dimension (≥41 mm) and volume (≥85 mL) for predicting postoperative severe LV dysfunction (ejection fraction<0.35) identified patients at high risk for event-free survival, but those of speckle-tracking parameters did not. CONCLUSIONS: Preoperative LV remodeling parameters, such as LV end-systolic dimension and volume, are superior to speckle tracking-derived deformation parameters in predicting LV dysfunction and clinical events after successful mitral valve repair in patients with severe mitral regurgitation.

Toxicity of cassia and cinnamon oil compounds and cinnamaldehyde-related compounds to Sitophilus oryzae (Coleoptera: Curculionidae).

The toxicity to adult Sitophilus oryzae (L.) (Coleoptera: Curculionidae) of two cassia oils (Especial and true), four cinnamon oils (technical, #500, bark, and green leaf), and (E)-cinnamaldehyde and its 41 structurally related compounds was examined by residual and vapor-phase toxicity bioassays. Results were compared with those of dichlorvos. In residual bioassays, cassia and cinnamon oils exhibited good insecticidal activity. Based on 48-h LD50 values, the toxicity of allyl cinnmate (0.0003 mg/cm2) was comparable with that of dichlorvos (0.00025 mg/cm2). Potent insecticidal activity also was observed with benzaldehyde, beta-caryophyllene, cinnamonitrile, hydrocinnamyl acetate, (E)-4-hydroxycinnamic acid, and alpha-terpineol (LD50 = 0.003-0.009 mg/cm2). Structure-activity relationships indicate that types of functional groups rather than hydrophobicity or vapor pressure parameters seem to play a role in determining the toxicities to adult S. oryzae. In vapor-phase toxicity tests with weevils, these compounds were more effective in closed containers than in open ones. These results indicate that the effect of the compounds was largely a result of action in the vapor phase. Cassia and cinnamon oils and test compounds described merit further study as potential fumigants for the control of S. oryzae because of their greater activity as a fumigant.