Synthesis of benzosuberone-tethered spirooxindoles: 1-3-dipolar cycloaddition of azomethine ylides and arylidene benzosuberones.

Expedient synthesis of benzosuberone-tethered spirooxindoles was accomplished by a three-component 1,3-dipolar cycloaddition reaction between azomethine ylide (generated in situ) and arylidene benzosuberone. This protocol offers good yield and wide functional group tolerance under mild reaction condition with high regio- and stereoselectivities.

Autoinducer 2 (AI-2) Production by Nontypeable Haemophilus influenzae 86-028NP Promotes Expression of a Predicted Glycosyltransferase That Is a Determinant of Biofilm Maturation, Prevention of Dispersal, and Persistence In Vivo.

Nontypeable Haemophilus influenzae (NTHi) is an extremely common human pathobiont that persists on the airway mucosal surface within biofilm communities, and our previous work has shown that NTHi biofilm maturation is coordinated by the production and uptake of autoinducer 2 (AI-2) quorum signals. To directly test roles for AI-2 in maturation and maintenance of NTHi biofilms, we generated an NTHi 86-028NP mutant in which luxS transcription was under the control of the xylA promoter (NTHi 86-028NP luxS xylA::luxS), rendering AI-2 production inducible by xylose. Comparison of biofilms under inducing and noninducing conditions revealed a biofilm defect in the absence of xylose, whereas biofilm maturation increased following xylose induction. The removal of xylose resulted in the interruption of luxS expression and biofilm dispersal. Measurement of luxS transcript levels by real-time reverse transcription-PCR (RT-PCR) showed that luxS expression peaked as biofilms matured and waned before dispersal. Transcript profiling revealed significant changes following the induction of luxS, including increased transcript levels for a predicted family 8 glycosyltransferase (NTHI1750; designated gstA); this result was confirmed by real-time RT-PCR. An isogenic NTHi 86-028NP gstA mutant had a biofilm defect, including decreased levels of sialylated matrix and significantly altered biofilm structure. In experimental chinchilla infections, we observed a significant decrease in the number of bacteria in the biofilm population (but not in effusions) for NTHi 86-028NP gstA compared to the parental strain. Therefore, we conclude that AI-2 promotes NTHi biofilm maturation and the maintenance of biofilm integrity, due at least in part to the expression of a probable glycosyltransferase that is potentially involved in the synthesis of the biofilm matrix.

Development of a novel air-liquid interface airway tissue equivalent model for in vitro respiratory modeling studies.

The human airways are complex structures with important interactions between cells, extracellular matrix (ECM) proteins and the biomechanical microenvironment. A robust, well-differentiated in vitro culture system that accurately models these interactions would provide a useful tool for studying normal and pathological airway biology. Here, we report the development and characterization of a physiologically relevant air-liquid interface (ALI) 3D airway 'organ tissue equivalent' (OTE) model with three novel features: native pulmonary fibroblasts, solubilized lung ECM, and hydrogel substrate with tunable stiffness and porosity. We demonstrate the versatility of the OTE model by evaluating the impact of these features on human bronchial epithelial (HBE) cell phenotype. Variations of this model were analyzed during 28 days of ALI culture by evaluating epithelial confluence, trans-epithelial electrical resistance, and epithelial phenotype via multispectral immuno-histochemistry and next-generation sequencing. Cultures that included both solubilized lung ECM and native pulmonary fibroblasts within the hydrogel substrate formed well-differentiated ALI cultures that maintained a barrier function and expressed mature epithelial markers relating to goblet, club, and ciliated cells. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. This study highlights the feasibility and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment.

Estimation of Step Length With Wearable Thigh Sensor Using an Unscented Kalman Filter.

The determination of step length, an important gait parameter, has been a challenging task. Although unobtrusive sensors (inertial measurement units) have been developed recently, they cannot facilitate the automatic estimation of step length. In this article, we use a model-based technique to determine the step length using the Unscented Kalman Filter with angular velocity from a gyroscope inside the thigh pocket. We then propose a novel covariance estimation algorithm based on a screening technique that performs a search for the optimal Process Noise Covariance matrix. Upon implementing the Unscented Kalman Filter, the step length is found using the horizontal position of the foot relative to the hip using a patient-independent robust peak detection algorithm. This research article paves the way for algorithms that are computationally much faster than black box methods, with more scope for the development of better algorithms for covariance estimation using the one proposed in this article as a foundation.

Step Length Estimation with Wearable Wrist Sensor using ANN.

Step Length is an important metric that can be used for the analysis and assessment of the gait. Proper dynamical models are not available in current literature associated with the wrist that can adequately determine the step length using recursive estimation techniques. This study presents a method to estimate the step length using angular velocity data from the wrist sensor. The technique maps the dynamical region corresponding to periods of activity of the gait manifested in angular velocity from the inertial measurement unit located at the wrist to that of the thigh using an artificial neural network, upon which an unscented Kalman filter is used to determine the horizontal position of the foot relative to the hip, and consequently, determine step length. The results for Step Length indicate an average accuracy of 81.8% and 91.1% for the young and elderly, respectively, when compared to a reference system, which, in our study, is data from a treadmill.

Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton.

A central hallmark of tumorigenesis is metabolic alterations that increase mitochondrial reactive oxygen species (mROS). In response, cancer cells upregulate their antioxidant capacity and redox-responsive signaling pathways. A promising chemotherapeutic approach is to increase ROS to levels incompatible with tumor cell survival. Mitochondrial peroxiredoxin 3 (PRX3) plays a significant role in detoxifying hydrogen peroxide (H2O2). PRX3 is a molecular target of thiostrepton (TS), a natural product and FDA-approved antibiotic. TS inactivates PRX3 by covalently adducting its two catalytic cysteine residues and crosslinking the homodimer. Using cellular models of malignant mesothelioma, we show here that PRX3 expression and mROS levels in cells correlate with sensitivity to TS and that TS reacts selectively with PRX3 relative to other PRX isoforms. Using recombinant PRXs 1-5, we demonstrate that TS preferentially reacts with a reduced thiolate in the PRX3 dimer at mitochondrial pH. We also show that partially oxidized PRX3 fully dissociates to dimers, while partially oxidized PRX1 and PRX2 remain largely decameric. The ability of TS to react with engineered dimers of PRX1 and PRX2 at mitochondrial pH, but inefficiently with wild-type decameric protein at cytoplasmic pH, supports a novel mechanism of action and explains the specificity of TS for PRX3. Thus, the unique structure and propensity of PRX3 to form dimers contribute to its increased sensitivity to TS-mediated inactivation, making PRX3 a promising target for prooxidant cancer therapy.

Noninvasive cuffless blood pressure estimation using pulse transit time, Womersley number, and photoplethysmogram intensity ratio.

OBJECTIVE: Continuous cuffless blood pressure (BP) monitoring research has emerged as blood pressure is one of the dynamic parameters which reflects cardiac arrhythmias and rheological disorders without the drawbacks of current techniques. All the existing measurement techniques are cuff-based with drawbacks such as being discontinuous in nature, being uncomfortable for the patient, etc. Therefore, the goal is to develop an algorithm to estimate BP accurately using the pulse transit time (PTT), photoplethysmogram intensity ratio (PIR) and Womersley number (α) in a noninvasive way. APPROACH: The PTT technique holds the promise of real-time, cuffless, continuous BP monitoring in clinical settings. However, the non-Newtonian fluid nature of blood is considered insignificant in the conventional PTT model-based BP modeling. In the proposed work, α, representing viscous effects, along with PTT and PIR, is included in the algorithm since it is also one of the parameters that affects the BP. MAIN RESULTS: The proposed algorithm is evaluated with 42 healthy and 39 diseased subjects and compared with the other conventional techniques to evaluate the importance of viscous effects in BP, using performance metrics like mean ± standard deviation, and 95% confidence interval for bias and limits of agreement; a better estimate is achieved with the proposed algorithm. SIGNIFICANCE: The proposed algorithm reflects the influences of vasomotor tone and baroflex as well, which makes it suitable for the assessment of BP in both healthy and diseased subjects, with improved accuracy than the current measurement techniques.

Endogenous Gs-coupled receptors in smooth muscle exhibit differential susceptibility to GRK2/3-mediated desensitization.

Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed G s-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gbetagamma sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via beta-agonist stimulation of the beta-2-adrenergic receptor (beta 2AR). Conversely, neither 5'-( N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE 2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE 2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE 2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the beta-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to G s-coupled receptors in ASM, GRK2/3 selectively attenuates beta 2AR signaling, yet relief of GRK2/3-dependent beta 2AR desensitization does not influence at least one important physiological function of the receptor.

Cooperative mitogenic signaling by G protein-coupled receptors and growth factors is dependent on G(q/11).

Previously we reported that the G protein-coupled receptor (GPCR) agonist thrombin potentiated the mitogenic effect of epidermal growth factor (EGF) on human airway smooth muscle (ASM) by promoting sustained late-phase activation of PI3K and p70S6K via a pathway dependent on Gbetagamma subunits of heterotrimeric G proteins. Here, we provide additional mechanistic insight and reveal the robustness of this phenomenon by demonstrating that H1 histamine and thromboxane receptors utilize the same mechanism to augment ASM growth via specific activation of the heterotrimeric G protein G(q/11). Thrombin, histamine, and U46619 all enhanced EGF-stimulated [3H]-thymidine incorporation as well as late-phase Akt and p70S6K phosphorylation in ASM cultures. Heterologous expression of Gbetagamma sequestrants (GRK2CT-GFP or Galpha(i)G203A), as well as GRK2NT-GFP (an RGS protein for G(q/11)) but neither p115RhoGEFRGS-GFP (an RGS for G(12/13)) nor pertussis toxin pretreatment (inactivating G(i/o)), attenuated the effects on both signaling and growth. Inhibition of Rho, Rho kinase, or Src, or modulation of arrestin expression did not significantly affect the cooperative signaling by EGF and any of the GPCR agonists. Thus, G(q/11)-coupled receptors are the principal GPCR subfamily mediating cooperative mitogenic signaling in ASM, acting through Gbetagamma-dependent, and Src/arrestin-independent activation of PI3K and p70S6K.

Unmasking of atrial repolarization waves using a simple modified limb lead system.

OBJECTIVE: In the present study, a modified limb lead (MLL) system was used to record the Ta wave in sinus rhythm and with AV block in male patients. METHODS: Eighty male subjects (mean age 36 ± 7 years) in sinus rhythm and 20 male patients with AV block (mean age 72 ± 5 years) were included in this study. Standard limb lead (SLL) ECGs and MLL ECGs were recorded for 60 seconds each with an EDAN SE-1010 PC ECG system. RESULTS: In sinus rhythm subjects, the observable Ta wave duration was 109 ± 4.7 ms, the P-Ta duration was 196 ± 5.1 ms, and the corrected P-Ta duration was 238 ± 7.2 ms. The Ta wave peak amplitude was -42 ± 8 µV. In AV block patients, the Ta wave duration was 314 ± 28 ms the P-Ta duration was 418 ± 29 ms and the corrected P-Ta duration was 46 ± 31 ms, while the Ta wave peak amplitude was -37 ± 9 µV. A correlation was found between the P and Ta wave amplitude, and no correlation was found between the P and Ta wave duration or the Ta amplitude and Ta duration in sinus rhythm and AV block subjects. CONCLUSION: The end of the Ta wave is not observable in sinus rhythm subjects, as it extends into the QRS complex and ST segment. In AV block patients, the Ta wave duration was generally three times longer than the observable Ta duration in sinus rhythm subjects.

Replication of type 5 adenovirus promotes middle ear infection by Streptococcus pneumoniae in the chinchilla model of otitis media.

Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyperinflammatory adenovirus mutant dl327 and the nonreplicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by S. pneumoniae as compared to nonadenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the nonreplicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease.

Aging and oxidative stress reduce the response of human articular chondrocytes to insulin-like growth factor 1 and osteogenic protein 1.

OBJECTIVE: To determine the effects of aging and oxidative stress on the response of human articular chondrocytes to insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1). METHODS: Chondrocytes isolated from normal articular cartilage obtained from tissue donors were cultured in alginate beads or monolayer. Cells were stimulated with 50-100 ng/ml of IGF-1, OP-1, or both. Oxidative stress was induced using tert-butyl hydroperoxide. Sulfate incorporation was used to measure proteoglycan synthesis, and immunoblotting of cell lysates was performed to analyze cell signaling. Confocal microscopy was performed to measure nuclear translocation of Smad4. RESULTS: Chondrocytes isolated from the articular cartilage of tissue donors ranging in age from 24 years to 81 years demonstrated an age-related decline in proteoglycan synthesis stimulated by IGF-1 and IGF-1 plus OP-1. Induction of oxidative stress inhibited both IGF-1- and OP-1-stimulated proteoglycan synthesis. Signaling studies showed that oxidative stress inhibited IGF-1-stimulated Akt phosphorylation while increasing phosphorylation of ERK, and that these effects were greater in cells from older donors. Oxidative stress also increased p38 phosphorylation, which resulted in phosphorylation of Smad1 at the Ser(206) inhibitory site and reduced nuclear accumulation of Smad1. Oxidative stress also modestly reduced OP-1-stimulated nuclear translocation of Smad4. CONCLUSION: These results demonstrate an age-related reduction in the response of human chondrocytes to IGF-1 and OP-1, which are 2 important anabolic factors in cartilage, and suggest that oxidative stress may be a contributing factor by altering IGF-1 and OP-1 signaling.