Quantitative histopathologic profiling of arterial dissection-related thrombi in acute ischemic stroke: etiological comparisons.

Introduction: Arterial dissection is an important etiology of stroke in young adults and may demonstrate distinct thrombus characteristics. While most studies have focused on compositional differences between cardioembolic (CE) and non-cardioembolic thrombi, systematic analyses of dissection-related thrombi remain scarce. This study characterized the compositions of dissection thrombi, compared them with those of non-dissection thrombi, and explored compositional variations among stroke etiologies.

Characterization of a Single-Molecule Sensitive Digital Flow Cytometer for Amplification-Free Digital Assays.

Digital assays such as digital PCR for nucleic acids and digital ELISA for proteins provide absolute quantitation and greater accuracy, sensitivity, and reproducibility than their analogue counterparts (real-time PCR and standard ELISA), but current digital assays involve amplification (e.g., DNA amplification in digital PCR and signal amplification in digital ELISA), which makes high multiplexing difficult, often requires complex and expensive sample compartmentalization, and adds reaction steps.

Comparison of EV characterization by commercial high-sensitivity flow cytometers and a custom single-molecule flow cytometer.

High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers.

Mechanisms of a novel regulatory light chain-dependent cardiac myosin inhibitor.

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the heart characterized by thickening of the left ventricle (LV), hypercontractility, and impaired relaxation. HCM is caused primarily by heritable mutations in sarcomeric proteins, such as β myosin heavy chain. Until recently, medications in clinical use for HCM did not directly target the underlying contractile changes in the sarcomere.

Late-life Rapamycin Treatment Enhances Cardiomyocyte Relaxation Kinetics and Reduces Myocardial Stiffness.

Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle.

Expression and significance of aquaporin-4 in thyroid carcinoma.

Objective: To investigate the expression of aquaporin-4 (AQP4) in thyroid carcinoma (TC) and explore its clinical significance.

Materials And Methods: The formalin-fixed paraffin-embedded specimens including 275 TC cancer tissues, 258 corresponding paracancerous thyroid tissues and their clinicopathologic data were retrospectively analyzed. Immunohistochemical EnVision two-step method was used to detect the expression of AQP4 in the cancer tissues and adjacent thyroid tissues, and its clinical significance was analyzed.

Long-term and inter-monthly dynamics of aquatic vegetation and its relation with environmental factors in Taihu Lake, China.

This paper discussed the long-term and inter-monthly variation in the distribution area of aquatic macrophytes in Taihu Lake, as well as the relationship between these variations and environmental factors. The findings were of great significance to the protection and environmental remediation of lake ecosystems. This paper presented data from 92 periods during 1980 to 2017 on the distribution area of aquatic macrophytes (including submerged macrophytes and floating-leaved macrophytes, but excluding emergent macrophytes) in Taihu Lake.

Effects of Cardiac Troponin I Mutation P83S on Contractile Properties and the Modulation by PKA-Mediated Phosphorylation.

cTnI(P82S) (cTnI(P83S) in rodents) resides at the I-T arm of cardiac troponin I (cTnI) and was initially identified as a disease-causing mutation of hypertrophic cardiomyopathy (HCM). However, later studies suggested this may not be true. We recently reported that introduction of an HCM-associated mutation in either inhibitory-peptide (cTnI(R146G)) or cardiac-specific N-terminus (cTnI(R21C)) of cTnI blunts the PKA-mediated modulation on myofibril activation/relaxation kinetics by prohibiting formation of intrasubunit contacts between these regions.

[Clinicopathologic features of succinate dehydrogenase-deficient gastrointestinal stromal tumor].

Objective: To investigate clinicopathologic features of succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GIST).

Methods: Immunohistochemical EnVision technique was used to assess the expression of succinate dehydrogenase subunit B (SDHB) in 192 cases of GIST. Cases of SDH-deficient GIST were further evaluated for the presence of CKIT exons 9, 11, 13 and 17 mutations and PDGFRA exons 12 and 18 mutations with clinical followed-up data.

Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility.

Cardiac troponin (cTn) acts as a pivotal regulator of muscle contraction and relaxation and is composed of three distinct subunits (cTnC: a highly conserved Ca(2+) binding subunit, cTnI: an actomyosin ATPase inhibitory subunit, and cTnT: a tropomyosin binding subunit). In this mini-review, we briefly summarize the structure-function relationship of cTn and its subunits, its modulation by PKA-mediated phosphorylation of cTnI, and what is known about how these properties are altered by hypertrophic cardiomyopathy (HCM) associated mutations of cTnI. This includes recent work using computational modeling approaches to understand the atomic-based structural level basis of disease-associated mutations.

2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function.

Contractile properties of developing human fetal cardiac muscle.

Key Points: The contractile properties of human fetal cardiac muscle have not been previously studied. Small-scale approaches such as isolated myofibril and isolated contractile protein biomechanical assays allow study of activation and relaxation kinetics of human fetal cardiac muscle under well-controlled conditions. We have examined the contractile properties of human fetal cardiac myofibrils and myosin across gestational age 59-134 days.

Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation.

Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI (KC-I) or contractile kinetics during β-adrenergic stimulation.

Effects of HCM cTnI mutation R145G on troponin structure and modulation by PKA phosphorylation elucidated by molecular dynamics simulations.

Cardiac troponin (cTn) is a key molecule in the regulation of human cardiac muscle contraction. The N-terminal cardiac-specific peptide of the inhibitory subunit of troponin, cTnI (cTnI(1-39)), is a target for phosphorylation by protein kinase A (PKA) during β-adrenergic stimulation. We recently presented evidence indicating that this peptide interacts with the inhibitory peptide (cTnl(137-147)) when S23 and S24 are phosphorylated.

2-Deoxy adenosine triphosphate improves contraction in human end-stage heart failure.

We are developing a novel treatment for heart failure by increasing myocardial 2 deoxy-ATP (dATP). Our studies in rodent models have shown that substitution of dATP for adenosine triphosphate (ATP) as the energy substrate in vitro or elevation of dATP in vivo increases myocardial contraction and that small increases in the native dATP pool of heart muscle are sufficient to improve cardiac function. Here we report, for the first time, the effect of dATP on human adult cardiac muscle contraction.

Computational studies of the effect of the S23D/S24D troponin I mutation on cardiac troponin structural dynamics.

During β-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease KCa and pCa50, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow), which speeds up the overall relaxation.

PKA phosphorylation of cardiac troponin I modulates activation and relaxation kinetics of ventricular myofibrils.

Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca(2+) levels vary.

A computationally designed inhibitor of an Epstein-Barr viral Bcl-2 protein induces apoptosis in infected cells.

Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity.

Unraveling the allosteric inhibition mechanism of PTP1B by free energy calculation based on umbrella sampling.

Protein tyrosine phosphatase 1B (PTP1B) is a promising target for the treatment of obesity and type II diabetes. Allosteric inhibitors can stabilize an active conformation of PTP1B by hindering the conformational transition of the WPD loop of PTP1B from the open to the closed state. Here, the umbrella sampling molecular dynamics (MD) simulations were employed to compute the reaction path of the conformational transition of PTP1B, and the snapshots extracted from the MD trajectory were clustered into 58 conformational groups based on the key conformational parameter.

Molecular dynamics simulations and MM/GBSA methods to investigate binding mechanisms of aminomethylpyrimidine inhibitors with DPP-IV.

The aminomethylpyrimidines were investigated as a novel class of DPP-IV inhibitors. In this Letter, the binding mechanisms of how slight change of substitution or position influences the binding affinity of five representative analogs was investigated by molecular dynamics simulation, free energy calculations and energy decomposition analysis. The conserved hydrogen bonds with Glu205 and Glu206 slightly favor the inhibitor binding; the van der Waals interactions, especially the two key contacts with Tyr547 and Tyr666, dominate in the binding free energy and play a crucial role on distinguishing the high active inhibitors from the low ones.

Structural basis of specific binding between Aurora A and TPX2 by molecular dynamics simulations.

In the present study, the impacts of G198N and W128F mutations on the recognition between Aurora A and targeting protein of Xenopus kinesin-like protein 2 (TPX2) were investigated using molecular dynamics (MD) simulations, free energy calculations, and free energy decomposition analysis. The predicted binding free energy of the wild-type complex is more favorable than those of three mutants, indicating that both single and double mutations are unfavorable for the Aurora A and TPX2 binding. It is also observed that the mutations alternate the binding pattern between Aurora A and TPX2, especially the downstream of TPX2.

The molecular mechanism studies of chirality effect of PHA-739358 on Aurora kinase A by molecular dynamics simulation and free energy calculations.

Aurora kinase family is one of the emerging targets in oncology drug discovery and several small molecules targeting aurora kinases have been discovered and evaluated under early phase I/II trials. Among them, PHA-739358 (compound 1r) is a 3-aminopyrazole derivative with strong activity against Aurora A under early phase II trial. Inhibitory potency of compound 1r (the benzylic substituent at the pro-R position) is 30 times over that of compound 1s (the benzylic substituent at the pro-S position).

Octathienyl/phenyl-substituted zinc phthalocyanines J-aggregated through conformational planarization.

A novel family of thiophene-decorated phthalocyanines (M-TPcs) was efficiently synthesized, during which the key intermediate of these compounds was purified through a chemical approach. In the optimized geometries of M-TPcs, the peripherally linked thiophene rings are tilted from the Pc core, which oppose aggregation considering the mutual steric hindrance. However, Zn-TPc formed J-aggregates in many solvents while Ni-TPc and Cu-TPc did not.

Studying the mechanism that enables paullones to selectively inhibit glycogen synthase kinase 3 rather than cyclin-dependent kinase 5 by molecular dynamics simulations and free-energy calculations.

Glycogen synthase kinase 3 (GSK-3) is an attractive target for the treatment of diabetes, and paullones have been reported to be effective inhibitors of GSK-3. However, it is still a challenging task to improve selectivity among protein kinases, especially cyclin-dependent kinases (CDKs). Here we investigated the mechanism that enables paullones to selectively inhibit GSK-3 rather than cyclin-dependent kinase 5 (CDK5) using sequence alignment, molecular dynamics simulations, free-energy calculations and free-energy decomposition analysis.