Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes.

Protein properties and interactions have been widely investigated by using external labels. However, the micromolar sensitivity of the current dyes limits their applicability due to the high material consumption and assay cost. In response to this challenge, we synthesized a series of cyanine5 (Cy5) dye-based quencher molecules to develop an external dye technique to probe proteins at the nanomolar protein level in a high-throughput one-step assay format. Several families of Cy5 dye-based quenchers with ring and/or side-chain modifications were designed and synthesized by introducing organic small molecules or peptides. Our results showed that steric hindrance and electrostatic interactions are more important than hydrophobicity in the interaction between the luminescent negatively charged europium-chelate-labeled peptide (Eu-probe) and the quencher molecules. The presence of substituents on the quencher indolenine rings reduces their quenching property, whereas the increased positive charge on the indolenine side chain improved the interaction between the quenchers and the luminescent compound. The designed quencher structures entirely altered the dynamics of the Eu-probe (protein-probe) for studying protein stability and interactions, as we were able to reduce the quencher concentration 100-fold. Moreover, the new quencher molecules allowed us to conduct the experiments using neutral buffer conditions, known as the peptide-probe assay. These improvements enabled us to apply the method in a one-step format for nanomolar protein-ligand interaction and protein profiling studies instead of the previously developed two-step protocol. These improvements provide a faster and simpler method with lower material consumption.

Protocol for performing and optimizing differential scanning fluorimetry experiments.

Differential scanning fluorimetry (DSF) is a widely used technique for determining the apparent melting temperature (Tma) of a purified protein. Here, we present a protocol for performing and optimizing DSF experiments. We describe steps for designing and performing the experiment, analyzing data, and optimization. We provide benchmarks for typical Tmas and ΔTmas, standard assay conditions, and upper and lower limits of commonly altered experimental variables. We also detail common pitfalls of DSF and ways to avoid, identify, and overcome them.

The Correlation Between Non-Invasive Ventilation Use and the Development of Dry Eye Disease.

The use of non-invasive ventilation (NIV) devices such as continuous positive airway pressure and bi-level positive airway pressure machines have been associated with an increased incidence of dry eye disease (DED). To understand how the use of these ventilation masks impacts the eyes, a review of the pathophysiology of DED and an evaluation of recent studies investigating the effects of NIV use on the severity and incidence of this condition were performed. It was found that the use of face masks associated with the ventilation devices exhibited a positive correlation to the incidence and severity of numerous ocular pathologies. However, the benefits of non-invasive mechanical ventilation are undeniable in treating conditions such as obstructive sleep apnea, chronic obstructive pulmonary disease, and respiratory failure; therefore, proper education, behavioral modifications, and treatment can help reduce or prevent the adverse effects that NIV have on the eyes.

A Rare Case of Hydrochlorothiazide-Induced Hemolytic Anemia.

Drug-induced immune hemolytic anemia is an exceedingly rare adverse drug event. Thiazide diuretics, commonly used in the treatment of primary hypertension, have been associated with this complication. In this case report, we present a 77-year-old male who developed acute hemolytic anemia two days after starting hydrochlorothiazide in the treatment of high blood pressure.

The Neurophysiology of Caffeine as a Central Nervous System Stimulant and the Resultant Effects on Cognitive Function.

Caffeine is one of the world's most consumed drugs. According to the Washington Post (2015), two billion cups of coffee are consumed per day worldwide. Caffeine is classified as a central nervous system (CNS) stimulant and an organic molecule called methylxanthine. Caffeine has three notable mechanisms of action on the CNS that produce a psychostimulant effect. These effects are responsible for the effect that caffeine has on cognitive function. The effects of caffeine consumption on cognitive function have been demonstrated across several studies involving humans and animals. With the immense number of people consuming caffeine around the world, it is of vital importance to study the effects that this drug has on people's cognitive function. This literature review provides useful insights on this question through the analysis of caffeine's effects on cognitive function, along with information on caffeine's three modes of action. The findings of recent studies show mixed results regarding the effects of caffeine on mood, attention, processing speed, and memory. Current research suggests that if caffeine does have an effect on mood, the most significant changes may be anxiety. Studies did not support caffeine as having any significant effect on attention, but that it did play a role in enhancing processing speed. The majority of the studies reviewed suggest caffeine as having a significant positive effect on both short and long-term memory in adults and the elderly. Current findings warrant continued research on the association of caffeine and the resultant effects on cognitive function.

FOXE3 mutations predispose to thoracic aortic aneurysms and dissections.

The ascending thoracic aorta is designed to withstand biomechanical forces from pulsatile blood. Thoracic aortic aneurysms and acute aortic dissections (TAADs) occur as a result of genetically triggered defects in aortic structure and a dysfunctional response to these forces. Here, we describe mutations in the forkhead transcription factor FOXE3 that predispose mutation-bearing individuals to TAAD. We performed exome sequencing of a large family with multiple members with TAADs and identified a rare variant in FOXE3 with an altered amino acid in the DNA-binding domain (p.Asp153His) that segregated with disease in this family. Additional pathogenic FOXE3 variants were identified in unrelated TAAD families. In mice, Foxe3 deficiency reduced smooth muscle cell (SMC) density and impaired SMC differentiation in the ascending aorta. Foxe3 expression was induced in aortic SMCs after transverse aortic constriction, and Foxe3 deficiency increased SMC apoptosis and ascending aortic rupture with increased aortic pressure. These phenotypes were rescued by inhibiting p53 activity, either by administration of a p53 inhibitor (pifithrin-α), or by crossing Foxe3-/- mice with p53-/- mice. Our data demonstrate that FOXE3 mutations lead to a reduced number of aortic SMCs during development and increased SMC apoptosis in the ascending aorta in response to increased biomechanical forces, thus defining an additional molecular pathway that leads to familial thoracic aortic disease.