Imaging Neurotransmitters with Small-Molecule Fluorescent Probes.

Neurotransmitters play a crucial role in regulating communication between neurons within the brain and central nervous system. Thus, imaging neurotransmitters has become a high priority in neuroscience. This minireview focuses on recent advancements in the development of fluorescent small-molecule fluorescent probes for neurotransmitter imaging and applications of these probes in neuroscience. Innovative approaches for probe design are highlighted as well as attributes which are necessary for practical utility, with a view to inspiring new probe development capable of visualizing neurotransmitters.

Synthesis of a Near-Infrared Fluorescent Probe for Imaging Catecholamines via a Tandem Nucleophilic Aromatic Substitution.

A near-infrared (NIR) fluorescent probe NS667 was developed using a novel synthetic strategy by integrating an electron-rich 1,2,3,4-tetrahydroquinoxaline (THQ) into the scaffold from NS510, which binds to catecholamines with high affinity. The fluorophore core was constructed with a tandem nucleophilic aromatic substitution. Upon binding to catecholamines, the fluorescence of this probe shifted, with the emission in the NIR region. Live cell imaging results demonstrate that NS667 can effectively image norepinephrine in chromaffin cells with shifted fluorescence, which highlights the potential of the probe for neuroimaging in tissues.

A Turn-On Fluorescent Amino Acid Sensor Reveals Chloroquine's Effect on Cellular Amino Acids via Inhibiting Cathepsin L.

Maintaining homeostasis of metabolites such as amino acids is critical for cell survival. Dysfunction of nutrient balance can result in human diseases such as diabetes. Much remains to be discovered about how cells transport, store, and utilize amino acids due to limited research tools. Here we developed a novel, pan-amino acid fluorescent turn-on sensor, NS560. It detects 18 of the 20 proteogenic amino acids and can be visualized in mammalian cells. Using NS560, we identified amino acids pools in lysosomes, late endosomes, and surrounding the rough endoplasmic reticulum. Interestingly, we observed amino acid accumulation in large cellular foci after treatment with chloroquine, but not with other autophagy inhibitors. Using a biotinylated photo-cross-linking chloroquine analog and chemical proteomics, we identified Cathepsin L (CTSL) as the chloroquine target leading to the amino acid accumulation phenotype. This study establishes NS560 as a useful tool to study amino acid regulation, identifies new mechanisms of action of chloroquine, and demonstrates the importance of CTSL regulation of lysosomes.

Spectroscopy, Morphology, and Electrochemistry of Electrospun Polyamic Acid Nanofibers.

Polyamic acid (PAA) nanofibers produced by using the electrospinning method were fully characterized in terms of morphology and spectroscopy. A PAA nanofiber-modified screen-printed carbon electrode was applied to the detection of selected sulfonamides by following an electroanalytical protocol. The polyamic acid (PAA) nanofibers were characterized using Fourier transform infrared (FTIR) spectroscopy to study the integrity of polyamic acid functional groups as nanofibers by comparing them to chemically synthesized polyamic acid. A scanning electron microscope (SEM) was used to confirm the morphology of the produced nanofibers and 3D arrangement at the electrode interface. The Brunauer-Emmett-Teller (BET) method was used to determine the surface area of the nanofibers. Atomic force microscopy (AFM) was used to study the porosity and surface roughness of the nanofibers. Electrochemical evaluation based on diffusion-controlled kinetics was applied to determine the number of electrons transferred in the system, the surface concentration of the deposited PAA thin film (2.14 × 10-6 mol/cm2), and the diffusion coefficient (De) for the PAA nanofiber-modified screen-printed carbon electrode (9.43 × 10-7 cm-2/s). The reported LODs for sulfadiazine and sulfamethazine detection are consistent with requirements for trace-level monitoring by early warning diagnostic systems.

Biliary hyperkinesia: an indication for cholecystectomy?

BACKGROUND: The main indications for laparoscopic cholecystectomy are stone-related diseases in adults. With a normal abdominal ultrasound (US), a hepatobiliary iminodiacetic acid (HIDA) scan with ejection fraction (EF) is recommended to evaluate gallbladder function. Biliary dyskinesia or low gallbladder EF (EF < 35%) is a recognized indication for cholecystectomy. Recent articles report long-term resolution of symptoms in children with high EFs on the HIDA scan. The purpose of this study is to evaluate the response of patients with biliary colic and hyperkinetic gallbladder to cholecystectomy. We suggest that laparoscopic cholecystectomy might be a considerable surgical option in a subset of the adult population whose workup for food-related biliary abdominal pain is negative except for the high-value EF on HIDA scan. METHODS: Data were consecutively collected from all patients who underwent laparoscopic cholecystectomy between June 2012 and June 2019 at a single institution. Cases were identified using Current Procedural Terminology codes. Patients older than 17 years of age with the negative US (no stone, no sludge, no gallbladder wall thickening) and EF greater than 80% on cholecystokinin (CCK)-HIDA scan were included in this study. All patients were seen at 2 weeks and 10-16 months after surgeries. RESULTS: Over 7 years from June 2012 until June 2019, of 2116 patients who underwent laparoscopic cholecystectomy, 59 patients (2.78%) met study criteria. Postprandial abdominal pain was the most common symptom (43, 72.90%) followed by nausea/vomiting. Forty-seven patients (74.6%) had a reproduction of symptoms with CCK infusion. The average EF was 88.51%. Final pathology showed chronic cholecystitis in 41 (69.5%) patients, cholesterolosis in 13 (22%), polyp in 2 (3.4%). Thirty-six (61%) patients had complete resolution of symptoms, 9 (15%) patients had partial resolution, and 14 (24%) patients had no change. There was a complete resolution rate of 61% and an improvement rate of 76%. CONCLUSIONS: In patients with biliary symptoms, negative ultrasound, and elevated EF on HIDA scan (EF > 80%), laparoscopic cholecystectomy led to a significant rate of symptomatic relief. Interestingly, 94% also had unexpected pathologic findings. This disease process requires further analysis, but this could represent a new indication for laparoscopic cholecystectomy in the adult population.

A novel water-soluble fluorescence probe based on ICT lighten for detecting hydrogen sulfide and its application in bioimaging.

Endogenous H2S, considered to be involved in many physiological processes, has attracted more attention in fluorescence detection and bioimaging. Therefore, it is necessary to design probes with good biocompatibility and high bioavailability. In this study, a novel fluorescent probe, QN-1, based on azide group and quinoline derivatives was developed for detecting H2S. QN-1 can detect H2S specifically in aqueous phase, which indicated QN-1 has excellent water solubility. Besides, QN-1 shows excellent properties of higher selectivity and 11-fold fluorescence enhancement at 533 nm. Therefore, QN-1 with excellent properties can be used for cell imaging.

A High-Affinity Fluorescent Sensor for Catecholamine: Application to Monitoring Norepinephrine Exocytosis.

A fluorescent sensor for catecholamines, NS510, is presented. The sensor is based on a quinolone fluorophore incorporating a boronic acid recognition element that gives it high affinity for catecholamines and a turn-on response to norepinephrine. The sensor results in punctate staining of norepinephrine-enriched chromaffin cells visualized using confocal microscopy indicating that it stains the norepinephrine in secretory vesicles. Amperometry in conjunction with total internal reflection fluorescence (TIRF) microscopy demonstrates that the sensor can be used to observe destaining of individual chromaffin granules upon exocytosis. NS510 is the highest affinity fluorescent norepinephrine sensor currently available and can be used for measuring catecholamines in live-cell assays.

Near-Infrared Fluorescent Rosol Dye Tailored toward Lymphatic Mapping Applications.

An optical molecular imaging contrast agent that is tailored toward lymphatic mapping techniques implementing near-infrared (NIR) fluorescence image-guided navigation in the planning and surgical treatment of cancers would significantly aid in enabling the real-time visualization of the potential metastatic tumor-draining lymph node(s) for their needed surgical biopsy and/or removal, thereby ensuring unmissed disease to prevent recurrence and improve patient survival rates. Here, the development of the first NIR fluorescent rosol dye (THQ-Rosol) tailored to overcome the limitations arising from the suboptimal properties of the generic molecular fluorescent dyes commonly used for such applications is described. In developing THQ-Rosol, we prepared a progressive series of torsionally restrictive N-substituted non-NIR fluorescent rosol dyes based on density functional theory (DFT) calculations, wherein we discerned high correlations amongst their calculated energetics, modeled N-C3' torsion angles, and evaluated properties. We leveraged these strong relationships to rationally design THQ-Rosol, wherein DFT calculations inspired an innovative approach and synthetic strategy to afford an uncharged xanthene core-based scaffold/molecular platform with an aptly elevated p Ka value alongside NIR fluorescence emission (ca.700-900 nm). THQ-Rosol exhibited 710 nm NIR fluorescence emission, a 160 nm Stokes shift, robust photostability, and an aptly elevated p Ka value (5.85) for affording pH-insensitivity and optimal contrast upon designed use. We demonstrated the efficacy of THQ-Rosol for lymphatic mapping with in vitro and in vivo studies, wherein it revealed timely tumor drainage and afforded definitive lymph node visualization upon its administration and accumulation. THQ-Rosol serves as a proof-of-concept for the effective tailoring of an uncharged xanthene core-based scaffold/molecular platform toward a specific imaging application using rational design.

Robotic retro-rectus repair of parastomal hernias.

OBJECTIVE: To present our technique of robotic retrorectus parastomal hernia repair. BACKGROUND: Parastomal hernias represent a significant problem with high recurrence and long-term complications. An estimated of 120,000 new stomas are created per year with a prevalence of up to 800,000 patients in the U.S. 40-60% of these ostomies will never be reversed. Parastomal hernias cause skin breakdown and make adherence of ostomy appliances difficult, creating the need for frequent bag exchanges. They can also cause pain, bowel obstruction and bowel incarceration or strangulation. All of these factors affect quality of life and represent a significant burden to our health care system. There is no definitive gold-standard technique to repair parastomal hernias. The use of prosthesis decreases the recurrence rates, yet using prosthetic material can result in long-term complications. Surgeons have developed techniques of pre-peritoneal mesh placement to provide long-lasting repairs and at the same time prevent complications associated with the mesh. We believe that a robotic retro-rectus approach provides a secure repair and avoids leaving prosthetic material in the abdominal cavity at the same time. METHODS: A three-arm technique is used, inserting ports opposite to the target anatomy. Hernia contents are reduced protecting the ostomy loop and mesentery. The contralateral retro-rectus space is entered and this space is developed extensively across the midline and around the ostomy. The hernia defect is approximated. Concomitant ventral hernias are also repaired. A polypropylene mesh with a keyhole is used and wide coverage is ensured in all directions. The leaflets of the mesh are stitched together and the mesh is sutured to the abdominal wall. Finally, the retro-rectus space is closed. RESULTS: We have performed this technique in two patients safely and at 1-year follow-up there were no recurrences in either. On conclusion, this is a novel minimally invasive technique to repair parastomal hernias that provides wide coverage of the defect and avoids leaving mesh intraperitoneally.

A novel near-infrared ratiometric fluorescent probe for cyanide and its bioimaging applications.

A new near-infrared ratiometric type fluorescent probe was prepared. 3-formyl BODIPY derivatives without substituent group in the 2, 6-position was obtained through DDQ oxidation reaction. Furthermore, it reacted with indole salt to produce probe. This probe bears indolium group as the recognition site and the 3-formyl-BODIPY as fluorophore. The specific detection of cyanide was conducted the nucleophilic attack of cyanide toward the indolium group of the probe, breaking the intramolecular charge transfer (ICT) effect and generating a ratio change in the fluorescence signal. The probe has high selectivity and sensitivity for cyanide. Moreover, cell experiments indicated this probe was benign to HepG-2 cells, and has the potential application in imaging CN- in living HepG-2 cells.

A Multi-Component Sensor System for Detection of Amphiphilic Compounds.

Many biologically important compounds are amphiphilic in character. Glycolipids, for example, represent a biologically important class of amphiphiles. Receptors and sensors for such compounds must also be amphiphilic making them a challenge to prepare. Here, a cucurbit[8]uril (CB[8])-based sensor system has been prepared and tested for detection of amphiphilic compounds. This multi-component system consists: a CB[8], which acts as a hydrophobic lipid receptor, a hydrophilic pyridinium-based carbohydrate receptor, and a fluorescent indicator. The system self-assembles in aqueous solution. The pyridinium quenches the fluorescence of the indicator giving a strong turn-on response when it is displaced by the analyte. The sensor system was characterized by NMR, X-ray crystallography, and fluorescence titrations.

A Two-Input Fluorescent Logic Gate for Glutamate and Zinc.

The direct visualization of neurotransmitters is a continuing problem in neuroscience; however, functional fluorescent sensors for organic analytes are still rare. Herein, we describe a fluorescent sensor for glutamate and zinc ions. The sensor acts as a fluorescent logic gate, giving a turn-off response to glutamate or zinc ion alone. The combination of analytes produces a large increase in fluorescence. This type of sensor will aid in the study of neurotransmission, in this case, for neurons that copackage high concentrations of zinc and glutamate.

Turn-On Near-Infrared Fluorescent Sensor for Selectively Imaging Serotonin.

A molecular imaging tool that provides for the direct visualization of serotonin would significantly aid in the investigation of neuropsychiatric disorders that are attributed to its neuronal dysregulation. Here, the design, synthesis, and evaluation of NeuroSensor 715 (NS715) is presented. NS715 is the first molecular sensor that exhibits a turn-on near-infrared fluorescence response toward serotonin. Density functional theory calculations facilitated the design of a fluorophore based on a coumarin-3-aldehyde scaffold that derives from an electron-rich 1,2,3,4-tetrahydroquinoxaline framework, which provides appropriate energetics to prevent the hydroxyindole moiety of serotonin from quenching its fluorescence emission. Spectroscopic studies revealed that NS715 produces an 8-fold fluorescence enhancement toward serotonin with an emission maximum at 715 nm. Accompanying binding studies indicated NS715 displays a 19-fold selective affinity for serotonin and a modest affinity for catecholamines over other primary-amine neurotransmitters. The utility of NS715 toward neuroimaging applications was validated by selectively labeling and directly imaging norepinephrine within secretory vesicles using live chromaffin cells, which serve as a model system for specialized neurons that synthesize, package, and release only a single, unique type of neurotransmitter. In addition, NS715 effectively differentiated between cell populations that express distinct neurotransmitter phenotypes.

A novel method for the synthesis of 1,2-benzisoxazoline-3-one and its application to hypochlorite recognition.

The reaction of salicylhydroxamic acid with hypochlorite produces 1,2-benzisoxazoline-3-one, a heterocycle that contains a fluorophore. As a result, this reaction was used as the basis for a new, selective and sensitive fluorescence system for the recognition of hypochlorite. The effectiveness of the method was demonstrated by its use to detect hypochlorite in a disinfectant solution as well as to image hypochlorite in cells.

Tunable Molecular Logic Gates Designed for Imaging Released Neurotransmitters.

Tunable dual-analyte fluorescent molecular logic gates (ExoSensors) were designed for the purpose of imaging select vesicular primary-amine neurotransmitters that are released from secretory vesicles upon exocytosis. ExoSensors are based on the coumarin-3-aldehyde scaffold and rely on both neurotransmitter binding and the change in environmental pH associated with exocytosis to afford a unique turn-on fluorescence output. A pH-functionality was directly integrated into the fluorophore π-system of the scaffold, thereby allowing for an enhanced fluorescence output upon the release of labeled neurotransmitters. By altering the pH-sensitive unit with various electron-donating and -withdrawing sulfonamide substituents, we identified a correlation between the pKa of the pH-sensitive group and the fluorescence output from the activated fluorophore. In doing so, we achieved a twelvefold fluorescence enhancement upon evaluating the ExoSensors under conditions that mimic exocytosis. ExoSensors are aptly suited to serve as molecular imaging tools that allow for the direct visualization of only the neurotransmitters that are released from secretory vesicles upon exocytosis.

A highly selective fluorescent sensor for glucosamine.

A new fluorescent chemical sensor for glucosamine is reported. The sensor is based on a boronic acid-containing coumarin aldehyde and shows excellent selectivity for glucosamine by forming a boronic ester with the sugar diol as well as an iminium ion with the amine group of glucosamine. The sensor successfully discriminates glucosamine over other similar biomolecules in terms of both fluorescence intensity and binding affinity. This method provides a new concept for the design and synthesis of very selective turn-on optical sensors for selective detection of multi-functional biomolecules.

Coumarin-3-aldehyde as a scaffold for the design of tunable PET-modulated fluorescent sensors for neurotransmitters.

NeuroSensor 521 (NS521) is a fluorescent sensor for primary-amine neurotransmitters based on a platform that consists of an aryl moiety appended to position C4 of the coumarin-3-aldehyde scaffold. We demonstrate that sensors based on this platform behave as a directly linked donor-acceptor system that operates through an intramolecular acceptor-excited photoinduced electron transfer (a-PET) mechanism. To evaluate the PET process, a series of benzene- and thiophene-substituted derivatives were prepared and the photophysical properties, binding affinities, and fluorescence responses toward glutamate, norepinephrine, and dopamine were determined. The calculated energy of the highest occupied molecular orbital (EHOMO ) of the pendant aryl substituents, along with oxidation and reduction potential values derived from the calculated molecular orbital energy values of the platform components, allowed for calculation of the fluorescence properties of the benzene sensor series. Interestingly, the thiophene derivatives did not fit the typical PET model, highlighting the limitations of the method. A new sensor, NeuroSensor 539, displayed enhanced photophysical properties aptly suited for biological imaging. NeuroSensor 539 was validated by selectively labeling and imaging norepinephrine in secretory vesicles of live chromaffin cells.

Three-input logic gates with potential applications for neuronal imaging.

Convenient methods for the direct visualization of neurotransmitter trafficking would bolster investigations into the development of neurodegenerative diseases. Here, tunable fluorescent molecular logic gates with applications to neuronal imaging have been developed. The three-input AND molecular logic gates are based on the coumarin-3-aldehyde scaffold and designed to give a turn-on fluorescence response upon the corelease of glutamate and zinc from secretory vesicles via exocytosis. Spectroscopic studies reveal an 11-fold fluorescence enhancement under conditions mimicking exocytosis. Methylation of the scaffold was used to optimize the spectral profile of the sensors toward desired excitation wavelengths. A binding study that elucidates the sensor-analyte interactions is presented. These sensors serve as a proof-of-concept toward the direct imaging of neurotransmitters released upon exocytosis using fluorescent molecular logic gates.

Clinicians' views on factors that trigger a sudden onset of low back pain.

PURPOSE: Little is known about what triggers an episode of low back pain (LBP) in those presenting to primary care. Previous studies of risk factors have focused on specific occupational settings and work conditions. No study has asked primary care clinicians to consider what triggers an episode of sudden-onset LBP in patients presenting to them for care. The purpose of this study, therefore, was to describe the short- and long-term factors that primary care clinicians consider important in triggering a sudden episode of acute LBP. METHODS: One hundred and thirty-one primary care clinicians who were recruiting patients with LBP to a large observational study were invited to participate. A questionnaire was designed to obtain information about the clinician's characteristics, profession and clinical experience. We also asked clinicians to nominate the five short- and five long-term exposure factors, most likely to trigger a sudden episode of acute LBP, based on their experience. Descriptive statistics and frequency distributions were used to describe clinician's characteristics and the frequencies of the main risk factor categories were reported. RESULTS: Based on the views of 103 primary care clinicians, biomechanical risk factors appear to be the most important short-term triggers (endorsed by 89.3% of clinicians) and long-term triggers (endorsed by 54.2% of clinicians) for a sudden episode of acute LBP. Individual risk factors were endorsed by 39% of clinicians as important long-term triggers, while only 6.4% of clinicians considered them important short-term triggers. Other risk factors, such as psychological/psychosocial and genetic factors, were not commonly endorsed as risk factors for an episode of LBP by primary care clinicians. CONCLUSIONS: This study shows that primary care clinicians believe that biomechanical risk factors are the most important short-term triggers, while biomechanical and individual risk factors are the most important long-term triggers for a sudden onset of LBP. However, other risk factors, such as psychological/psychosocial and genetic, were not commonly endorsed as risk factors for an episode of LBP by primary care clinicians. Results of this study are based on primary care clinicians' views and further investigation is needed to test the validity of these suggested risk factors.