Type 2 Diabetes Mellitus (T2DM) is linked to multiple complications, including cognitive impairment, and the prevalence of memory-related neurodegenerative diseases is higher in T2DM patients. One possible theory is the alteration of the microvascular and macrovascular environment of the blood-brain barrier (BBB). In this study, we employed different approaches, including RT-PCR, functional pharmacokinetic studies using sodium fluorescein (NaFL), and confocal microscopy, to characterize the functional and molecular integrity of the BBB in a T2DM animal model, leptin receptor-deficient mutant mice (Leprdb/db mice). As a result, VCAM-1, ICAM-1, MMP-9, and S100b (BBB-related markers) dysregulation was observed in the Leprdb/db animal model compared to littermate wild-type mice. The brain concentration of sodium fluorescein (NaFL) increased significantly in Leprdb/db untreated mice compared to insulin-treated mice. Therefore, the permeability of NaFL was higher in Leprdb/db control mice than in all remaining groups. Identifying the factors that increase the BBB in Leprdb/db mice will provide a better understanding of the BBB microvasculature and present previously undescribed findings of T2DM-related brain illnesses, filling knowledge gaps in this emerging field of research.

Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Protein (CRISPR-Cas) systems have evolved several mechanisms to specifically target foreign DNA. These properties have made them attractive as biosensors. The primary drawback associated with contemporary CRISPR-Cas biosensors is their weak signaling capacity, which is typically compensated for by coupling the CRISPR-Cas systems to nucleic acid amplification. An alternative strategy to improve signaling capacity is to engineer the reporter, i.e., design new signal-generating substrates for Cas proteins. Unfortunately, due to their reliance on custom synthesis, most of these engineered reporter substrates are inaccessible to many researchers. Herein, we investigate a substrate based on a fluorescein (FAM)-tetramethylrhodamine (TAMRA) Förster resonant energy-transfer (FRET) pair that functions as a seamless \"drop-in\" replacement for existing reporters, without the need to change any other aspect of a CRISPR-Cas12a-based assay. The reporter is readily available and employs FRET to produce two signals upon cleavage by Cas12a. The use of both signals in a ratiometric manner provides for improved assay performance and a decreased time-to-result for several CRISPR-Cas12a assays when compared to a traditional FAM-Black Hole Quencher (BHQ) quench-based reporter. We comprehensively characterize this reporter to better understand the reasons for the improved signaling capacity and benchmark it against the current standard CRISPR-Cas reporter. Finally, to showcase the real-world utility of the reporter, we employ it in a Recombinase Polymerase Amplification (RPA)-CRISPR-Cas12a DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) assay to detect Human papillomavirus in patient-derived samples.

Controlled release of low molecular weight hydrophilic drugs, administered locally, allows maintenance of high concentrations at the target site, reduces systemic side effects, and improves patient compliance. Injectable hydrogels are commonly used as a vehicle. However, slow release of low molecular weight hydrophilic drugs is very difficult to achieve, mainly due to a rapid diffusion of the drug out of the drug delivery system. Here we present an injectable and self-healing hydrogel based entirely on the self-assembly of liposomes. Gelation of liposomes, without damaging their structural integrity, was induced by modifying the cholesterol content and surface charge. The small hydrophilic molecule, sodium fluorescein, was loaded either within the extra-liposomal space or encapsulated into the aqueous cores of the liposomes. This encapsulation strategy enabled the achievement of controlled and adjustable release profiles, dependent on the mechanical strength of the gel. The hydrogel had a high mechanical strength, minimal swelling, and slow degradation. The liposome-based hydrogel had prolonged mechanical stability in vivo with benign tissue reaction. This work presents a new class of injectable hydrogel that holds promise as a versatile drug delivery system. STATEMENT OF SIGNIFICANCE: The porous nature of hydrogels poses a challenge for delivering small hydrophilic drug, often resulting in initial burst release and shorten duration of release. This issue is particularly pronounced with physically crosslinked hydrogels, since their matrix can swell and dissipate rapidly, but even in cases where the polymers in the hydrogel are covalently cross-linked, small molecules can be rapidly released through its porous mesh. Here we present an injectable self-healing hydrogel based entirely on the self-assembly of liposomes. Small hydrophilic molecules were entrapped inside the extra-liposomal space or loaded into the aqueous cores of the liposomes, allowing controlled and tunable release profiles.

UNASSIGNED: Awake craniotomy (AC) maximizes the resection of lesions in eloquent brain areas while preserving functionality. Tumor delineation with intraoperative use of sodium fluorescein (NaFl) facilitates total resection. When used with AC, it may allow for safe resection without increasing the risk of postoperative neurologic deficits. This study investigated the efficacy and safety of the combined use of NaFl and AC for maximum safe resection in patients with brain metastases.
UNASSIGNED: Patients who underwent AC due to brain metastasis in the Department of Neurosurgery of Uludağ University\'s Faculty of Medicine between January 1, 2018 and August 1, 2022, were retrospectively analyzed. The study comprised 2 patient groups: plain AC (pAC) and NaFl-guided AC (NaFlg-AC). Surgical outcomes related to fluorescence intensity, degree of resection, perioperative complications, and postoperative neurological factors were evaluated.
UNASSIGNED: The pAC group included 16 patients (12 males, 4 females), and the NaFlg-AC group comprised 21 (13 males, 7 females). The mean patient ages for males and females were 61.4 years (61.4 ± 9.5 years) and 60.4 years (60.6 ± 12 years), respectively. The most common origin of the metastatic lesion was the lung in both the pAC and NaFlg-AC groups (n = 12 vs. n = 14, respectively). Gross total resection (GTR) was achieved in 85.7% of the patients in the NaFlg-AC group, whereas the GTR rate was 68.7% in the pAC group. There was no significant difference in GTR rates between the 2 groups (p = 0.254). The mean duration of the resection time was significantly shorter in the NaFlg-AC group (45.95 ± 7.00 min vs. 57.5 ± 12.51 min; p = 0.002). The patients\' Karnofsky Performance Status (KPS) score did not reach statistical significance at 6-month follow-up in either group compared to their preoperative baseline scores (p = 0.374). KPS did not show a significant difference between the 2 groups at any time.
UNASSIGNED: Fluorescence-guided resection in AC for metastatic tumors in sensory, motor, and cognitive areas is a feasible, safe, and convenient technique that significantly increases GTR rates and shortens operative time compared to conventional white light surgery without fluorescence guidance. It also does not increase the incidence of postoperative complications. With the combined use of AC and NaFl, ensuring clear and visible tumor margins during surgery and controlling patients\' neurological function in real-time are possible.

OBJECTIVE: The vital function of eloquent and deep brain areas necessitates precise treatment for tumors located in these regions. Fluorescein-guided surgery (FGS) has been widely used for high-grade gliomas (HGGs) resection. Nevertheless, the safety and efficacy of utilizing this technique for resecting brain tumors located in eloquent and deep-seated areas remain uncertain. This study aims to assess the safety and extent of resection of HGGs in these challenging tumors with fluorescein and explore its impact on patient survival.
METHODS: A retrospective analysis was conducted on the clinical and radiological data of 67 consecutive patients with eloquent or deep-seated HGGs who underwent surgery between January 2020 and June 2023. Lacroix functional location grade was used to determine the eloquence of the tumors. The comparison between the fluorescence-guided surgery group (FGS, n = 32) and the conventional white-light microscopic surgery group (non-FGS, n = 35) included assessments of extent of resection (EOR), rates of gross total resection (GTR, 100%) and near-total resection (NTR, 99 to 98%), postoperative Neurologic Assessment in Neuro-Oncology (NANO) scores, overall survival (OS), and progression-free survival (PFS), to evaluate the safety and efficacy of fluorescein-guided technology in tumor resection at these specific locations.
RESULTS: Baseline of demographics, lesion location, and pathology showed no significant difference between the two groups. GTR of the FGS group was higher than the non-FGS group (84.4% vs. 60.0%, OR 3.60, 95% CI 1.18-10.28, p < 0.05). The FGS group also showed higher GTR + NTR (EOR ≥ 98%) than the non-FGS group (93.8% vs. 65.7%, OR 7.83, 95% CI 1.86-36.85, p < 0.01). 87.0% of eloquent tumors (Lacroix grade III) in the FGS group achieved GTR + NTR, compared to 52.2% of control group (OR 6.11, 95% CI 1.50-22.78, p < 0.05). For deep-seated tumors, the rate of GTR + NTR in the two groups were 91.7% and 53.3%, respectively (OR 9.62, 95% CI 1.05-116.50, p < 0.05). No significant difference of the preoperative NANO score of the two groups was found. The postoperative NANO score of the FGS group was significantly lower than the non-FGS group (2.56 ± 1.29 vs. 3.43 ± 1.63, p < 0.05). Median OS of the FGS group was 4.2 months longer than the non-FGS group despite no statistical difference (18.2 months vs. 14.0 months, HR 0.63, 95% CI 0.36-1.11, p = 0.112), while PSF was found significantly longer in FGS patients than those of the non-FGS group (11.2 months vs. 7.7 months, HR 0.59, 95% CI 0.35-0.99, p < 0.05).
CONCLUSIONS: Sodium fluorescein-guided surgery for high-grade gliomas in eloquent and deep-seated brain regions enables more extensive resection while preserving neurologic function and improve patient survival.

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer-fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.

Amorphous silica particles (ASPs) have been reported to exhibit bioactive properties and are becoming the focus of attention as bioceramics. However, their interactions with proteins in living organisms remain to be understood and need to be investigated in order to achieve wider applications. Our research group found that chlorine (Cl)-containing ASPs are useful for protein immobilization. Photofunctional dyes (fluorescein (FS-), methylene blue (MB+)) that have the carboxy and amino groups as the main functional groups were immobilized on the Cl-containing ASPs via the mechanochemical method as the model molecule and their spectral properties were used to investigate and discuss the organic/inorganic interfacial bonding states. In FS-, the oxygen atoms of the carboxy groups in the molecule were immobilized by the hydrogen bonds with the silanol groups on the ASPs surfaces, indicating that there is an optimum Cl content for the immobilization as the monomer state. In the case of MB+, as the Cl concentration in the ASPs increases, the immobilization via the electrostatic interactions between the Cl in the ASPs and the terminal dimethylamino group, and the hydrogen bonding between the N atoms of the MB+ hetero ring and the particle silanol group were enhanced. These results mainly suggest that the protein adsorption system occurs through the hydrogen bonding between the carboxy groups of the protein and the silanol groups on the particles and via electrostatic interactions between the amino groups of the protein and the dissociated silanol groups and the contained Cl at the particles. Thus, the spectral characterization using dyes as probes is expected to predict the protein interactions with the amorphous silica particles.

Oral ingestion of fluorescein can be done in ambulatory pediatric clinics. We show that oral ultra-widefield fluorescein angiography is a non-invasive approach to rapidly diagnose and manage a diverse set of pediatric retinal vascular diseases.

Gold nanoparticles (AuNPs) are good candidates for donor material in energy transfer systems and can easily be functionalized with various ligands on the surface with Au-S bonding. Cyclodextrin (CD) forms inclusion complexes with fluorophores due to its unique structure for host-guest interaction. In this study, we fabricated βCD-functionalized AuNPs using different lengths of thiol ligands and recognized cholesterol to confirm the energy-transfer-based turn-on fluorescence mechanism. AuNP-βCD conjugated with various thiol ligands and quenched the fluorescein (Fl) dye, forming βCD-Fl inclusion complexes. As the distance between AuNPs and βCD decreased, the quenching efficiency became higher. The quenched fluorescence was recovered when the cholesterol replaced the Fl because of the stronger binding affinity of the cholesterol with βCD. The efficiency of cholesterol recognition was also affected by the energy transfer effect because the shorter βCD ligand had a higher fluorescence recovery. Furthermore, we fabricated a liposome with cholesterol embedded in the lipid bilayer membrane to mimic the cholesterol coexisting with lipids in human serum. These cellular cholesterols accelerated the replacement of the Fl molecules, resulting in a fluorescence recovery higher than that of pure lipid. These discoveries are expected to give guidance towards cholesterol sensors or energy-transfer-based biosensors using AuNPs.

BACKGROUND: In recent years, insulin eye drops have attracted increasing attention from researchers and ophthalmologists. The aim of this study was to investigate the efficacy and possible mechanism of action of insulin eye drops in diabetic mice with corneal wounds.
METHODS: A type 1 diabetes model was induced, and a corneal epithelial injury model of 2.5 mm was established. We used corneal fluorescein staining, hematoxylin-eosin (H-E) staining and the Cochet-Bonnet esthesiometer to examine the process of wound healing. Subsequently, the expression levels of Ki-67, IL-1β, β3-tubulin and neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP), were examined at 72 h after corneal injury.
RESULTS: Fluorescein staining demonstrated an acceleration of the recovery of corneal epithelial injury in diabetic mice compared with the saline treatment, which was further evidenced by the overexpression of Ki-67. Moreover, 72 h of insulin application attenuated the expression of inflammatory cytokines and neutrophil infiltration. Remarkably, the results demonstrated that topical insulin treatment enhanced the density of corneal epithelial nerves, as well as neuropeptide SP and CGRP release, in the healing cornea via immunofluorescence staining.
CONCLUSIONS: Our results indicated that insulin eye drops may accelerate corneal wound healing and decrease inflammatory responses in diabetic mice by promoting nerve regeneration and increasing levels of neuropeptides SP and CGRP.