Polyamidoamine-Remdesivir Conjugate: Physical Stability and Cellular Uptake Enhancement

Nucleoside analogue antiviral remdesivir works by inhibiting the RNA-dependent RNA polymerase enzyme and terminating the viral replication. Currently, remdesivir is under a clinical trial for its activity against SARS-CoV-2. In the blood, remdesivir will undergo an enzymatic reaction to become monophosphate analogue form which is difficult to penetrate into the cell membrane. PAMAM (polyamidoamine) dendrimer is a good carrier to encapsulate remdesivir as a water-insoluble drug (0,339 mg/mL). Entrapment of remdesivir in the PAMAM cavity avoided remdesivir molecules to not undergo the enzymatic reactions. This study aimed to synthesize, characterize and evaluate cellular uptake of PAMAM-Remdesivir conjugate. PAMAM-Remdesivir was prepared with various stirring times (3, 6, 12, 24, and 48 hours). The conjugates were characterized to observe the size and particle distribution using Particle Size Analyzer, encapsulating efficiency using UV-Vis Spectroscopy, interaction between PAMAM and remdesivir particle using Fourier Transform Infrared Spectroscopy and cellular uptake of PAMAM-RDV using Fluorescence Microscope. The optimized stirring time of PAMAM-Remdesivir conjugate was 24 hours wich resulted the particles charge of + 23,07 mV of zeta potential, 1008 nm of particle size, 0,730 of PDI, and 69% entrapment efficiency. In addition, the FTIR analysis showed that remdesivir molecules successfully conjugated to PAMAM. Thus, through strring optimization time, the remdesivir molecules were successfully entrapped to PAMAM cavity. The cellular uptake in Vero Cell of PAMAM-RDV conjugated fluorescein isothiocyanate was observed with fluorescence microscope and had a stronger intensity than remdesivir only solution.

Long-chain PUFA ameliorate ETEC-induced intestinal inflammation and cell injury by modulating pyroptosis and necroptosis signaling pathways in IPEC-1 cells

This study was aimed to investigate whether eicosapentaenoic acid (EPA) and arachidonic acid (ARA), the representative n-3 or n-6 polyunsaturated fatty acids (PUFA), could alleviate enterotoxigenic Escherichia coli (ETEC) K88-induced inflammation and injury of intestinal porcine epithelial cells 1 (IPEC-1) by modulating pyroptosis and necroptosis signaling pathways. IPEC-1 cells were cultured with or without EPA or ARA in the presence or absence of ETEC K88. EPA and ARA reduced ETEC K88 adhesion and endotoxin content in the supernatant. EPA and ARA increased transepithelial electrical resistance (TEER) and decreased permeability of fluorescein isothiocyanate-labeled dextran (FD4), and increased membrane protein expression of occludin, ZO-1 and claudin-1, and relieved disturbed distribution of these proteins. EPA and ARA also reduced cell necrosis ratio. EPA or ARA reduced mRNA and concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8, and decreased mRNA abundances of intestinal toll-like receptors 4 (TLR4) and its downstream signals. Moreover, EPA and ARA downregulated mRNA expression of nod-like receptor protein 3 (NLRP3), caspase 1 and IL-18, and inhibited protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D and caspase-1. Finally, EPA and ARA reduced mRNA expression of fas-associated death domain protein (FADD), caspase 8, receptor interacting protein kinase (RIP) 1, mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase 5 (PGAM5), motility related protein 1 (Drp1) and high mobility protein 1 (HMGB1), and inhibited protein expression of phosphorylated-RIP1 (p-RIP1), p-RIP3, p-MLKL and HMGB1. These data demonstrate that EPA and ARA prevent ETEC K88-induced cell inflammation and injury, which is partly through inhibiting pyroptosis and necroptosis signaling pathways.

Perfusion of Brain Preautonomic Areas in Hypertension: Compensatory Absence of Capillary Rarefaction and Protective Effects of Exercise Training

Remodeling of capillary rarefaction and deleterious arteries are characteristic hallmarks of hypertension that are partially corrected by exercise training. In addition, experimental evidence showed capillary rarefaction within the brain cortex and reduced cerebral blood flow. There is no information on hypertension- and exercise-induced effects on capillary profile and function within preautonomic nuclei. We sought now to evaluate the effects of hypertension and exercise training (T) on the capillary network within hypothalamic paraventricular (PVN) and solitary tract (NTS) nuclei, and on the remodeling of brain arteries. Age-matched spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY), submitted to moderate T or kept sedentary (S) for three months, were chronically cannulated for hemodynamic recordings at rest. Rats were anesthetized for i.v. administration of fluorescein isothiocyanate (FITC)-dextran (capillary volume/density measurements) or 4% paraformaldehyde perfusion (basilar, middle, and posterior arteries' morphometry) followed by brain harvesting and processing. Other groups of conscious rats had carotid blood flow (CBF, ultrasound flowmeter) acquired simultaneously with hemodynamic recordings at rest and exercise. SHR-S exhibited elevated pressure and heart rate, reduced CBF, increased wall/lumen ratio of arteries, but no capillary rarefaction within the PVN and NTS. T improved performance gain and caused resting bradycardia in both groups; reduction of pressure and sympathetic vasomotor activity and normalization of the wall/lumen ratio were only observed in SHR-T. T groups responded with marked PVN and NTS capillary angiogenesis and augmented CBF during exercise; to avoid overperfusion at rest, reduced basal CBF was observed only in WKY-T. Data indicated that the absence of SHR-S capillary rarefaction and the intense SHR-T angiogenesis within autonomic areas associated with correction of deleterious arteries' remodeling are essential adjustments to hypertension and exercise training, respectively. These adaptive responses maintain adequate baseline perfusion in SHR-S and SHR-T preautonomic nuclei, augmenting it in exercised rats when a well-coordinated autonomic control is required.

Pharmacokinetics and Excretion Study of Lycium barbarum Polysaccharides in Rats by FITC-Fluorescence Labeling

A high-performance gel permeation chromatography fluorescence detection (HPGPC-FD) method combined with fluorescein isothiocyanate (FITC) labeling was established for the microanalysis of L. barbarum polysaccharides (LBP). The calibration curves linear over the range of 0.2–20 µg/mL in rat plasma, and 0.25–500 μg/mL in urine and feces samples with correlation coefficients greater than 0.99. The inter-day and intra-day precisions (RSD, %) of the method were under 15% with the relative recovery ranging from 84.6% to 104.0% and the RSD ranging from 0.47% to 7.28%. The concentration–time curve of LBP-FITC in plasma following intragastric administration at 100, 50 and 25 mg/kg well fitted to a nonlinear model. LBP-FITC slowly eliminated from plasma according to the long half-lives (t1/2 = 31.39, 38.09, and 45.76 h, respectively) and mean retention times (MRT0–t = 18.38, 19.15 and 20.07 h, respectively; AUC0–∞ = 230.49, 236.18 and 242.57 h, respectively) after administration of LBP-FITC at doses of 100, 50, and 25 mg/kg, respectively. After intragastric administration at 50 mg/kg for 72 h, the concentration of LBP-FITC in urine and feces was 0.09 ± 0.04% and 92.18 ± 3.61% respectively; the excretion rate of urine was the highest in 0–4 h period and decreased continuously in 4–24 h period. The excretion rate of feces was the highest in 4–10 h, 48.28 ± 9.349% in feces within 4–10 h, and decreased rapidly in 10–24 h. The present study showed that LBP was absorbed as its prototype and most proportion of LBP was excreted from feces, indicating a long time remaining in intestine.

Novel role of zonulin in the pathophysiology of gastro-duodenal transit: a clinical and translational study

We examined the relationship between zonulin and gastric motility in critical care patients and a translational mouse model of systemic inflammation. Gastric motility and haptoglobin (HP) 2 isoform quantification, proxy for zonulin, were examined in patients. Inflammation was triggered by lipopolysaccharide (LPS) injection in C57Bl/6 zonulin transgenic mouse (Ztm) and wildtype (WT) mice as controls, and gastro-duodenal transit was examined by fluorescein-isothiocyanate, 6 and 12 h after LPS-injection. Serum cytokines and zonulin protein levels, and zonulin gastric-duodenal mRNA expression were examined. Eight of 20 patients [14 years, IQR (12.25, 18)] developed gastric dysmotility and were HP2 isoform-producing. HP2 correlated with gastric dysmotility (r = − 0.51, CI − 0.81 to 0.003, p = 0.048). LPS injection induced a time-dependent increase in IL-6 and KC-Gro levels in all mice (p < 0.0001). Gastric dysmotility was reduced similarly in Ztm and WT mice in a time-dependent manner. Ztm had 16% faster duodenal motility than WT mice 6H post-LPS, p = 0.01. Zonulin mRNA expression by delta cycle threshold (dCT) was higher in the stomach (9.7, SD 1.4) than the duodenum (13.9, SD 1.4) 6H post-LPS, p = 0.04. Serum zonulin protein levels were higher in LPS-injected mice compared to vehicle-injected animals in a time-dependent manner. Zonulin correlated with gastric dysmotility in patients. A mouse model had time-dependent gastro-duodenal dysmotility after LPS-injection that paralleled zonulin mRNA expression and protein levels.

A Fluorescent Biosensor for Sensitive Detection of Salmonella Typhimurium Using Low-Gradient Magnetic Field and Deep Learning via Faster Region-Based Convolutional Neural Network

In this study, a fluorescent biosensor was developed for the sensitive detection of Salmonella typhimurium using a low-gradient magnetic field and deep learning via faster region-based convolutional neural networks (R-CNN) to recognize the fluorescent spots on the bacterial cells. First, magnetic nanobeads (MNBs) coated with capture antibodies were used to separate target bacteria from the sample background, resulting in the formation of magnetic bacteria. Then, fluorescein isothiocyanate fluorescent microspheres (FITC-FMs) modified with detection antibodies were used to label the magnetic bacteria, resulting in the formation of fluorescent bacteria. After the fluorescent bacteria were attracted against the bottom of an ELISA well using a low-gradient magnetic field, resulting in the conversion from a three-dimensional (spatial) distribution of the fluorescent bacteria to a two-dimensional (planar) distribution, the images of the fluorescent bacteria were finally collected using a high-resolution fluorescence microscope and processed using the faster R-CNN algorithm to calculate the number of the fluorescent spots for the determination of target bacteria. Under the optimal conditions, this biosensor was able to quantitatively detect Salmonella typhimurium from 6.9 × 101 to 1.1 × 103 CFU/mL within 2.5 h with the lower detection limit of 55 CFU/mL. The fluorescent biosensor has the potential to simultaneously detect multiple types of foodborne bacteria using MNBs coated with their capture antibodies and different fluorescent microspheres modified with their detection antibodies.

The Influence of Electronic Acupuncture At A Specific Frequency In Facilitating The Passage of NGF Through The Blood-Brain Barrier And Its Effect On Learning And Memory In MCAO/R Rats

The blood-brain barrier (BBB) maintains the balance of the internal environment of the brain and strictly controls substance exchange between the brain and blood dynamically but stably. Transient increases in the permeability of the BBB plays an important role in helping macromolecular drugs enter the brain to exert their pharmacological effects. Previous research has revealed that electronic acupuncture (EA) stimulation at a specific frequency can enhance the permeability of the BBB and induce the entry of 20 kDa fluorescein isothiocyanate-dextran (FITC-dextran) into the cerebral cortex, but whether it can also allow drugs to pass the BBB remains unknown. We hypothesized that EA at a specific frequency could open the BBB and induce the entry of nerve growth factor (NGF) into the brain to exert its therapeutic effect. To simulate the clinical apoplexy sequelae observed in patients and determine the basic timing of BBB repair under pathological conditions, we employed the middle cerebral artery occlusion (MCAO) model and assessed changes in the permeability and structure of the BBB by measuring both the intensity of Evans blue (EB) staining and the cerebral infarction volume and evaluating the ultrastructure of the BBB. Then, we used a laser spectrometer and immunofluorescence to observe entry of NGF into the brain. Finally, we assessed the learning and memory ability of rats and used the DeadEnd TM Fluorometric TUNEL System to assess apoptosis in the hippocampus. Our results showed that the BBB was essentially repaired three weeks after MCAO, indicating that EA stimulation at a specific frequency can enhance BBB permeability and induce NGF uptake by prefrontal neurons. In the presence of EA stimulation, entry of NGF into the brain promoted learning and memory in rats and inhibited the apoptosis of neurons in the hippocampus. In this study, the MCAO model was used to determine the timing of BBB repair under pathological conditions and assess the EA stimulation-induced entry of NGF into the brain to exert its therapeutic effect. EA could serve as a new strategy for delivering therapeutics to the central nervous system (CNS), given that EA stimulation at a specific frequency was shown to increase the permeability of the BBB. Further study of the mechanism underlying the opening of the BBB and its timing is needed.