Proteoid biodynamers for safe mRNA transfection via pH-responsive nanorods enabling endosomal escape.

The recent success of mRNA vaccines using lipid-based vectors highlights the importance of strategies for nucleotide delivery under the pandemic situation. Although current mRNA delivery is focused on lipid-based vectors, still they need to be optimized for increasing stability, targeting, and efficiency, and for reducing toxicity. In this regard, other vector systems featuring smart strategies such as pH-responsive degradability and endosomal escape ability hold the potential to overcome the current limitations. Here, we report pH-responsive polymeric nanorods made of amino acid-derivatives connected by dynamic covalent bonds called proteoid-biodynamers, as mRNA vectors. They show excellent biocompatibility due to the biodegradation, and outstanding transfection. The biodynamers of Lys, His, and Arg or monomer mixtures thereof were shown to form nanocomplexes with mRNA. They outperformed conventional transfection agents three times regarding transfection efficacy in three human cell lines, with 82-98% transfection in living cells. Also, we confirmed that the biodynamers disrupted the endosomes up to 10-fold more in number than the conventional vectors. We discuss here their outstanding performance with a thorough analysis of their nanorod structure changes in endosomal microenvironments.

Evaluation of Corneal Alterations After Short-Term Silicone Hydrogel Contact Lens Use by Confocal Microscopy.

Objectives: To evaluate the corneal subbasal nerve morphology, corneal sensitivity, and anterior segment alterations in short-term silicone hydrogel contact lens (SiHCL) users by confocal microscopy. Materials and Methods: The study included 25 right eyes of 25 male volunteers aged 25-30 years who had never used SiHCLs before. ocular surface disease index (OSDI), tear break-up time, Schirmer test, tear meniscus area, strip meniscometry tube, corneal sensitivity, and corneal subbasal nerve morphology were evaluated before and after 1 month of CL use. Results: OSDI was 10.6±1.1 before CL use and 17.2±1.2 after 1 month of CL use (p<0.01). Schirmer test distance was 16.3±2.3 mm before and 14.3±1.9 mm after 1 month of CL use (p>0.05). Tear film break-up time was 7.1±0.4 s before and 6.2±0.3 s after CL use (p>0.05). The tear meniscus area was 0.026±0.002 mm2 before and 0.024±0.001 mm2 after 1 month of CL use (p>0.05). Strip meniscometry tube results were 5.4±0.9 mm before and 4.9±0.8 mm after 1 month of CL use (p>0.05). Corneal sensitivity values were 3.2±0.4 mm before and 2.95±0.3 mm after 1 month of CL use (p>0.05). Dendritic cell density evaluated by confocal microscopy was 14.84±3.1 cells/mm2 before and 32.57±4.2 cells/mm2 after 1 month of CL use (p<0.01). Subbasal nerve tortuosity was 0.92±0.2 before and 1.03±0.2 after 1 month of CL use (p>0.05). Subbasal nerve density was measured as 4726±310 pixels/frame before and 4570±272 pixels/frame after 1 month of CL use (p>0.05). Conclusion: After a month of SiHCL use, no significant changes were observed in tear secretion, corneal sensitivity, tear meniscus volume, subbasal corneal nerve density, reflectivity, or tortuosity, while a significant increase was found in OSDI and dendritic cell density.

Comparison of Central Corneal Thickness Measurements with Four Different New Devices and Ultrasound Pachymetry.

Objectives: To assess the central corneal thickness (CCT) with 5 different devices, evaluate the repeatability of the devices, and determine the possible relationship between thickness values and sex. Materials and Methods: The study included 308 eyes of 154 patients (76 women, 78 men) between the ages of 18-30 who presented to the Ophthalmology Clinic of Mugla Sitki Koçman University Training and Research Hospital. Autorefractor (Topcon, Japan), ultrasound pachymetry (UP) (Ceniscan, USA), high-resolution Pentacam (Oculus, USA), anterior segment-optical coherence tomography (AS-OCT) (Optovue, USA), and Spectralis AS-OCT (Heidelberg, Germany) measurements were assessed. Results: The mean age of the study participants was 23.2±0.2 years and the mean CCT was 540±14.1 µm, with no statistically significant difference in CCT between sexes (p>0.05). Mean CCT values were 557.0±26.7 µm with the autorefractor, 543.6±32.9 µm with UP, 533.8±30.2 µm with the Oculus Pentacam, 519.8±30.1 µm with Optovue AS-OCT, and 547.5±31.6 µm with Heidelberg AS-OCT. Pairwise comparisons between devices showed that the Optovue AS-OCT gave significantly lower CCT measurements than the autorefractor and Heidelberg AS-OCT device (p=0.027 and p=0.033, respectively). The coefficient of repeatability for autorefractor, UP, high-resolution Pentacam, Optovue AS-OCT, and Heidelberg AS-OCT CCT measurements were 1.51%, 2.46%, 3.72%, 2.57%, and 3.34%, respectively. Conclusion: Measurements made with five different devices showed that CCT was comparable and clinically usable. However, it was determined that the Optovue AS-OCT showed lower CCT values compare to other devices. When compared in terms of repeatability, it was found to be lower in the Pentacam than other devices.

Identification of RAD51-BRCA2 Inhibitors Using N-Acylhydrazone-Based Dynamic Combinatorial Chemistry.

RAD51 is an ATP-dependent recombinase, recruited by BRCA2 to mediate DNA double-strand breaks repair through homologous recombination and represents an attractive cancer drug target. Herein, we applied for the first-time protein-templated dynamic combinatorial chemistry on RAD51 as a hit identification strategy. Upon design of N-acylhydrazone-based dynamic combinatorial libraries, RAD51 showed a clear templating effect, amplifying 19 N-acylhydrazones. Screening against the RAD51-BRCA2 protein-protein interaction via ELISA assay afforded 10 inhibitors in the micromolar range. Further 19F NMR experiments revealed that 7 could bind RAD51 and be displaced by BRC4, suggesting an interaction in the same binding pocket of BRCA2. These results proved not only that ptDCC could be successfully applied on full-length oligomeric RAD51, but also that it could address the need of alternative strategies toward the identification of small-molecule PPI inhibitors.

Structure-Based Design of α-Substituted Mercaptoacetamides as Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa.

Antivirulence therapy has become a widely applicable method for fighting infections caused by multidrug-resistant bacteria. Among the many virulence factors produced by the Gram-negative bacterium Pseudomonas aeruginosa, elastase (LasB) stands out as an important target as it plays a pivotal role in the invasion of the host tissue and evasion of the immune response. In this work, we explored the recently reported LasB inhibitor class of α-benzyl-N-aryl mercaptoacetamides by exploiting the crystal structure of one of the compounds. Our exploration yielded inhibitors that maintained inhibitory activity, selectivity, and increased hydrophilicity. These inhibitors were found to reduce the pathogenicity of the bacteria and to maintain the integrity of lung and skin cells in the diseased state. Furthermore, two most promising compounds increased the survival rate of Galleria mellonella larvae treated with P. aeruginosa culture supernatant.

N-Aryl Mercaptopropionamides as Broad-Spectrum Inhibitors of Metallo-ß-Lactamases.

Drug-resistant pathogens pose a global challenge to public health as they cause diseases that are extremely difficult to cure. Metallo-ß-lactamases (MBLs) are a diverse set of zinc-containing enzymes that catalyze the hydrolysis of ß-lactam drugs, including carbapenems, which are considered as the last resort to fight severe infections. To restore the activity of current ß-lactam antibiotics and to offer an orthogonal strategy to the discovery of new antibiotics, we have identified a series of polar N-aryl mercaptopropionamide derivatives as potent inhibitors of several class B1 MBLs. We have identified a hit structure with high selectivity restoring the effect of imipenem and reducing minimum inhibitory concentration (MIC) values up to 256-fold in resistant isolates from Escherichia coli. Furthermore, the combination of imipenem with our inhibitor showed in vivo efficacy in a Galleria mellonella model, increasing the survival rate of infected larvae by up to 31%.

Targeting Extracellular Bacterial Proteases for the Development of Novel Antivirulence Agents.

As resistance to clinically available antibiotics persistently increases, applying new strategies to target pathogenic bacteria are paramount to design effective drugs. Bacterial proteases play vital roles in cell viability and stress response, contributing to the pathogenicity of the resistant bacteria. Targeting these extracellular enzymes by antivirulence therapy is a prominent strategy in combating multi-drug resistant bacteria. By preventing the colonization and infiltration of the host, this method can lower selection pressure and reduce resistance development significantly. Here, we review the role of bacterial proteases, the rise of antivirulence therapy and we report on the development of novel antivirulence agents targeting two key virulence factors: elastase B (LasB) from Pseudomonas aeruginosa and collagenase H (ColH) from Clostridium histolyticum.

Substrate-Inspired Fragment Merging and Growing Affords Efficacious LasB Inhibitors.

Extracellular virulence factors have emerged as attractive targets in the current antimicrobial resistance crisis. The Gram-negative pathogen Pseudomonas aeruginosa secretes the virulence factor elastase B (LasB), which plays an important role in the infection process. Here, we report a sub-micromolar, non-peptidic, fragment-like inhibitor of LasB discovered by careful visual inspection of structural data. Inspired by the natural LasB substrate, the original fragment was successfully merged and grown. The optimized inhibitor is accessible via simple chemistry and retained selectivity with a substantial improvement in activity, which can be rationalized by the crystal structure of LasB in complex with the inhibitor. We also demonstrate an improved in vivo efficacy of the optimized hit in Galleria mellonella larvae, highlighting the significance of this class of compounds as promising drug candidates.

Sensitivity and Specificity of the Optical Coherence Tomography Angiography for Detection of Neovascularization and Evaluation of Peripheral Ischemia in Diabetic Retinopathy.

Objectives: The purpose of this study was to identify the sensitivity and specificity of optical coherence tomography angiography (OCTA) parameters for the presence of neovascularization elsewhere (NVE) and to investigate the relationship between ischemic areas. Methods: This study included 59 eyes with non-proliferative diabetic retinopathy (NPDR) and 36 eyes with proliferative diabetic retinopathy (PDR). The foveal avascular zone (FAZ), vessel density (VD) for the superficial and the deep capillary plexus (DCP), choriocapillaris flow area (CCP), and non-perfusion area (unit²) were recorded. The area under the curve (AUC) under the receiver operating characteristic curves, sensitivity and specificity were calculated for statistically significant outcomes. Later, based on visual acuity, PDR group was subdivided into group 2A: PDR eyes with VA ≤0.2 logMAR and group 2B: PDR eyes with VA>0.2 logMAR. Non-perfusion area and OCTA features were compared between the subgroups. Results: The VD in DCP was significantly lower, FAZ and non-perfusion area were larger in PDR group (p=0.001, p<0.001, and p<0.001). The AUC for presence of NVE, for the VD, was 0.710 (p=0.012) with sensitivity and specificity of 64% and 65%, for the FAZ was 0.746 (p<0.001) with sensitivity and specificity of 72% and 72.7%. There was a significant positive correlation between the FAZ and non-perfusion area (For NPDR, p=0.025, for PDR p<0.001). There was a significant negative correlation between the VD in DCP and ischemic area in PDR group. (p<0.001) In group 2B, non-perfusion area and FAZ were larger than group 2A. The VD and CCP flow area were also lower in group 2B (All, p<0.05). Conclusion: In cases with decreased VD in DCP and increased FAZ, the probability of PDR increases. Despite the sensitivity and specificity of the OCTA indices for the prediction of NVE being moderate, the OCTA is very useful in evaluating the microvascular structure in DR.

"Off-on" switching of intracellular singlet oxygen release under biocompatible conditions.

Precise spatiotemporal control of singlet oxygen generation is of immense importance considering its involvement in photodynamic therapy. In this work, we present a rational design for an endoperoxide which is highly stable at ambient temperatures yet, can rapidly be converted into a highly labile endoperoxide, thus releasing the "stored" singlet oxygen on demand. The "off-on" chemical switching from the stable to the labile form is accomplished by the reaction with fluoride ions. The potential utility of controlled singlet oxygen release was demonstrated in cell cultures.

Uroprotective effect of ambroxol in cyclophosphamide-induced cystitis in mice.

PURPOSE: Hemorrhagic cystitis (HC) is defined as any types of acute or chronic inflammation of urinary bladder with several reasons. One of the most common causes of HC is cyclophosphamide (CYP), an effective antineoplastic agent, due to its urotoxic potential. Ambroxol (AMB) is a mucoactive drug that has been used for numerous respiratory diseases. Besides its mucolytic activity, AMB is a potent antioxidant and antiinflammatory agent that is becoming more attractive for the treatment of several oxidative/inflammatory disorders. The aim of this study was to evaluate the uroprotective potential of AMB in CYP-induced HC. METHOD: Male Balb/c mice were pretreated with AMB (30, 70, and 100 mg/kg) once a day for 3 consecutive days before HC induction with CYP (300 mg/kg). Mesna (30 mg/kg;i.p.), only drug in the management of CYP-induced HC, was administered 20 min before; 4 and 8 h after cystitis induction. The urinary bladders were harvested and evaluated in functional, biochemical, and histological studies. RESULTS: CYP-induced HC markedly reduced acetylcholine (ACh)-induced contractions in detrusor strips and AMB at 100 mg/kg caused a significant increase in the responsiveness to ACh. Pretreatment with AMB prevented the elevation of malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-α) level, reduction of total glutathione (GSH) that induced by CYP. However, treatment with AMB did not improve the bladder weight and some histological parameters. CONCLUSION: These results suggest that AMB pretreatment could improve CYP-induced HC via antioxidant and antiinflammatory activities.

Uroprotective effect of pantoprazole against cyclophosphamide-induced cystitis in mice.

PURPOSE: The aim of the present study was to evaluate the potential uroprotective effect of pantoprazole (PPZ) in a mouse model of cyclophosphamide (CP)-induced hemorrhagic cystitis (HC) due to its antioxidant and anti-inflammatory properties. METHODS: Balb/c mice received a single intraperitoneal (i.p.) injection of CP (300 mg/kg) to induce HC. PPZ (20, 50, and 100 mg/kg/day;i.p.) was administered for 3 consecutive days before the induction of HC. Mesna (30 mg/kg;i.p.) was administered 20 min before, 4 and 8 h after CP injection to compare the protective effects of PPZ. After 24 h of HC induction, the bladders were removed for functional studies, biochemical analyses, and histopathological examination. RESULTS: In vitro contractility studies demonstrated that CP-induced HC decreased the responsiveness of detrusor muscle strips to acetylcholine (ACh), which was reversed by PPZ pretreatment at all doses tested. However, mesna treatment was not able to improve responsiveness to ACh. Biochemical analyses showed that CP caused significant elevation of malondialdehyde (MDA), reduction of total glutathione (GSH), and increment of proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) level, which were measured in bladder homogenates. PPZ pretreatment at three doses found to be effective in reducing the CP-induced elevation of MDA and TNF-α levels. The highest dose of PPZ (100 mg/kg) caused a significant increase in GSH level. CP induced severe HC with marked bladder edema and histological disturbances which were partially abolished by PPZ pretreatment. CONCLUSIONS: Our results indicate that PPZ pretreatment could attenuate CP-induced HC by interfering with oxidative stress and modulating proinflammatory cytokines.

Protective effects of L-theanine against doxorubicin-induced nephrotoxicity in rats.

BACKGROUND/AIM: L-theanine is the unique amino acid found in tea plants, has antioxidant and antiinflammatory activities, and functions in mental concentration and sleep quality. In this study, it is aimed to investigate the effects of L-theanine on doxorubicin (DOX, a chemotherapeutic agent) induced nephrotoxicity in rats, especially via GSH related enzymes. MATERIALS AND METHODS: 32 male Sprague Dawley rats weighing 300-400 g were randomly assigned into 4 groups (n = 8) and the substances were given intraperitoneally to them: Control group (saline for 5 days); Theanine group (200 mg/kg/day theanine for 5 days); DOX group (single dose of 20 mg/kg DOX); DOX + Theanine group (20 mg/kg DOX at first day and 200 mg/kg/day theanine for 5 days). Kidney tissues were evaluated by histopathological analysis. Serum levels of blood urea nitrogen (BUN) and creatinine by spectrophotometrically; percentage of apoptosis indexes (AI%) in the tissues by TUNEL method; caspase-3 levels, reduced and oxidized glutathione (GSH and GSSG), gamma-glutamyltransferase 1 (GGT1), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST), nuclear factor kappa B p65 (NF-kB p65) by commercial kits; malondialdehyde (MDA) by spectrophotometrically were determined in plasma and kidney tissues. RESULTS: According to DOX group, the DOX + Theanine group has much lower tissue and plasma GSSG, GGT1, NF-κB p65 levels and tissue AI%, whereas significantly higher GSH levels and GPx, GR, GST activities (P < 0.05). CONCLUSION: It is suggested that L-theanine may have protective effects by enhancing effects on the antioxidant system of GSH and GSH-related enzymes against DOX-induced nephrotoxicity in rats. But this finding needs to be supported with further studies.