Effect of low dose honey on the apoptosis and inflammation gene expression in corneal limbal stem cells and keratocytes and its efficacy as an ophthalmic formulation in the treatment of dry eye: in-vitro and clinical study.

Background: The use of honey as an eye treatment encounters challenges due to its high osmolarity, low pH, and difficulties in sterilization. This study addresses these issues by employing a low concentration of honey, focusing on both in-vitro experiments and clinical trials for treating dry eye disease in corneal cells. Methods: In the in-vitro experiment, we investigated the impact of a 1% honey-supplemented medium (HSM) on limbal stem cells (LSCs) and keratocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and real-time polymerase chain reaction (PCR) for BCL-2, BAX, and IL-1ß gene expression. Simultaneously, in the clinical trial, 80 participants were divided into two groups, receiving either a 1% w/v honey ophthalmic formulation or a placebo for 3 months. Study outcomes included subjective improvement in dry eye symptoms, tear break-up time (TBUT), and Schirmer's test results. Results: MTT results indicated that 1% HSM did not compromise the survival of corneal cells and significantly reduced the expression of the IL-1ß gene. Additionally, participants in the honey group demonstrated a higher rate of improvement in dry eye symptoms and a significant enhancement in TBUT values at the three-month follow-up. However, there was no significant difference between the study groups in terms of Schirmer's test values. No adverse events were observed or reported. Conclusion: In conclusion, 1% honey exhibits anti-inflammatory and anti-infective properties, proving effective in ameliorating dry eye symptoms and enhancing tear film stability in patients with dry eye disease.Clinical Trial Registration: https://irct.behdasht.gov.ir/trial/63800.

A Review on the Role of Stem Cells against SARS-CoV-2 in Children and Pregnant Women.

Since the COVID-19 outbreak was acknowledged by the WHO on 30 January 2020, much research has been conducted to unveil various features of the responsible SARS-CoV-2 virus. Different rates of contagion in adults, children, and pregnant women may guide us to understand the underlying infection conditions of COVID-19. In this study, we first provide a review of recent reports of COVID-19 clinical outcomes in children and pregnant women. We then suggest a mechanism that explains the curious case of COVID-19 in children/pregnant women. The unique stem cell molecular signature, as well as the very low expression of angiotensin-converting enzyme 2 and the lower ACE/ACE2 ratio in stem cells of children/pregnant women compared to adults might be the cause of milder symptoms of COVID-19 in them. This study provides the main molecular keys on how stem cells can function properly and exert their immunomodulatory and regenerative effects in COVID-19-infected children/pregnant women, while failing to replicate their role in adults. This can lay the groundwork for both predicting the pattern of spread and severity of the symptoms in a population and designing novel stem cell-based treatment and prevention strategies for COVID-19.

Design, Synthesis, EPR-Studies and Conformational Bias of Novel Spin-Labeled DCC-Analogues for the Highly Regioselective Labeling of Aliphatic and Aromatic Carboxylic Acids.

Novel types of spin-labeled N,N'-dicyclohexylcarbodiimides (DCC) are reported that bear a 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO) residue on one side and different aromatic and aliphatic cyclohexyl analogues on the other side of the diimide core. These readily available novel reagents add efficiently to aliphatic and aromatic carboxylic acids, forming two possible spin-labeled amide derivatives with different radical distances of the resulting amide. The addition of aromatic DCC analogues proceeds with excellent selectivity, giving amides where the carboxylic acid is exclusively connected to the aromatic residue, while little or no selectivity was observed for the aliphatic congeners. The usefulness of these adducts in structural studies was demonstrated by EPR (electron paramagnetic resonance) measurements of biradical adducts of biphenyl-4,4'-dicarboxylic acids. These analyses also reveal high degrees of conformational bias for aromatic DCC derivatives, which further underlines the powerfulness of these novel reagents. This observation was further corroborated by quantum chemical calculations, giving a detailed understanding of the structural dynamics, while detailed information on the solid state structure of all novel reagents was obtained by X-ray structure analyses.

EPR studies on the kinetics of the α-hydroxyethyl radical generated by Fenton-like chemistry.

Flow systems, either stopped or continuous, have long been at the core of kinetic studies of chemical reactions. Such flow systems need to be coupled with appropriate spectroscopic or otherwise techniques for the detection of the chemical species studied. If paramagnetic species are formed or consumed during the investigated reaction, electron paramagnetic resonance (EPR) with its nanomolar sensitivity can be the spectroscopic method of choice. However, not much literature is available on the application of EPR to quantitatively study kinetics of chemical reactions in the liquid state. Herein, we report the characterisation of the commercially available mixing resonator ER 4117 MX from Bruker using the TEMPO-dithionite reaction as a standard. Furthermore, this setup was used to study the kinetics of the Fenton-like system of TiCl3/H2O2 and ethanol forming theα-hydroxyethyl radical. Potential contributions of reactions with O2, H2O2, Ti(3+/4+), and self-recombination in the decay of theα-hydroxyethyl radical were investigated and the bimolecular decay was shown to be the dominant decay pathway, with a decay rate constant of 6.6×10(8) M(-1) s(-1). This study shows the effectiveness and capabilities of EPR as a direct, sensitive and in-situ method in kinetic studies.

A radical intermediate in tyrosine scission to the CO and CN- ligands of FeFe hydrogenase.

The radical S-adenosylmethionine (SAM) enzyme HydG lyses free l-tyrosine to produce CO and CN(-) for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of (2)H, (13)C, and (15)N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster. Heterolytic cleavage of this tyrosine radical at the Cα-Cß bond forms a transient 4-oxidobenzyl (4OB(•)) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB(•) radical forms p-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN(-), a key intermediate in the assembly of the 2Fe subunit of the H cluster.

Nitric oxide synthase stabilizes the tetrahydrobiopterin cofactor radical by controlling its protonation state.

Nitric oxide synthase (NOS), a homodimeric enzyme with a flavin reductase domain and a P450-type heme-containing oxygenase domain, catalyzes the formation of NO from L-arginine, NADPH, and O(2) in a two-step reaction sequence. In the first step, a tetrahydrobiopterin (H(4)B) cofactor bound near one of the heme propionate groups acts as an electron donor to the P450-type heme active site, yielding a one-electron oxidized radical that is subsequently re-reduced. In solution, H(4)B undergoes two-electron oxidation, showing that the enzyme significantly alters the proton- and electron-transfer properties of the cofactor. Multifrequency EPR and ENDOR spectroscopy were used to determine magnetic parameters, and from them the (de)protonation state of the H(4)B radical in the oxygenase domain dimer of inducible NO synthase that was trapped by rapid freeze quench. From 9.5 and 330-416 GHz EPR and from 34 GHz (1)H ENDOR spectroscopy, the g tensor of the radical and the hyperfine tensors of several N and H nuclei in the radical were obtained. Density functional theory calculations at the PBE0/EPR-II level for H(4)B radical models predict different spin density distributions and g and hyperfine tensors for different protonation states. Comparison of the predicted and experimental values leads to the conclusion that the radical is cationic H(4)B(*+), suggesting that NOS stabilizes this protonated form to utilize the cofactor in a unique dual one-electron redox role, where it can deliver an electron to the active site for reductive oxygen activation and also remove an electron from the active site to generate NO and not NO(-). The protein environment also prevents further oxidation and subsequent loss of function of the cofactor, thus enabling the enzyme to perform the unusual catalytic one-electron chemistry.

Pterin-centered radical as a mechanistic probe of the second step of nitric oxide synthase.

The enzyme nitric oxide synthase is both medically relevant and of particular interest from a basic sciences perspective due to the complex nature of the chemical mechanism used to generate NO. The enzyme utilizes multiple redox-active cofactors and substrates to catalyze the five-electron oxidation of substrate l-arginine to citrulline and nitric oxide. Two flavins, a cysteine-coordinated heme cofactor and, uniquely, a tetrahydrobiopterin cofactor, are used to deliver electrons from the cosubstrate NADPH to molecular oxygen, analogous to other P450s. The unprecedented involvement of the pterin cofactor as a single electron donor is unique among P450s and pterin utilizing proteins alike and adds to the complexity of this enzyme. In this report, the peroxide shunt with both Mn- and Fe-containing heme domain constructs of iNOS(heme) was used to characterize the formation of HNO as the initial inorganic product produced when oxygen activation occurs without pterin radical formation. To recover NO formation, preturnover of the iron-containing enzyme with l-arginine was used to generate the pterin-centered radical, followed by peroxide shunt chemistry. Comparison of NO produced by this reaction with reactions that do not undergo preturnover, do not have peroxide added, or are performed with a pterin unable to generate a radical shows NO production to be dependent on both a pterin-centered radical and activated oxygen. Finally, the chemical HNO donor, Angeli's salt, was used to form the ferrous nitrosyl in the presence of the pterin radical intermediate. Under these conditions, the rate of pterin radical decay was increased as monitored by EPR spectroscopy. In comparison to pterin that aerobically decays, the Angeli's salt treated sample is also significantly protected from oxidation, suggesting ferrous-nitrosyl-mediated reduction of the radical. Taken together, these results support a dual redox cycling role for the pterin cofactor during NOS turnover of NHA with particular importance for the proper release of NO from a proposed ferrous nitrosyl intermediate.