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© 2015 Choudhury et al.We present a 3D model of the four transmembrane (TM) helical regions of bilitranslocase (BTL), a structurally uncharacterized protein that transports organic anions across the cell membrane. The model was computed by considering heli

This work reports the surface photochemistry study of alloxazine adsorbed within nanochannels of MFI zeolites, namely in a series of Na橷 H攣5 zeolites where the hydrophobic and hydrophilic character of the host varies systematically. Laser-induced room temperature and 77 K luminescence of air-equilibrated solid powdered samples of alloxazine adsorbed onto the two sets of zeolites, which we will name NaZSM-5 and HZSM-5, revealed the existence of a single emission of alloxazine as a broad band centred at about 450 nm in some cases, while in others an emission with a maximum at about 510 nm was detected. The decay times of the alloxazine emission vary greatly going from solution to entrapment within the nanochannels of the ZSM-5 zeolites. In the latter case a lifetime distribution analysis has shown that the longest lifetime for the alloxazine fluorescence emission exists in the case where an isoalloxazine-type emission was detected, i.e.whenever the hydrophobic character of the host increases. Alloxazine entrapped in the more acidic zeolites exists in the form of emissive monomers. However, alloxazine emits both from monomeric and aggregated emissive forms in the case of the hydrophobic zeolites. These data indicate the formation of planar dimers of alloxazine whenever the number of active sites in the zeolite decreases. These dimers have to be formed at the intersections of the zig–zag and linear nanochannels of the zeolite since there is no space available for their formation inside the zeolite channels. The isoalloxazine tautomers are formed due to the existence of alloxazine dimers which may undergo double proton transfer in the excited state, following laser excitation. Delayed fluorescence of alloxazine was also detected for the HZSM-5 and NaZSM-5 entrapment both at room temperature and at 77 K. The present study is paradigmatic as regards the host influence on the photochemistry of the guest. [ABSTRACT FROM AUTHOR]

The composition of isolated natural organic matter (NOM) fractions was studied by means of high pressure size-exclusion chromatography (HP-SEC), specific ultraviolet absorbance (SUVA) at 254 nm, total luminescence spectroscopy (TLS) and synchronous scanning fluorescence measurements. The HPSEC, SUVA and TLS studies revealed that humic acids (HA) and hydrophobic acids (HOA) are composed of complex, aromatic and high molecular weight subunits. Hydrophobic neutrals (HON) consist of few groups of compounds with relatively low molecular sizes and a degree of condensed aromatic moieties. Similar characteristics were observed for hydrophilic acids (HIA) and bases (HIB). Obtained results also demonstrated that hydrophilic neutrals (HIN) are made of non-humic, aliphatic and low molecular weight components. Furthermore, the synchronous excitation-emission spectra indicated the presence of protein-like constituents in HIB, HIN and HON fractions as well as lignin-derived constituents with relatively low molecular sizes in HIA. [ABSTRACT FROM AUTHOR]

Conformational studies of three agonists of V2 receptor modified with 1-aminocyclohexane-1-carboxylic acid (Acc), [Acc2,DArg8]VP, [Acc3]AVP and [Cpa1,Acc3]AVP, using 2D NMR and theoretical calculations are presented in this paper. It is believed that α, α-disubstituted amino acids, such as Acc, affect the formation of either 310 or α-helical conformation. Moreover, a peptide with Acc may adopt either the γ- or an inverse γ-turn over it. Thus, incorporation of Acc into the arginine-vasopressin sequence induced C7-membered ring conformation with Acc at the top of it, and additional formation of β-bend involving residues in the 2–5 fragment of the peptides. Furthermore, the analogues are also characterized by type I of β-turn involving residues Acc3-Cys6 in [Acc3]AVP and [Cpa1,Acc3]AVP, and by type IV or II′ in [Acc2,DArg8]VP. Replacement of Tyr at position 2 of [Acc2,DArg8]VP with Acc afforded a hydrogen bond between the guanidine moiety of DArg8 and the side chain of either Asn5 or Gln4. In the remaining analogues, the β-turn comprising the Cys6-Gly9 residues allows the positively charged side chain of Arg8 to be directed toward Tyr2. The substitution of Cys1 with Cpa1 enhances hydrophobic properties of N-terminal part of the molecule strengthening thereby the affinity to the binding pocket of receptors. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) display advantages over traditional antibiotics due to their broad spectrum of activity against various pathogens, and may even overcome bacterial drug resistance. However, despite their potential therapeutic benefits, widespread application of AMPs is limited by their instability, sensitivity to high salt concentrations, toxicity, and immunogenicity. Lipidation is a promising strategy in overcoming these drawbacks and potential problems for drug candidates. While N-terminal lipidation is a well-studied form of acylation of biologically active peptides, fatty acylation of the lysine side chain has still been poorly explored. In this study, we examined systematic introduction of octanoic (C 8 ) or decanoic (C 10 ) acid into the sequences of three antimicrobial α-helical peptides, namely LL-I, LK6, and ATRA-1, by acylation of subsequent lysine residues, resulting in 17 lipopeptides. Fatty acid lengths optimal for antimicrobial activity were selected based on a previous study on the N-terminal lipidated counterparts of these peptides. Shuffling the position of the fatty acid tails in the sequences of the peptides preserved high activity against Gram-positive bacteria, increased activity against Gram-negative strains and reduced cytotoxicity, compared to the N-terminal acylated counterparts. In the case of the LL-I and LK6 conjugates, the interactions with artificial negatively charged membranes induced formation of an α-helical structure but without a direct correlation between helicity and amphipathicity. Unexpectedly, the ATRA-1 derivatives showed only a small tendency, if any, to adopt a helical structure upon binding to POPG vesicles, which may indicate a non-helical active conformation. A more detailed study of the selected analogues, namely LL-I-4C 8 , LK6-7C 8 , and ATRA-1-11C 10 , provided evidence of a tendency to self-assemble into clumped and/or isolated fibrils, micelles or clusters of micelles, and proved that the lipid bilayer is the main target of action of the tested lipopeptides. In summary, the results of the present study highlight that alternative conjugation sites for lipid modification of AMPs, rather than the commonly applied N-terminal conjugation site, may improve the selectivity of action and be feasible in testing for the development of new lipid-peptide conjugates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

A phospholipid bilayer is a typical structure that serves crucial functions in various cells and organelles. However, it is not unusual for it to take part in pathological processes. The cell membrane may be a binding target for amyloid-forming proteins, becoming a factor modulating the oligomerization process leading to amyloid deposition-a hallmark of amyloidogenic diseases-e.g., Alzheimer's disease. The information on the mechanisms governing the oligomerization influenced by the protein-membrane interactions is scarce. Therefore, our study aims to describe the interactions between DPPA, a cell membrane mimetic, and amyloidogenic protein human cystatin C. Circular dichroism spectroscopy and differential scanning calorimetry were used to monitor (i) the secondary structure of the human cystatin C and (ii) the phase transition temperature of the DPPA, during the protein-membrane interactions. NMR techniques were used to determine the protein fragments responsible for the interactions, and molecular dynamics simulations were applied to provide a molecular structure representing the interaction. The obtained data indicate that the protein interacts with DPPA, submerging itself into the bilayer via the AS region. Additionally, the interaction increases the content of α-helix within the protein's secondary structure and stabilizes the whole molecule against denaturation.

The PD-1/PD-L1 complex belongs to the group of inhibitory immune checkpoints and plays a critical role in immune regulation. The PD-1/PD-L1 axis is also responsible for immune evasion of cancer cells, and this complex is one of the main targets of immunotherapies used in oncology. Treatment using immune checkpoint inhibitors is mainly based on antibodies. This approach has great therapeutic potential; however, it also has major drawbacks and can induce immune-related adverse events. Thus, there is a strong need for alternative, non-antibody-based therapies using small molecules, peptides, or peptidomimetics. In the present study, we designed, synthesized, and evaluated a set of PD-1-targeting peptides based on the sequence and structure of PD-L1. The binding of these peptides to PD-1 was investigated using SPR and ELISA. We also assessed their ability to compete with PD-L1 for binding to PD-1 and their inhibitory properties against the PD-1/PD-L1 complex at the cellular level. The best results were obtained for the peptide PD-L1(111-127) (Y112C-I126C) , named (L11), which displaced PD-L1 from binding to PD-1 in the competitive assay and inhibited the formation of the PD-1/PD-L1 complex. The (L11) peptide also exhibited strong affinity for PD-1. NMR studies revealed that (L11) does not form a well-defined secondary structure; however, MD simulation indicated that (L11) binds to PD-1 at the same place as PD-L1. After further optimization of the structure, the peptide inhibitor obtained in this study could also be used as a potential therapeutic compound targeting the PD-1/PD-L1 axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: trans-Resveratrol (TR) is a well-known phytochemical compound with important biological properties. It can be recovered from agri-food by-products or wastes, such as vine shoots. Once recovered, its concentration should be measured, possibly in a green, non-destructive, and efficient manner. With these premises, this work aimed to explore the feasibility of excitation-emission fluorescence spectroscopy combined with chemometrics for the analysis of TR in raw extracts obtained from vine shoots. A total of 75 extracts were produced and analyzed by ultra-performance liquid chromatography method with diode array detection (UPLC-DAD) and spectrofluorimetry. Then, the feasibility of two calibration strategies for TR quantitation was assessed - a parallel factor analysis (PARAFAC)-based calibration and the N-way partial least squares (NPLS) regression., Results: The extracts showed variable TR content, the excitation/emission maxima of which were at around 305/390 nm, respectively. The best PARAFAC-based calibration allowed a root mean square error of prediction (RMSEP) of 22.57 mg L -1 , and a relative prediction deviation (RPD) of 2.91 to be obtained but a large number of PARAFAC components should be considered to improve the predictions. The results of the NPLS regression were slightly better, with a RMSEP of 19.47 mg L -1 , and an RPD of 3.33 in the best case., Conclusion: Fluorescence could be an alternative analytical technique to measure TR in complex samples. Chemometric tools allowed the identification of the TR signal in the fluorescence landscapes, which could be further used for its non-destructive quantitation. The need for a more accurate criterion for optimal PARAFAC complexity emerged. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Physiological adaptation of the eye to the visual perception of near objects consists of the "near triad": convergence, accommodation, and pupil miosis. Normally, these tend to revert when one stops fixating on a near object. Spasm of the near reflex (SNR) is a pathological phenomenon, which manifests itself by the persistence of the above-mentioned adjustments, which prevents the eye from returning to its relaxed state. In this narrative review, we aim to summarize the etiology, diagnostics, treatment, and prevention of SNR. The literature review was performed by searching online databases. The clinical presentation of SNR is diverse; it presents as isolated accommodative spasm more frequently than impairment of all three components of the near triad. Patients usually present with fluctuations in visual acuity, blurred vision, diplopia, and asthenopia. The etiology is not fully understood. Potential causes include neuroanatomic, organic, and psychogenic disorders. The diagnosis is clinical, based on the constellation of symptoms and assessment of the near triad. The diagnostic golden standard is a cycloplegic examination of refraction, preferably using cyclopentolate hydrochloride (1%, 0.5%, or 0.1% solution). The first-line treatment requires the administration of a cycloplegic drug in combination with plus lenses, flipper lenses, optical fogging, or miotics. For secondary cases, causal treatment should be implemented. Prevention of SNR should be based on eliminating modifiable risk factors. We propose including screening for SNR symptoms in every ophthalmic examination, especially among patients with psychogenic or neural disorders, after brain trauma, or young adults spending much time in front of computer screens., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

A biological membrane is a structure characteristic for various cells and organelles present in almost all living organisms. Even though, it is one of the most common structures in organisms, where it serves crucial functions, a phospholipid bilayer may also take part in pathological processes leading to severe diseases. Research indicates that biological membranes have a profound impact on the pathological processes of oligomerization of amyloid-forming proteins. These processes are a hallmark of amyloid diseases, a group of pathological states involving, e.g., Parkinson's or Alzheimer's disease. Even though amyloidogenic diseases reap the harvest in modern societies, especially in elderly patients, the mechanisms governing the amyloid deposition are not clearly described. Therefore, the presented study focuses on the description of interactions between a model biological membrane (POPG) and one of amyloid forming proteins - human cystatin C. For the purpose of the study molecular dynamics simulations were applied to confirm interactions between the protein and POPG membrane. Next the NMR techniques were used to verify how the data obtained in solution compared to MD simulations and determine fragments of the protein responsible for interactions with POPG. Finally, circular dichroism was used to monitor the changes in secondary structure of the protein and size exclusion chromatography was used to monitor its oligomerization process. Obtained data indicates that the protein interacts with POPG submerging itself into the bilayer with the AS region. However, the presence of POPG bilayer does not significantly affect the structure or oligomerization process of human cystatin C., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Salicylic and acetylsalicylic acids and ethacridine have complementary bioactive properties. They can be combined to obtain double-drug multicomponent crystals. Their reactivity in different environments was explored to obtain the possible compounds, stable at different hydration degrees. Solution, liquid-assisted grinding, and dry preparation approaches were applied to the couples of reactants in different stoichiometric ratios. Four compounds were obtained, and three out of them were stable and reproducible enough to determine their structures using SCXRD or PXRD methods. When coupled to ethacridine, salicylic acid gave two stable structures (1 and 3, both showing 1:1 ratio but different hydration degree) and a metastable one (5), while acetylsalicylic acid only one structure from solution (2 in 1:1 ratio), while LAG caused hydrolysis and formation of the same compound obtained by LAG of ethacridine with salicylic acid. While solution precipitation gave dihydrated (1) or monohydrated (2) structures with low yields, LAG of salicylic acid and ethacridine allowed obtaining an anhydrous salt complex (3) with a yield close to 1. The structures obtained by solution crystallizations maximize π (acridine) -π (acridine) contacts with a less compact packing, while the LAG structure is more compact with a packing driven by hydrogen bonds. For all compounds, NMR, ATR-FTIR, and Hirshfeld surface analysis and energy framework calculations were performed., (© 2024. The Author(s).)

Extracellular Neutrophils Traps (NETs) and their formation, known as NETosis, have become pivotal in the pathogenesis of aortic aneurysm development. This study investigates the NETosis markers with the assessment of selected parameters of inflammation and coagulation system in patients with thoracoabdominal aortic aneurysms in the pre-and postop period undergoing t-Branch stent-graft implantation. The study included 20 patients with thoracoabdominal aortic aneurysms. Three markers double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and citrullinated H3 histones (Cit-H3) were tested at three-time points from patients' blood. The parameters of NETosis, inflammation, and coagulation system were examined in the preoperative period (within 24 h before surgery) and in the postoperative period (on the 3rd and 5th postoperative day). Free-circulating DNA (cfDNA) was isolated from the blood using the MagMAXTM Cell-Free DNA Extraction Kit. Double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) were then quantified using the Qubit dsDNA HS Assay Kit and the Qubit ssDNA Assay Kit. Cit-H3 concentration was determined by enzyme immunoassay ELISA (Cayman). The results revealed the significance of NETs secretion in response to the complex processes after stent-graft implantation. All NET markers increased shortly after surgery, with histones being the first to return to preoperative levels. The lack of normalization of dsDNA and ssDNA levels to preoperative levels by the last postoperative blood collection demonstrates NETs reorganization. The increase in the number of neutrophils was not related to the expansion of postoperative NETosis. The study reveals a new marker of NETosis, ssDNA, that has not been studied so far. The implantation of a stent graft in a patient with TAAA triggers an inflammatory response manifested by an increase in inflammatory parameters. One of the hallmarks of inflammation is the activation of neutrophil extracellular traps., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Michalska, Grochowiecki, Wyczałkowska-Tomasik, Pączek, Jakimowicz, Cacko, Jama, Stec, Sikorska, Nazarewski and Gałązka.)

Immune checkpoints can be divided into co-stimulatory and co-inhibitory molecules that regulate the activation and effector functions of T cells. The co-inhibitory pathways mediated by ICPs are used by cancer cells to escape from immune surveillance, and therefore the blockade of these receptor/ligand interactions is one of the strategies used in the treatment of cancer. The two main pathways currently under investigation are CTLA-4/CD80/CD86 and PD-1/PD-L1, and the monoclonal Abs targeting them have shown potent immunomodulatory effects and activity in clinical environments. Another interesting target in cancer treatment is the BTLA/HVEM complex. Binding of BTLA protein on T cells to HVEM on cancer cells leads to inhibition of T cell proliferation and cytokine production. In the presented work, we focused on blocking the HVEM protein using BTLA-derived peptides. Based on the crystal structure of the BTLA/HVEM complex and MM/GBSA analysis performed here, we designed and synthesized peptides, specifically fragments of BTLA protein. We subsequently checked the inhibitory capacities of these compounds using ELISA and a cellular reporter platform. Two of these peptides, namely BTLA(35-43) and BTLA(33-64) C58Abu displayed the most promising properties, and we therefore performed further studies to evaluate their affinity to HVEM protein, their stability in plasma and their effect on viability of human PBMCs. In addition, the 3D structure for the peptide BTLA(33-64) C58Abu was determined using NMR. Obtained data confirmed that the BTLA-derived peptides could be the basis for future drugs and their immunomodulatory potential merits further examination., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Tetramethylalloxazines (TMeAll) have been found to have a high quantum yield of singlet oxygen generation when used as photosensitizers. Their electronic structure and transition energies (S 0  → S i , S 0  → T i , T 1  → T i ) were calculated using DFT and TD-DFT methods and compared to experimental absorption spectra. Generally, TMeAll display an energy diagram similar to other derivatives belonging to the alloxazine class of compounds, namely π,π* transitions are accompanied by closely located n,π* transitions. Photophysical data such as quantum yields of fluorescence, fluorescence lifetimes, and nonradiative rate constants were also studied in methanol (MeOH), acetonitrile (ACN), and 1,2-dichloroethane (DCE). The transient absorption spectra were also analyzed. To assess cytotoxicity of new compounds, a hemolytic assay was performed using human red blood cells (RBC) in vitro. Subsequently, fluorescence lifetime imaging experiments (FLIM) were performed on RBC under physiological and oxidative stress conditions alone or in the presence of TMeAll allowing for pinpointing changes caused by those compounds on the intracellular environment of these cells., (© 2023. Springer Nature Limited.)

Flavins are a unique class of compounds that combine the features of singlet oxygen generators and redox-dependent fluorophores. From a broad family of flavin derivatives, deazaalloxazines are significantly underdeveloped from the point of view of photophysical properties. Herein, we report photophysics of 5-deazaalloxazine (1a) in water, acetonitrile, and some other solvents. In particular, triplet excited states of 1a in water and in acetonitrile were investigated using ultraviolet-visible (UV-Vis) transient absorption spectroscopy. The measured triplet lifetimes for 1a were all on the microsecond time scale (≈ 60 μs) in deoxygenated solutions. The quantum yield of S 1  → T 1 intersystem crossing for 1a in water was 0.43 based on T 1 energy transfer from 1a to indicaxanthin (5) acting as acceptor and on comparative actinometric measurements using benzophenone (6). 1a was an efficient photosensitizer for singlet oxygen in aerated solutions, with quantum yields of singlet oxygen in methanol of about 0.76, compared to acetonitrile ~ 0.74, dichloromethane ~ 0.64 and 1,2-dichloroethane ~ 0.54. Significantly lower singlet oxygen quantum yields were obtained in water and deuterated water (Ф Δ  ~ 0.42 and 0.44, respectively). Human red blood cells (RBC) were used as a cell model to study the antioxidant capacity in vitro and cytotoxic activity of 1a. Fluorescence-lifetime imaging microscopy (FLIM) data were analyzed by fluorescence lifetime parameters and distribution for different parts of the emission spectrum. Comparison of multidimensional fluorescent properties of RBC under physiological-like and oxidative-stress conditions in the presence and absence of 1a suggests its dual activity as probe and singlet-oxygen generator and opens up a pathway for using FLIM to analyze complex intracellular behavior of flavin-like compounds. These new data on structure-property relationship contribute to the body of information required for a rational design of flavin-based tools for future biological and biochemical applications., (© 2023. The Author(s).)