Evaluation of perioperative pachymetry of thin corneas during corneal crosslinking using hypo-osmolar riboflavin and hydroxypropyl methylcellulose

ABSTRACT Purpose: This study aimed to analyze variations in intraoperative corneal thickness during corneal cross-linking in patients with keratoconus and to investigate its possible correlation with presurgical maximal keratometry (Kmax) and pachymetry. Methods: This was a prospective case series. We used a method similar to the Dresden protocol, with the application of hydroxypropyl methylcellulose 0.1% hypo-osmolar riboflavin in corneas between 330 and 400 µm after epithelium removal. Corneal thickness was measured using portable calipers before and immediately after epithelium removal, and 30 and 60 min after the procedure. Results: The 30 patients in this study were followed up for one year. A statistically significant difference was observed in pachymetry values during the intraoperative period (p<0.0001) and an increase of 3.05 µm (95%C1: 0.56-5.54) for each diopter was seen after epithelium removal (p0.019). We found an average Kmax difference of —2.12 D between men and women (p0.013). One year after treatment, there was a statistically significant reduction in pachymetry (p<0.0001) and Kmax (p0.0170) values. Conclusions: A significant increase in pachymetry measurements was seen during the procedure, and most patients showed a regression in Kmax and pachymetry values one year after surgery.

Recent Applications of Sulfonium Salts in Synthesis and Catalysis.

The use of sulfonium salts in organic synthesis has experienced a dramatic increase during the last years that can arguably be attributed to three main factors; the development of more direct and efficient synthetic methods that make easily available sulfonium reagents of a wide structural variety, their intrinsic thermal stability, which facilitates their structural modification, handling and purification even on large scale, and the recognition that their reactivity resembles that of hypervalent iodine compounds and therefore, they can be used as replacement of such reagents for most of their uses. This renewed interest has led to the improvement of already existing reactions, as well as to the discovery of unprecedented transformations; in particular, by the implementation of photocatalytic protocols. This review aims to summarize the most recent advancements on the area focusing on the work published during and after 2020. The scope of the methods developed will be highlighted and their limitations critically evaluated.

Autophagic cell death induced by pH modulation for enhanced iron-based chemodynamic therapy.

Iron-based chemodynamic therapy (CDT) exhibits commendable biocompatibility and selectivity, but its efficacy is constrained by the intracellular pH of tumors. To overcome this obstacle, we constructed a silica delivery platform loaded with autophagy-inducing reagents (rapamycin, RAPA) and iron-based Fenton reagents (Fe3O4). This platform was utilized to explore a novel strategy that leverages autophagy to decrease tumor acidity, consequently boosting the effectiveness of CDT. Both in vitro and in vivo experiments revealed that RAPA prompted the generation of acidic organelles (e.g., autophagic vacuoles and autophagosomes), effectively changing the intracellular pH in the tumor microenvironment. Furthermore, RAPA-induced tumor acidification significantly amplified the efficacy of Fe3O4-based Fenton reactions, consequently increasing the effectiveness of Fe3O4-based CDT. This innovative approach, which leverages the interplay between autophagy induction and iron-based CDT, shows promise in overcoming the limitations posed by tumor pH, thus offering a more efficient approach to tumor treatments.

A set of pretreatment reagents including improved formula fixation and decalcification facilitating immunohistochemistry and DNA analyses of formalin-fixed paraffin-embedded bone marrow trephine biopsy.

Bone marrow biopsy depends on tissue morphology, immunohistochemical staining, and moleculardetection. Tissue pretreatment is required for bone marrow samples, from clinical specimen acquisition to pathological reporting, but during the process, proteins and nucleic acids are often altered because of the acid in fixation and decalcification solutions. In our study, we present an easy and effective pretreatment protocol and compared this novel pretreatment protocol (Set 2) with an existing traditional pretreatment process (Set 1) using tissue morphology, IHC staining, and molecular pathological analyses. Granulocytic IHC markers showed more intensive staining in samples of Set 2 than in those of Set 1. The Set 2 protocol provided a higher DNA yield and less fragmentation; moreover, samples processed with the Set 2 protocol could be subsequently used in FISH and DNA sequencing assays. Our optimized novel pretreatment protocol could better protect proteins and DNA molecules while maintaining good cell morphology compared to traditional pretreatment The novel pretreatment reagents could role as a reference by more laboratories for pretreating bone marrow biopsy samples and scientific research.

Stability of immunohaematological reagents used for blood typing of recipients in the tube technique.

BACKGROUND AND OBJECTIVES: The storage temperature of immunohaematological reagents generally ranges from 2 to 8°C, and they should be utilised at room temperature. This study aimed to analyse the stability of immunohaematological reagents used in ABO and RhD typing. METHODS: The evaluation encompassed the potency, specificity, and integrity of anti-A, anti-B, anti-D, RhD control sera, and A1 and B red blood cells (RBC) reagents after long (8 h) and short (4 h) daily periods of exposure to room temperature (20-24°C), 5 days a week for 4 weeks. Additionally, the A1 and B RBC reagents were exposed daily for 11 h and 30 min at room temperature, including 30 more minutes at room temperature with simultaneous homogenisation through equipment. For the control, an aliquot of each reagent was constantly stored at refrigeration temperature, while another was exposed to room temperature for 12 h daily. Tests conducted included reaction intensity, titration, and avidity for antisera, reaction intensity, free haemoglobin determination, and electrical conductivity for the RBC reagents. RESULTS: The antisera maintained the reaction intensity. The titre and avidity of the antisera satisfied the minimum Brazilian requirements after different exposure periods. A higher free haemoglobin concentration was noted in the RBC reagents subjected to room temperature and simultaneous homogenisation, although this did not affect the potency and specificity. The electrical conductivity average of the RBC reagent remained consistent. CONCLUSION: The findings indicate that the immunohaematological reagents from a specific manufacturer are stable under the tested temperature, ensuring the quality of the results under these conditions.

Stereoselective Hydroxyallylation of Cyclopropenes with Cyclopropanols via NHC Catalysis of Transient Organozinc Species.

A stereoselective hydroxyallylation reaction of cyclopropenes with cyclopropanols is achieved under zinc-mediated conditions, affording densely functionalized cyclopropanes with excellent diastereocontrol over three contiguous stereocenters within and outside the cyclopropane ring. A racemic variant of the reaction is synergistically promoted by catalytic N-heterocyclic carbene (NHC) and organic base, whereas chiral amino alcohol-derived bifunctional NHC enables a catalytic enantioselective variant. The reaction likely involves the generation of enolized zinc homoenolate via ring-opening of zinc cyclopropoxide and enolization of the resulting homoenolate, followed by its addition to the cyclopropene as a prochiral allylzinc nucleophile. Our mechanistic investigations highlighted the transient nature of enolized homoenolate, which, once generated from thermodynamically predominant cyclopropoxide, immediately proceeds to allylzincation with cyclopropene. The NHC not only promotes the rate-determining generation of enolized homoenolate but also engages in the allylzincation process. The resulting cyclopropylzinc species undergoes partial in situ protonation while partially remaining intact, thereby leaving an opportunity for trapping with an external electrophile.

Experimental Observation of a Terminal Borylene-Dinitrogen Adduct via Cleavage of a 1,2,3,4,5-Diboratriazoline.

While azides do not react with simple alkenes except under harsh conditions, a diboron alkene analogue, the doubly cyclic alkyl(amino)carbene (CAAC)-stabilized dicyanodiborene 1, reacts spontaneously with organic azides (7-10 equiv.) at room temperature to yield two equivalents of stable CAAC-stabilized imino(cyano)boranes (2-R). NMR-spectroscopic monitoring of the reaction mixtures shows the initial formation of a 1:1 mixture of 2-R and a relatively long-lived intermediate (Int), which in the presence of excess azide is converted into a second equivalent of 2-R. In the absence of excess azide, however, Int decomposes to 3, the product of an intramolecular C‒H activation by a putative dicoordinate borylene intermediate "(CAAC)B(CN)". Mechanistic insights from trapping experiments, NMR-spectroscopic and high-resolution mass spectrometry data, as well as DFT computations reveal that Int is the terminal borylene end-on-dinitrogen adduct [(CAAC)B(CN)(η1-N2)]. The formation of the iminoboranes 2-R from diborene 1 and RN3 proceeds via an azide-diborene Huisgen-type [3+2] cycloaddition reaction, followed by a retro-[3+2] cycloaddition, yielding 2-R and [(CAAC)B(CN)(η1-N2)]. The latter then undergoes either N2 extrusion and intramolecular C‒H activationto generate 3, or a Staudinger-type reaction with a second equivalent of azide to generate a second equivalent of the iminoborane 2-R.

Overriding Cage Effect in Electron Donor-Acceptor Photoactivation of Diaryliodonium Reagents: Synthesis of Chalcogenides.

In recent times, diaryliodonium reagents (DAIRs) have witnessed a resurgence as arylating reagents, especially under photoinduced conditions. However, reactions proceeding through electron donor-acceptor (EDA) complex formation with DAIRs are restricted to electron-rich reacting partners serving as donors due to the well-known cage effect. We discovered a practical and high-yielding visible-light-induced EDA platform to generate aryl radicals from the corresponding DAIRs and use them to synthesize key chalcogenides. In this process, an array of DAIRs and dichalcogenides react in the presence of 1,4 diazabicyclo[2.2.2]octane (DABCO) as a cheap and readily available donor, furnishing a variety of di(hetero)aryl and aryl/alkyl chalcogenides in good yields. The method is scalable, features a broad scope with good yields, and operates under open-to-air conditions. The photoinduced chalcogenation technology is suitable for late-stage functionalizations and disulfide bioconjugations and facilitates access to biologically relevant thioesters, dithiocarbamates, sulfoximines, and sulfones. Moreover, the method applies to synthesizing diverse pharmaceuticals, such as vortioxetine, promazine, mequitazine, and dapsone, under amenable conditions.

Long term investigation of formulation buffers to mitigate stability issues of conjugated critical reagents.

Stability of conjugated critical reagents supporting ligand binding assays to enable biotherapeutic drug development is a universal concern. Formulation buffer employed for long-term cold storage may be key to mitigate protein aggregation issues. We investigated biophysical and functional attributes of murine mAb and human multispecific drug labeled with biotin, ruthenium, and Alexa fluor 647 frozen at -80 °C in PBS or a protein storage buffer for 3-15 months. Aggregation was observed at 4 months in mAb A-Ru (11.2%) and -Alexa (10%) in PBS followed by precipitation and reduced biological binding at 15 months. Increased aggregation in drug Ru (11.7%, 6 months) and Alexa (6.9%, 15 months) were noted but without impact on performance. There were no observations with biotin labeled reagents.

Predicting bond dissociation energies of cyclic hypervalent halogen reagents using DFT calculations and graph attention network model.

Although hypervalent iodine(III) reagents have become staples in organic chemistry, the exploration of their isoelectronic counterparts, namely hypervalent bromine(III) and chlorine(III) reagents, has been relatively limited, partly due to challenges in synthesizing and stabilizing these compounds. In this study, we conduct a thorough examination of both homolytic and heterolytic bond dissociation energies (BDEs) critical for assessing the chemical stability and functional group transfer capability of cyclic hypervalent halogen compounds using density functional theory (DFT) analysis. A moderate linear correlation was observed between the homolytic BDEs across different halogen centers, while a strong linear correlation was noted among the heterolytic BDEs across these centers. Furthermore, we developed a predictive model for both homolytic and heterolytic BDEs of cyclic hypervalent halogen compounds using machine learning algorithms. The results of this study could aid in estimating the chemical stability and functional group transfer capabilities of hypervalent bromine(III) and chlorine(III) reagents, thereby facilitating their development.

Progress and Remaining Opportunities to Increase the Use of Animal-free Antibodies in the USA.

The scientific and ethical issues associated with the use of animal-derived antibodies in research can be overcome by the use of animal-free, sequence-defined recombinant antibodies, whose benefits are well documented. Here, we describe progress made following a 2019 expert meeting focused on improving the quality and reproducibility of biomedical research by accelerating the production and use of animal-free recombinant antibodies in the USA. In the five intervening years since the meeting, participants have established multifaceted initiatives to tackle the next steps outlined during the meeting. These initiatives include: prioritising the replacement of ascites-derived and polyclonal antibodies; distributing educational materials describing recombinant antibodies; fostering public-private partnerships to increase access to recombinant antibodies; and increasing the availability of funding for recombinant antibody development. Given the widescale use of antibodies across scientific disciplines, a transition to modern antibody production methods relies on a commitment from government agencies, universities, industry and funding organisations, to initiatives such as those outlined here.

Critical reagent considerations for immunogenicity assay development for bispecific biotherapeutic candidates.

Aim: To demonstrate the importance of critical reagent characterization for immunogenicity assay development for multi-specific drugs using two case studies. Methods: Bridging anti-drug antibody (ADA) assay with acid-dissociated samples were used for both cases. Results: In the first case study, the unexpected interference in an ADA assay from clinical samples was identified; a model was created to replicate the issue, and an anti-target antibody was identified to mitigate the target interference. In the second case study, an issue due to non-specific binding of a domain-specific confirmatory reagent was identified, and various mitigation techniques were evaluated. Conclusion: A thorough characterization of the critical reagents helped identify the issues with these ADA case studies and provided strategies for resolving them.

[Box: see text].

Fmoc Solid-Phase Peptide Synthesis.

Synthetic peptides are important as drugs and in research. Currently, the method of choice for producing these compounds is solid-phase peptide synthesis. Here, we describe the scope and limitations of Fmoc solid-phase peptide synthesis. Furthermore, we provide a detailed protocol for Fmoc peptide synthesis.

Co(II) Macrocyclic Complexes with Amide-Glycinate Pendants as ParaCEST and Liposomal CEST Agents.

Macrocyclic Co(II) complexes with appended amide-glycinate groups were prepared to develop paramagnetic Co(II) chemical exchange saturation transfer (CEST) agents of reduced overall charge. Complexes with reduced charge and lowered osmolarity are important for their loading into liposomes and to provide complexes that are highly water soluble and well tolerated in animals. Co(L1) has two non-coordinating benzyl groups and two amide-glycinate pendants, whereas Co(L2) has two unsubstituted amide pendants and two amide-glycinate pendants on cyclam (1,4,8,11-tetraazacyclododecane). The 1H NMR spectrum of Co(L1) is consistent with a single cis-pendant isomer with both amide protons in the trans-configuration, as supported by an X-ray crystal structure. Co(L2) has a mixture of different isomers in solution, including the trans-1,4 and 1,8 pendant isomers. The Z-spectrum of Co(L1) shows one highly-shifted CEST peak, whereas Co(L2) exhibits six CEST peaks. Encapsulation of 40 mM Co(L1) in a liposome with osmotically-induced shrinking at 300 mOsm/L produces a liposomal CEST agent with saturation frequency offset of 3 ppm. Addition of the amphiphilic 1,4,7-triazacyclononane-based complex Co(L5) to the liposomal bilayer at 18 mM with Co(L1) encapsulated in the liposome at 50 mM changes the sign and increases the magnitude of the saturation frequency offset to -7.5 ppm at 300 mOsm/L.

Bilateral Unsymmetrical Disulfurating Reagent Design for Polysulfide Construction.

Polysulfides are significant compounds in life science, pharmaceutical science, and materials science. Therefore, polysulfide construction is in great demand. The controllable sequential installation of groups on both ends of a S-S motif faces an enormous challenge owing to the reversible nature of the covalent S-S bond. A library was established with two divergent mask groups for bilateral unsymmetrical disulfurating reagents (R1O-SS-SO2R2). Sequential coupling with preferential activation of the S-SO2 bond (37.6 kcal/mol) and controllable activation of the S-O bond (54.8 kcal/mol) in the presence of the S-S bond (62.0 kcal/mol) enabled successive reactions at each end of the S-S motif to afford unsymmetrical disulfides and trisulfides, even for the cross-linkage of natural products, pharmaceuticals, peptides, and a protein (bovine serum albumin).

Removal enhancement of persistent basic fuchsin dye from wastewater using an eco-friendly, cost-effective Fenton process with sodium percarbonate and waste iron catalyst.

In this comprehensive investigation, we evaluate the efficacy of the Fenton process in degrading basic fuchsin (BF), a resistant dye. Our primary focus is on the utilization of readily available, environmentally benign, and cost-effective reagents for the degradation process. Furthermore, we delve into various operational parameters, including the quantity of sodium percarbonate (SPC), pH levels, and the dimensions of waste iron bars, to optimize the treatment efficiency. In the course of our research, we employed an initial SPC concentration of 0.5 mM, a pH level of 3, a waste iron bar measuring 3.5 cm in length and 0.4 cm in diameter, and a processing time of 10 min. Our findings reveal the successful elimination of the BF dye, even when subjected to treatment with diverse salts and surfactants under elevated temperatures and acidic conditions (pH below 3). This underscores the robustness of the Fenton process in purifying wastewater contaminated with dye compounds. The outcomes of our study not only demonstrate the efficiency of the Fenton process but highlight its adaptability to address dye contamination challenges across various industries. Critically, this research pioneers the application of waste iron bars as a source of iron in the Fenton reaction, introducing a novel, sustainable approach that enhances the environmental and economic viability of the process. This innovative use of recycled materials as catalysts represents a significant advancement in sustainable chemical engineering practices.

Tritium concentration in the modern commercial D2O reagents.

We investigated the tritium concentration in commercial modern D2O reagents frequently used in nuclear magnetic resonance analysis for analytical chemistry and in environmental tracer testing. The concentration of tritium in 11 D2O and 1 H218O reagents ranged from 61 Bq/L (5 × 102 TU) to 2.5 × 103 Bq/L (2 × 104 TU) in order of magnitude. The tritium concentration in the D2O reagents have increased with the increasing purity of D2O. The tritium concentration in all reagents was an order of magnitude greater than that in the surface waters at the Fukushima off-site of the Fukushima Daiichi Nuclear Power Plant after the accident in 2011 and in precipitation during the nuclear test era. However, the concentration of the tritium was lower than the regulatory limit for the concentration of tritium in drinking water accepted by the World Health Organization guidelines. The internal exposure effects from drinking the tritium water, which is contaminated by the tritium condensed in the reagent production processes, were negligible, even if the reagent was used in the environmental tracer test.

Gene silencing in the aedine cell lines C6/36 and U4.4 using long double-stranded RNA.

BACKGROUND: RNA interference (RNAi) is a target-specific gene silencing method that can be used to determine gene functions and investigate host-pathogen interactions, as well as facilitating the development of ecofriendly pesticides. Commercially available transfection reagents (TRs) can improve the efficacy of RNAi. However, we currently lack a product and protocol for the transfection of insect cell lines with long double-stranded RNA (dsRNA). METHODS: We used agarose gel electrophoresis to determine the capacity of eight TRs to form complexes with long dsRNA. A CellTiter-Glo assay was then used to assess the cytotoxicity of the resulting lipoplexes. We also measured the cellular uptake of dsRNA by fluorescence microscopy using the fluorophore Cy3 as a label. Finally, we analyzed the TRs based on their transfection efficacy and compared the RNAi responses of Aedes albopictus C6/36 and U4.4 cells by knocking down an mCherry reporter Semliki Forest virus in both cell lines. RESULTS: The TRs from Biontex (K4, Metafectene Pro, and Metafectene SI+) showed the best complexing capacity and the lowest dsRNA:TR ratio needed for complete complex formation. Only HiPerFect was unable to complex the dsRNA completely, even at a ratio of 1:9. Most of the complexes containing mCherry-dsRNA were nontoxic at 2 ng/µL, but Lipofectamine 2000 was toxic at 1 ng/µL in U4.4 cells and at 2 ng/µL in C6/36 cells. The transfection of U4.4 cells with mCherry-dsRNA/TR complexes achieved significant knockdown of the virus reporter. Comparison of the RNAi response in C6/36 and U4.4 cells suggested that C6/36 cells lack the antiviral RNAi response because there was no significant knockdown of the virus reporter in any of the treatments. CONCLUSIONS: C6/36 cells have an impaired RNAi response as previously reported. This investigation provides valuable information for future RNAi experiments by showing how to mitigate the adverse effects attributed to TRs. This will facilitate the judicious selection of TRs and transfection conditions conducive to RNAi research in mosquitoes.

New Monomer Capable of Dual Chemical Binding with Dentin to Improve Bonding Durability.

The water-rich nature of the dentin bonding microenvironment, coupled with the stresses on the bonding interface, contributes to the hydrolytic degradation of the hybrid layer, resulting in a decline in bonding durability and, ultimately, restoration failure. Currently, the 3-step etch-and-rinse technique remains the gold standard for dentin bonding, and the bonding mechanism mainly involves a physical interaction with little chemical bonding. In this study, we have developed a siloxane-modified polyurethane monomer (SPU) with acrylate and siloxane modifications that chemically binds to both collagen and hydroxyapatite in dentin. Formulated as a bisphenol A-glycidyl methacrylate alternative, the SPU monomer-based adhesive was designed to improve dentin bonding quality and durability. Attenuated total reflection Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscope, and hydroxyproline release assays were performed on SPU-treated collagen, hydroxyapatite, and acid-etched dentin slices to dentin. The physicochemical properties of the configured SPU adhesives were profiled for polymerization behavior, water contact angle, and tensile strain and strength. The bonding effectiveness was assessed through micro-tensile strength, nano-leakage tests conducted on the bonded samples before and after thermal cycle aging. Finally, we further conducted in vivo and in vitro experiments to assess the biocompatibility of adhesives. The results showed that the siloxane groups of SPU monomer could covalently bind to dentin collagen and hydroxyapatite. The incorporation of SPU in the adhesive led to a significant increase in adhesive polymerization (P < 0.05) and tensile strain at break up to 134.11%. Furthermore, the SPU adhesive significantly improved dentin bond strength (P < 0.05), reduced interfacial nano-leakage (P < 0.05), and displayed good biocompatibility. In conclusion, the application of SPU, which achieves dual chemical bonding with dentin, can improve the quality of the hybrid layer, buffer the interfacial stresses, enhance the interfacial resistance to hydrolysis, and provide a feasible strategy to extend the service life of adhesive restorations.

Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay.

Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC).Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target.Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.

Many of the drugs available today are produced by a living organism and these are called biologics. Biologics are larger than chemical drugs and the human body can detect them as foreign and create antibodies against them. This is called immunogenicity. When the antibodies created against the biologic blocks the drug's ability to work correctly, they are called neutralizing antibodies (NAbs). Testing for NAbs is one of the requirements of regulatory agencies for biologics. Here we describe challenges encountered developing an assay to test for NAbs against a biologic.