Methylene blue-assisted molecularly-imprinted film modified nitrogen and sulfur co-doped molybdenum carbide for simultaneous electrochemical determination of two hepatotoxic drugs.

A novel electrochemical sensor with a dual-template molecular imprinting technology was fabricated for the simultaneous detection of paracetamol (PAR) and isoniazid (INZ). The sensor was constructed using nitrogen and sulfur co-doped molybdenum carbide (N, S@Mo2C) and a thin layer of electro-polymerized methylene blue was applied onto the surface of the N, S@Mo2C. The electrochemical sensor demonstrated remarkable analytical efficiency for the concurrent PAR and INZ quantification under optimal circumstances. The system achieved an exceptionally low limit of detection (S/N = 3) of  3.7 nM for PAR, with a concentration range  of  0.013 and 140 µM.  A LOD of 7.6 nM was attained for INZ, with a linear range  between 0.025 and 140 µM. Furthermore, the platform's selectivity was evaluated using differential pulse voltammetry  (DPV). The designed platform successfully detected PAR and INZ in authentic samples with recoveries varying between 98.3% and 104.9%. The relative standard deviations (RSD) for these measurements ranged from 2.7 to 4.0%, demonstrating that the proposed sensor is extremely stable, repeatable, and reproducible. These promising results suggest that the sensor holds potential for the detection of various (bio) molecules, paving the way for future applications in sensing fields.

Sensing the invisible: Ultrasensitive and selective colorimetric detection of E. coli O157:H7 based on masking the peroxidase-mimetic activity of aptamer-modified Au/Fe3O4.

Escherichia coli O157:H7 (E. coli O157:H7) emerges as a significantly worrisome pathogen associated with foodborne illnesses, emphasizing the imperative for creating precise detection tools. In this investigation, we developed a sensitive colorimetric biosensor for detecting E. coli O157:H7. It was constructed using a nanozyme comprised of Au@Fe3O4 NPs, which was fabricated and subsequently modified with an aptamer (Apt). The nanozyme harnesses its inherent peroxidase-like activity to facilitate the transformation of reduced TMB into its oxidized form in the presence of H2O2, resulting in a noticeable shift to a blue color. However, the presence of E. coli O157:H7 effectively diminished the absorbance of oxidized TMB. Consequently, the normalized absorbance at 652 nm demonstrates a linear decrease corresponding to concentrations of E. coli O157:H7 within the range of 101 to 108 CFU mL-1 with a low limit of detection (LOD, S/N = 3) of 3 CFU mL-1.

Sonochemical synthesis of lanthanum ferrite nanoparticle-decorated carbon nanotubes for simultaneous electrochemical determination of acetaminophen and dopamine.

A new nanocomposite consisting of lanthanum ferrite nanoparticles (LaFeO3 NPs) integrated with carbon nanotubes (CNTs) was fabricated via facile sonochemical approach. The engineered nanocomposite was applied to simultaneously determine acetaminophen (ACP) and dopamine (DA) in a binary mixture. The LaFeO3 NPs@CNT probe possesses several advantages such as superior conductivity, large surface area, and more active sites, improving its electrocatalytic activity towards ACP and DA. Under optimized conditions, the anodic peak currents (Ipa) linearly increased with increasing concentration of ACP and DA in the range 0.069-210 µM and 0.15-210 µM, respectively. The sensitivity of LaFeO3 NPs@CNTs/glassy carbon electrode (GCE) for detecting ACP and DA is 7.456 and 5.980 µA·µM-1·cm-2, respectively. The detection limits (S/N = 3) for ACP and DA are 0.02 µM and 0.05 µM, respectively. Advantages of LaFeO3 NPs@CNTs/GCE for the detection of ACP and DA include wide linear ranges, low-detection limits, good selectivity, and long-term stability. The as-fabricated electrode was applied to determine ACP and DA in pharmaceutical formulations and human serum samples with recoveries ranging from 97.7 to 103.3% and an RSD that did not exceed 3.7%, confirming the suitability of the proposed sensor for the determination of ACP and DA in real samples. This study not only presents promising opportunities for enhancing the sensitivity and stability of electrochemical sensors used in the detection of bioanalytes but also significantly contributes to the progress of unique and comprehensive biochemical detection methodologies.

Colorimetric and fluorometric dual-mode determination of hypochlorite based on redox-mediated quenching.

We have successfully created a dual-modal probe, labeled as of iron(ii)-ortho-phenanthroline/N, S@g-CDs, which combines both fluorometric and colorimetric techniques for the accurate and sensitive detection of hypochlorite (ClO-). The mechanism behind this probe involves the fluorescence quenching interaction between nitrogen and sulfur co-doped green emissive carbon dots (N, S@g-CDs) and the iron(ii)-ortho-phenanthroline chelate, utilizing both the inner filter effect and redox processes. As the concentration of ClO- increases, the iron(ii) undergo oxidation to iron(iii) as follows: Fe(ii) + 2HClO = Fe(iii) + Cl2O + H2O, leading to the restoration of N, S@g-CDs fluorescence. Simultaneously, the color of the system transitions gradually from red to colorless, enabling colorimetric measurements. In the fluorometric method with an excitation wavelength of 370 nm, the ClO- concentration exhibits a wide linear correlation with fluorescence intensity ranging from 0.07 to 220 µM. The detection limit achieved in this method is 0.02 µM (S/N = 3). In contrast, the colorimetric method exhibits a linear range of 1.12 to 200 µM, with a detection limit of 0.335 µM (S/N = 3). The proposed selective absorbance for this method is 510 nm. The probe has been effectively utilized for the detection of ClO- in various samples, including water and milk samples. This successful application showcases its potential for determining ClO- in complex matrices, highlighting its broad range of practical uses.

Taxifolin inhibits NETosis through activation of Nrf2 and provides protective effects in models of lupus and antiphospholipid syndrome.

OBJECTIVES: Taxifolin (dihydroquercetin) is a bioactive plant flavonoid that exhibits anti-inflammatory and anti-oxidative properties. We hypothesized that taxifolin might be an effective dietary supplement to ameliorate symptoms arising from thrombo-inflammatory diseases such as lupus and antiphospholipid syndrome (APS). METHODS: We used in vitro assays and a mouse model to determine mechanisms by which taxifolin inhibits neutrophil extracellular trap (NET) formation (i.e., NETosis) and venous thrombosis in lupus and APS. RESULTS: At doses ranging from 0.1 to 1 µg/ml, taxifolin inhibited NETosis from control neutrophils stimulated with autoantibodies isolated from lupus and APS patients, and its suppressive effects were mitigated by blocking the antioxidant transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Furthermore, taxifolin at a dose as low as 20 mg/kg/day reduced in vivo NETosis in thrombo-inflammatory mouse models of lupus and APS while also significantly attenuating autoantibody formation, inflammatory cytokine production, and large-vein thrombosis. CONCLUSION: Our study is the first to demonstrate the protective effects of taxifolin in the context of lupus and APS. Importantly, our study also suggests a therapeutic potential to neutralize neutrophil hyperactivity and NETosis that could have relevance to a variety of thrombo-inflammatory diseases.

Ginger intake suppresses neutrophil extracellular trap formation in autoimmune mice and healthy humans.

We previously reported that treatment of mice with 6-gingerol, the most abundant phytochemical in ginger root, leads to phosphodiesterase inhibition that counteracts neutrophil hyperactivity in models of antiphospholipid syndrome (APS) and lupus. Here, we explored the extent to which oral intake of a whole-ginger extract would similarly impact neutrophils in both autoimmune mice and healthy humans. In vitro, a solubilized ginger extract was able to attenuate neutrophil extracellular trap formation (NETosis) by human neutrophils through a mechanism that was dependent upon the cyclic AMP-dependent kinase, protein kinase A. When mice with features of either APS or lupus were administered a ginger extract orally, they demonstrated reduced circulating NETs, as well as the tempering of other disease outcomes, such as large-vein thrombosis (APS) and autoantibody production (lupus). In a pilot clinical trial, which was validated in a second cohort, daily intake of a ginger supplement for 7 days by healthy volunteers boosted neutrophil cAMP, inhibited NETosis in response to disease-relevant stimuli, and reduced circulating plasma NET levels. In summary, this work demonstrates that ginger intake restrains neutrophil hyperactivity in autoimmune mouse models and that ginger consumption by healthy individuals makes their neutrophils more resistant to NETosis.

HPLC/UV approach method for the first simultaneous estimation of molnupiravir and ertapenem as a binary mixture in human plasma and dosage form as a regimen for COVID-19 treatments.

COVID-19 is a serious virus that can have a lot of effects, one of which is a secondary bacterial infection that can be more life-threatening and even lethal than the initial viral infection. Hence a fast and sensitive HPLC/UV method was developed and validated for the first estimation of a binary mixture of molnupiravir (MOL) and ertapenem (ERT) as a co-administrated medicine for the management of COVID-19 in pharmaceutical dosage forms, and human plasma samples. The drug combination was separated within 5 min via RP-ODS column using isocratic elution with a mobile phase of 0.05 M phosphate buffer (pH 3.5): acetonitrile with a 76: 24% ratio v/v. The presented method provided a linear response ranging from 0.03 to 17.0 and 0.05-20 µg mL-1 with LOD values of 0.009 and 0.008 µg mL-1 for MOL and ERT respectively. The good separation and high sensitivity of the HPLC method provide the determination of the cited drugs in human plasma without matrix interference with a percent of recovery ranging from 94.97 ± 2.05 to 98.44 ± 1.92. Based on the results, this method could be utilized to monitor cited drugs in quality control and therapeutic laboratories.

Protein Expression of NEK2, JMJD4, and REST in Clear Cell Renal Cell Carcinoma (ccRCC): Clinical, Pathological, and Prognostic Findings.

Background & Objective: Cells of renal cell carcinoma (RCC) are resistant to the most currently used chemotherapeutic agents and targeted therapies; hence, we evaluated the expression of NEK2, JMJD4, and REST in cases of clear cell renal cell carcinoma (ccRCC) and benign adjacent tissues of kidney to detect associations between their expression and clinicopathological features, prognostic data, tumor recurrence, and survival rates. Methods: We collected 200 samples including tumoral and adjacent non-neoplastic tissues related to 100 ccRCC patients. All samples were evaluated for the expression of NEK2, JMJD4, and REST, and the patients were followed up for about 5 years. Tumor recurrence and survival data were documented and analyzed. Results: NEK2 and JMJD4 expression showed increase in ccRCC tissues (P=0.002 and 0.006), while REST was downregulated (P<0.001). The elevated expression of NEK2 was positively related ro the tumor size (P=0.015), higher grades (P=0.002), higher stages (P=0.013), distant spread (P=0.004), tumor recurrence, shorter progression-free survival (PFS) rate, and overall survival (OS) rate (P<0.001). Likewise, the high expression of JMJD4 showed positive correlation with the tumor size (P=0.047), higher grades (P=0.003), higher stages (P=0.043), distant spread (P=0.001), tumor recurrence, shorter PFS rate, and OS rate (P<0.001). Conversely, low expression of REST demonstrated positive relationship with the tumor size, higher grades, higher stages, distant spread, tumor recurrence, and shorter PFS and OS rates (P<0.001). Conclusion: Overexpression of NEK2 and JMJD4 and downregulation of REST may be noted in malignant renal tissues compared to benign renal tissues and may be correlated with unfavorable pathological findings, poor clinical parameters, and poor patient outcomes.

Facile fabrication of boron and nitrogen co-doped carbon dots for "ON-OFF-ON" fluorescence sensing of Al3+ and F- ions in water samples.

Water contamination with harmful ions has grown to be a significant environmental issue on a global scale. Therefore, the fabrication of simple, cost-effective, and reliable sensors is essential for identifying these ions. Herein, co-doping of carbon dots with new caffeine and H3BO3-derived boron (B) and nitrogen (N) was performed (BN@CDs). The as-prepared BN@CDs probe was used for the tandem fluorescence sensing of Al3+ and F- based on "ON-OFF-ON" switches. The BN@CDs nanoswitch has a high quantum yield of 44.8% with λexc. and λem. of 360 nm and 440 nm, respectively. The probe exhibited good stability with different pH, ionic-strengths, and irradiation times. The fluorescence emission of BN@CDs was decreased as the Al3+ concentration was increased with a linear range of 0.03-90 µM and a limit of detection (S/N = 3) equal to 9.0 nM. Addition of F- restored the BN@CDs emission as F- ions form a strong and stable complex with Al3+ ions [Al(OH)3F]-. Therefore, the ratio response (F/F°) was raised by raising the F- ion concentration to the range of 0.18-80 µM with a detection limit (S/N = 3) of 50.0 nM. The BN@CDs sensor exhibits some advantages over other reported methods in terms of simplicity, high quantum yield, and low detection limit. Importantly, the sensor was successfully applied to determine Al3+ and F- in various ecological water specimens with accepted results.

A dual-recognition-controlled electrochemical biosensor for selective and ultrasensitive detection of acrylamide in heat-treated carbohydrate-rich food.

A synergistic hybrid was fabricated for the electrochemical aptasensing of acrylamide (AAM) via molecularly imprinted technology. The aptasensor depends on the modification of glassy carbon electrode with AuNPs and reduced graphene oxide (rGO)/multiwalled carbon nanotubes (MWCNTs) {Au@rGO-MWCNTs/GCE}. The aptamer (Apt-SH) and AAM (template) were incubated with the electrode. After that, the monomer was electro-polymerized to fabricate molecular imprinted polymeric film (MIP) over the surface of Apt-SH/Au@rGO/MWCNTs/GCE. The modified electrodes were characterized using different morphological and electrochemical techniques. Under optimum conditions, the aptasensor exhibited a linear relationship between AAM concentration and anodic peak current difference (ΔIpa) in the range of 1-600 nM with a limit of quantitation (LOQ, S/N = 10) and a limit of detection (LOD, S/N = 3) of 0.346 and 0.104 nM, respectively. The aptasensor was successfully applied for the determination of AAM in potato fries samples with recoveries % in the range of 98.7-103.4 % and RSDs did not exceed 3.2 %. The advantages of MIP/Apt-SH/Au@rGO/MWCNTs/GCE are low detection limit, high selectivity, and satisfactory stability towards AAM detection.

An innovative dual recognition aptasensor for specific detection of Staphylococcus aureus based on Au/Fe3O4 binary hybrid.

Pathogenic bacteria cause disease outbreaks and threaten human health, prompting the research on advanced detection assays. Herein, we developed a selective molecular imprinted aptasensor for sensitive and prompt quantitation of Staphylococcus aureus (S. aureus) bacteria. The aptasensor was constructed by immobilization of aptamer on gold nanoparticles modified magnetic nanoparticles (apt-AuNPs@ Fe3O4). A functional monomer (o-phenylenediamine, o-phen) was electro-polymerized on the surface of the as-synthesized nanocomposite in the presence of a template (S. aureus). After removing S. aureus, the formed imprinted sites were available to extract pathogenic bacteria from complicated matrices. The surface morphology of the as-fabricated nanocomposites was characterized using different spectroscopic and electrochemical methods. Moreover, we thoroughly evaluated factors affecting the synthesis and determination procedures. The molecular imprinted aptasensor exhibited a wide linear range of 101-107 CFU mL-1 with a Limit of Detection, LOD (signal to noise = 3) of 1 CFU mL-1. The aptasensor detected S. aureus in milk, conduit water, and apple juice samples with good recoveries % and satisfactory relative standard deviations (RSDs %) values.

Surgical roles in the management of metachronus para-aortic lymph node recurrence and synchronous para-aortic lymph node metastasis in colorectal cancer patients

Background: The relative rarity of synchronous para-aortic lymph node (PALN) metastasis (SPM) and metachronous PALN recurrence (MPR) in colorectal carcinoma (CRC) patients leads to a limited number of studies on patient management, and no treatment guidelines have been established to date. Objective: To assess the prognostic, predictive roles, and long-term outcomes of different management strategies for isolated MPR and SPM in CRC patients to establish the best one. Materials and Methods: We included 35 CRC patients with isolated MPR and 25 patients with isolated SPM who underwent curative R0 resection. We performed PALN dissection (PALND) in 15 cases in MPR group and in 10 cases in the SPM group; all remaining patients in both groups underwent chemoradiotherapy (CRT) without further surgical intervention. During the study period of about 5 years, we compared the patients who underwent PALND and those who underwent CRT. Results: The overall survival and recurrence-free survival rates were significantly longer in patients who underwent PALND (p = 0.049 and 0.036 respectively). (AU)

A simple single jar "on-off fluorescence" designed system for the determination of mitoxantrone using an eosin Y dye in raw powder, vial, and human biofluids.

In this work, a direct, simple, one-pot, and green spectrofluorimetric approach was applied to measure mitoxantrone, a chemotherapeutic agent, through a green validated method. The suggested approach focused on establishing an easy association complex combining mitoxantrone and the eosin Y reagent in a slightly acidic solution. The fluorometric analysis was dependent on off-mitoxantrone action on the emission intensity of the dye (eosin Y) at 544.5 nm (excitation = 301 nm). The devised system has a linear range of 0.07-2.5 µg mL-1 and a detection limit of 0.016 µg mL-1. All system parameters for the formation of mitoxantrone-eosin Y complexes were modulated analytically. Also, the system was reviewed in agreement with ICH criteria. Furthermore, the proposed model was approached to quantify mitoxantrone in its pharmaceutical vial dosage form with high recoveries. Also, the proposed spectroscopic design was efficiently employed to detect the investigated drug in body fluids (blood and urine). Lastly, the designed method was evaluated from a greenness point of view according to eco-scale.

Double-signal quantification of amoxicillin based on interaction with 4-aminoantipyrine at copper and nitrogen co-doped carbon quantum dots as an artificial nanozyme.

An one-pot hydrothermal method was developed for synthesis of carbon quantum dots co-doped with copper and nitrogen (Cu, N@CQDs). The synthesized Cu, N@CQDs has unique advantages such as high fluorescence quantum yield (39.1%) and high catalytic activity. Oxidative coupling of amoxicillin (AMX) with 4-aminoantipyrine (4-NH2-APE) in the presence of H2O2 as an oxidant to produce pink quinoneimine chromogen was carried out with the aid of Cu, N@CQDs as a peroxidase-like catalyst. This system was used for the colorimetric and fluorometric assays of AMX with reliable results. Colorimetric method is based on the measurement of a pink-colored product at λmax = 505 nm while the fluorometric assay is based on the quenching of the fluorescence emission of Cu, N@CQDs at 440 nm after excitation at 370 nm. For the colorimetric method, the absorption intensity linearly increased over the concentration range 4.3-110.0 µM with LOD (S/N = 3) of 1.3 µM. For the fluorometric method, the emission intensity of Cu, N@CQDs linearly decreased upon addition of AMX in the concentration range 0.2-120.0 µM with a limit of detection (LOD, S/N = 3) of 0.06 µM. The proposed system was applied to the determination of AMX in different real samples such as pharmaceutical capsules, human serum, milk, and conduit water samples with recoveries in the range 95.8-104.1% and relative standard deviation (RSD %) less than 4.1%.

A comparison between different management surgical approaches in the treatment of splenic flexure colon cancer

Background: There are many surgical approaches which described extent of resection of the colon for adequate surgicalmanagement of splenic flexure cancer, but up till now there is no established surgical procedure, this is because the presence of double lymphatic drainage of themesenteric vessels. Segmental resection of the colon for the management of splenic flexure cancer was a recently accepted surgical procedure. Objective: In the present study, we aimed to compare three surgical management techniques to clarify the best management approach of Egyptian patients with splenic flexure cancer regarding operative, clinical, and oncological outcomes: segmental resection, and extended left or right hemicolectomy,. Materials and Methods In the present study, we included 90 patients with splenic flexure cancer. Cases were divided into 3 groups. Each group included 30 patients in order to compare three surgical techniques: segmental resection, extended left hemicolectomy, and extended right hemicolectomy. Results: We have found no statistically significant differences between the three included groups regarding operative findings, postoperative complications, local recurrence, distant recurrence, disease progression, recurrence-free survival rate, progression-free survival rate, and overall survival rate. The operative time was longer, and the number of lymph nodes was higher in the extended right hemicolectomy group (p<0.001). Conclusion: We have shown that segmental resection of the splenic flexure is surgically and clinically suitable for the adequate management of operable cases of carcinoma of the splenic flexure. (AU)

Defibrotide Inhibits Antiphospholipid Antibody-Mediated Neutrophil Extracellular Trap Formation and Venous Thrombosis.

OBJECTIVE: Defibrotide is a heterogenous mixture of polyanionic oligonucleotides currently approved for treatment of transplant-associated venoocclusive disease. While defibrotide has a known role in limiting endothelial cell activation, some studies have also demonstrated anti-leukocyte properties. In a recent study, we found that neutrophil extracellular traps (NETs) play a role in the thrombotic complications of antiphospholipid syndrome (APS). In the present study, we investigated the hypothesis that defibrotide might act to mitigate APS-relevant NET formation in vitro and in mouse models. METHODS: We used in vitro assays and a mouse model to determine the mechanisms by which defibrotide inhibits NET formation and venous thrombosis in APS. RESULTS: At doses ranging from 1 to 10 µg/ml, defibrotide significantly suppressed NET formation from control neutrophils stimulated with IgG isolated from patients with APS. Defibrotide increased levels of intracellular cyclic AMP in neutrophils, and its suppressive effects on NET formation were mitigated by blocking adenosine A2A receptor or by inhibiting the cyclic AMP-dependent kinase protein kinase A. Defibrotide at doses ranging from 15 to 150 mg/kg/day inhibited NET formation and venous thrombosis in a model of antiphospholipid antibody-accelerated thrombosis-an effect that was reduced in adenosine A2A receptor-knockout mice. CONCLUSION: This study is the first to demonstrate mechanisms by which defibrotide counteracts neutrophil-mediated thrombotic inflammation inherent to APS.

Mitogenomic Features and Evolution of the Nile River Dominant Tilapiine Species (Perciformes: Cichlidae).

To better understand the diversity and evolution of cichlids, we sequenced, assembled, and annotated the complete mitochondrial genomes of three Nile tilapiine species (Coptodon zillii, Oreochromis niloticus, and Sarotherodon galilaeus) dominating the Nile River waters. Our results showed that the general mitogenomic features were conserved among the Nile tilapiine species. The genome length ranged from 16,436 to 16,631 bp and a total of 37 genes were identified (two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), 13 protein-coding genes (PCGs), and 1 control region). The ND6 was the only CDS that presented a negative AT skew and a positive GC skew. The most extended repeat sequences were in the D-loop followed by the pseudogenes (trnSGCU). The ND5 showed relatively high substitution rates whereas ATP8 had the lowest substitution rate. The codon usage bias displayed a greater quantity of NNA and NNC at the third position and anti-bias against NNG. The phylogenetic relationship based on the complete mitogenomes and CDS was able to differentiate the three species as previously reported. This study provides new insight into the evolutionary connections between various subfamilies within cichlids while providing new molecular data that can be applied to discriminate between Nile tilapiine species and their populations.

Two facile approaches based on association complex with erythrosine-B for nano-level analysis of duloxetine: application to content uniformity.

Duloxetine is an antidepressant that exhibits its action by preventing the reuptake of serotonin and norepinephrine by neurons. In this analytical study, we developed two facile, sensitive methods for duloxetine analysis. Both methods rely on the formation of binary association complex between erythrosine-B and duloxetine in an acidic medium using spectrofluorimetric and resonance Rayleigh scattering (RRS) techniques. Spectrofluorimetric method simply uses the quenching property of the formed complex on the native fluorescence of erythrosine-B at an emission wavelength of 557.2 nm (λ ex = 528.6), while RRS is based on detecting the enhancement in the RRS signal at 357.2 nm. The proposed methods have been validated according to the International Conference on Harmonization guidelines. The approaches provide linear assay of duloxetine hydrochloride over 0.1-2.4 µg ml-1 and 0.2-2.0 µg ml-1 for spectrofluorimetric and RRS methods, respectively. Variables affecting methods and complex formation were studied and optimized. The limit of detection values were 0.03 and 0.056 µg ml-1 for spectrofluorimetric and RRS methods, respectively. Both approaches were applied with acceptable results for formulation analysis and evaluation of cymbatex capsule content uniformity.

Investigation of the association complex formed between dapoxetine and erythrosine-B for facile dapoxetine assay in pharmaceutical formulation using resonance Rayleigh scattering and spectrofluorimetric techniques.

Premature ejaculation is a male sexual problem that is marked by rapid ejaculation and quick attainment of orgasm. Dapoxetine belongs to the antidepressant category and modulates its action by preventing the reuptake of serotonin by neurons. Dapoxetine is marketed as an off-label therapy for premature ejaculation. Here, two facile, sensitive, and green compatible approaches were established for dapoxetine assay. The approaches chemically rely on association complex formed between erythrosine-B and dapoxetine in an acidic buffered medium. The quenching effect of the formed complex on the native erythrosine fluorescence at emission 553.5 nm is simply the main idea of spectrofluorimetric assay, while resonance Rayleigh scattering methodology uses augmentation of resonance Rayleigh scattering spectrum at 345 nm by the added dapoxetine. The current approaches exhibit linearity between response and dapoxetine concentration over 0.2-2.5 µg/ml and 0.3-3.0 µg/ml for spectrofluorimetric and resonance Rayleigh scattering (RRS) methods, respectively. All variables affecting methods and complex formation were studied and precisely optimized. The criteria of validation were performed by the directives provided by International Conference on Harmonization's (ICH) Guidelines and limits of detection were 0.06 and 0.05 µg/ml for spectrofluorimetric and RRS techniques, respectively. Finally, the approaches were applied with acceptable results for pharmaceutical formulation analysis.

Endothelium-protective, histone-neutralizing properties of the polyanionic agent defibrotide.

Neutrophil-mediated activation and injury of the endothelium play roles in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap-derived [NET-derived] histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the US Food and Drug Administration-approved polyanionic agent defibrotide (a pleiotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro, defibrotide counteracted endothelial cell activation and pyroptosis-mediated cell death, whether triggered by purified NETs or recombinant histone H4. In vivo, defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.