Hemoglobin Binding Research Articles

2-Methoxy-4-formylphenol suppresses methylglyoxal glycation mediated structural alterations and esterase activity of hemoglobin – A multi spectroscopic, biophysical and in-silico study

Glycation is the non-enzymatic reaction of glucose or its metabolites to proteins, causing irreversible changes. Methylglyoxal, a dicarbonyl, affects the structure and function of physiologically important proteins. Being a major circulatory protein, hemoglobin is highly prone to glycation. Current research focuses on identifying potent glycation inhibitors to prevent glycation and their impact on protein structure and function. The present study investigates the Advanced Glycation Endproducts (AGEs) inhibitory effects of 2-methoxy-4-formylphenol (Vanillin) against methylglyoxal mediated glycation of hemoglobin. The hemoglobin-vanillin glycation model exhibited inhibition of AGE formation, amyloid fibrils, aggregates and reduction in esterase activity. The fluorescence spectroscopic technique revealed efficient binding of vanillin and hemoglobin, with Stern Volmer plot indicating the presence of static quenching. The conformational stability of the vanillin and hemoglobin interaction was also evident from the molecular docking and dynamics studies. The proximal orientation of residues (H2 and K82 associated in esterase activity) of hemoglobin β1 chain and vanillin, supports the noted effect of reduced esterase activity in the presence of vanillin in glycated hemoglobin and the inhibition of the overall formation of AGE of hemoglobin in the presence of vanillin.

Genetic Polymorphisms Associated with Fetal Hemoglobin (HbF) Levels and F-Cell Numbers: A Systematic Review of Genome-Wide Association Studies.

Elevated fetal hemoglobin (HbF), which is partly controlled by genetic modifiers, ameliorates disease severity in β hemoglobinopathies. Understanding the genetic basis of this trait holds great promise for personalized therapeutic approaches. PubMed, MedRxiv, and the GWAS Catalog were searched up to May 2024 to identify eligible GWAS studies following PRISMA guidelines. Four independent reviewers screened, extracted, and synthesized data using narrative and descriptive methods. Study quality was assessed using a modified version of the Q-Genie tool. Pathway enrichment analysis was conducted on gene lists derived from the selected GWAS studies. Out of 113 initially screened studies, 62 underwent full-text review, and 16 met the inclusion criteria for quality assessment and data synthesis. A total of 939 significant SNP-trait associations (p-value < 1 × 10-5) were identified, mapping to 133 genes (23 with overlapping variant positions) and 103 intergenic sequences. Most SNP-trait associations converged around BCL11A (chr.2), HBS1L-MYB, (chr.6), olfactory receptor and beta globin (HBB) gene clusters (chr.11), with less frequent loci including FHIT (chr.3), ALDH8A1, BACH2, RPS6KA2, SGK1 (chr.6), JAZF1 (chr.7), MMP26 (chr.11), COCH (chr.14), ABCC1 (chr.16), CTC1, PFAS (chr.17), GCDH, KLF1, NFIX, and ZBTB7A (chr.19). Pathway analysis highlighted Gene Ontology (GO) terms and pathways related to olfaction, hemoglobin and haptoglobin binding, and oxygen carrier activity. This systematic review confirms established genetic modifiers of HbF level, while highlighting less frequently associated loci as promising areas for further research. Expanding research across ethnic populations is essential for advancing personalized therapies and enhancing outcomes for individuals with sickle cell disease or β-thalassemia.

DNA and hemoglobin binding activities: Investigation of coumarin-thiosemicarbazone hybrids

Coumarin and coumarin-thiosemicarbazone hybrids were synthesized and characterized by various techniques such as FT-IR, 1H NMR, 13C NMR, MALDI-TOF-MS spectroscopy, and single crystal X-Ray diffractometer (XRD). The photochemical and photophysical properties of the compounds, such as solvatochromism, solubility, and chemical reactivity, were analyzed using UV–vis spectroscopy in different solvents. Due to the potential biological activities of the synthesized compounds, their binding affinity and mechanisms with calf thymus DNA (ct-DNA) and bovine hemoglobin (BHb) were determined using several useful spectrophotometric and theoretical approaches such as UV–vis absorption and fluorescence spectroscopy, molecular docking, and density functional theory (DFT). The experimental results showed that the compounds exhibited strong binding interactions with DNA and BHb. Additionally, the compounds demonstrated predominantly binding modes, such as intercalation and groove binding with DNA and π–π stacking interactions with BHb.To better understand the thermodynamics of these interactions, quenching constants, binding constants, and Gibbs free energy changes (ΔG°) were calculated. Molecular docking and DFT results supported the experimental data regarding the binding affinity and mechanisms of the compounds to DNA and BHb. Overall, this comprehensive study on coumarin and coumarin-thiosemicarbazone hybrids provides valuable insights into their interaction mechanisms with critical biomolecules, highlighting their potential in therapeutic applications as multifunctional agents.

Investigating the importance of selected surface-exposed loops in HpuB for hemoglobin binding and utilization by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the etiological agent of the sexually transmitted infection gonorrhea. The pathogen is a global health challenge since no protective immunity results from infection, and far fewer treatment options are available with increasing antimicrobial resistance. With no efficacious vaccines, researchers are exploring new targets for vaccine development and innovative therapeutics. The outer membrane TonB-dependent transporters (TdTs) produced by N. gonorrhoeae are considered promising vaccine antigens as they are highly conserved and play crucial roles in overcoming nutritional immunity. One of these TdTs is part of the hemoglobin transport system comprised of HpuA and HpuB. This system allows N. gonorrhoeae to acquire iron from hemoglobin (hHb). In the current study, mutations in the hpuB gene were generated to better understand the structure-function relationships in HpuB. This study is one of the first to demonstrate that N. gonorrhoeae can bind to and utilize hemoglobin produced by animals other than humans. This study also determined that when HpuA is absent, mutations targeting extracellular loop 7 of HpuB led to defective hHb binding and utilization. However, when the lipoprotein HpuA is present, these loop 7 mutants recovered their ability to bind hHb, although the growth phenotype remained significantly impaired. Interestingly, loop 7 contains putative heme-binding motifs and a hypothetical α-helical region, both of which may be important for the use of hHb. Taken together, these results highlight the importance of loop 7 in the functionality of HpuB in binding hHb and extracting and internalizing iron.

Hemoglobin Binding to the Red Blood Cell (RBC) Membrane Is Associated with Decreased Cell Deformability.

The deformability of red blood cells (RBCs), expressing their ability to change their shape as a function of flow-induced shear stress, allows them to optimize oxygen delivery to the tissues and minimize their resistance to flow, especially in microcirculation. During physiological aging and blood storage, or under external stimulations, RBCs undergo metabolic and structural alterations, one of which is hemoglobin (Hb) redistribution between the cytosol and the membrane. Consequently, part of the Hb may attach to the cell membrane, and although this process is reversible, the increase in membrane-bound Hb (MBHb) can affect the cell's mechanical properties and deformability in particular. In the present study, we examined the correlation between the MBHb levels, determined by mass spectroscopy, and the cell deformability, determined by image analysis. Six hemoglobin subunits were found attached to the RBC membranes. The cell deformability was negatively correlated with the level of four subunits, with a highly significant inter-correlation between them. These data suggest that the decrease in RBC deformability results from Hb redistribution between the cytosol and the cell membrane and the respective Hb interaction with the cell membrane.

Aqueous humor perturbations in chronic smokers: a proteomic study

The detrimental effects of smoking are multisystemic and its effects on the eye health are significant. Smoking is a strong risk factor for age-related nuclear cataract, age-related macular degeneration, glaucoma, delayed corneal epithelial healing and increased risk of cystoid macular edema in patients with intermediate uveitis among others. We aimed to characterize the aqueous humor (AH) proteome in chronic smokers to gain insight into its perturbations and to identify potential biomarkers for smoking-associated ocular pathologies. Compared to the control group, chronic smokers displayed 67 (37 upregulated, 30 downregulated) differentially expressed proteins (DEPs). Analysis of DEPs from the biological point of view revealed that they were proteins involved in complement activation, lymphocyte mediated immunity, innate immune response, cellular oxidant detoxification, bicarbonate transport and platelet degranulation. From the molecular function point of view, DEPs were involved in oxygen binding, oxygen carrier activity, hemoglobin binding, peptidase/endopeptidase/cysteine-type endopeptidase inhibitory activity. Several of the upregulated proteins were acute phase reactant proteins such as clusterin, alpha-2-HS-glycoprotein, fibrinogen, alpha-1-antitrypsin, C4b-binding protein and serum amyloid A-2. Further research should confirm if these proteins might serve as biomarkers or therapeutic target for smoking-associated ocular diseases.

Effect of Garlic Extract on the Erythrocyte as a Simple Model Cell.

Garlic is known to have diverse effects on mammalian cells, being cytotoxic, especially to cancer cells, but also protect against oxidative stress. Mammalian erythrocyte is a simple cell devoid of intracellular organelles, protein synthesis ability, and most signaling pathways. Therefore, examination of the effects of garlic on erythrocytes allows for revealing primary events in the cellular action of garlic extract. In this study, human erythrocytes or erythrocyte membranes were exposed to garlic extract at various dilutions. Hemoglobin oxidation to methemoglobin, increased binding of hemoglobin to the membrane, and formation of Heinz bodies were observed. Garlic extract depleted acid-soluble thiols, especially glutathione, and induced a prooxidative shift in the cellular glutathione redox potential. The extract increased the osmotic fragility of erythrocytes, induced hemolysis, and inhibited hemolysis in isotonic ammonium chloride, indicative of decreased membrane permeability for Cl- and increased the membrane fluidity. Fluorescent probes indicated an increased level of reactive oxygen species and induction of lipid peroxidation, but these results should be interpreted with care since the extract alone induced oxidation of the probes (dichlorodihydrofluorescein diacetate and BODIPY C11). These results demonstrate that garlic extract induces oxidative changes in the erythrocyte, first of all, thiol and hemoglobin oxidation.

The Administration of Intradialytic Parenteral Nutrition Does Not Affect the Anemia Status of Chronic Kidney Disease Patients Undergoing Hemodialysis

Background: Hemodialysis (HD) patients are susceptible to malnutrition, and there is a close relationship between malnutrition and the incidence of anemia. Parenteral nutrition plays a role in treating malnutrition. Objective: This study aims to determine the effect of parenteral nutrition on anemia in HD patients. Methods: Quasi-experimental research which is part of the nutritional research tree at the Hemodialysis Unit of RSUP Dr. Soetomo Surabaya for 3 months. A total of 45 malnourished CKD patients (SGA B &amp; C) undergoing routine HD were included in this study (n=26 received regular diet &amp; education, n=17 received regular diet, education and intradialytic parenteral nutrition, and n=2 dropped out of school due to blood transfusion). Measurements of body mass index (BMI), hemoglobin (Hb), serum iron (SI), and total iron binding capacity (TIBC) were carried out before and 8 weeks after therapy. Between groups used the Mann-Whitney test, while pre and post used the Wilcoxon matched-pairs sign rank test. Results: The treatment group was older than the control group. There was no difference in duration of HD between groups. Intradialytic parenteral nutrition had no effect on BMI (24.71±3.939 vs 24.71±4.026; p=0.3802), Hb (9.746±1.309 vs 9.162±1.960; p= 0.3525), SI (62.33±34.74 vs. 53.78±24.89; p=0.3594), and TIBC (242.8±119.0 vs. 197.3±43, 65; p=0.4258). Conclusion: In HD patients, intradialytic parenteral nutrition for 8 weeks did not affect Hb, SI and TIBC levels. Long-term observations with larger samples are needed to confirm these findings.

Positive association between blood ethylene oxide levels and metabolic syndrome: NHANES 2013-2020.

The exposure of Ethylene oxide (EO) is linked to systemic inflammatory response and various cardiovascular risk factors. Hemoglobin's binding to ethylene oxide (HbEO) was used to measure serum EO level. This research aims to explore the association between metabolic syndrome (MetS) and HbEO, and between HbEO and components of metabolic syndrome. This research included 1842 participants from 2013 to 2020 in National Health and Nutrition Examination Survey (NHANES) database. Weighted logistic regression models were used to analyze the relationship between HbEO and metabolic syndrome risk, using odds ratio (OR) and 95% confidence interval (CI). The restricted cubic spline plot explores whether there is a dose-response relationship between HbEO and MetS risk. Subgroup analysis was performed to analyze study heterogeneity. Significant differences were found in gender, educational level, marital status, diabetes status and hypertension among different groups (P < 0.001, P = 0.007, P = 0.003, P < 0.001, P < 0.001, respectively). The serum HbEO level exhibited positive correlation with metabolic syndrome risk in Q2 level (OR=1.64, 1.04~2.48), Q3 level (OR=1.99, 1.29~3.08), and Q4 level (OR=2.89, 1.92~4.34). The dose-response association suggested a possible linear association between serum HbEO and metabolic syndrome risk (P-overall=0.0359, P-non-linear=0.179). L-shaped association was found between HbEO and the risk of MetS in female population, obese population and mid-age and elder population (P-overall<0.001, P-non-linear=0.0024; P-overall=0.0107, P-non-linear=0.0055 P-overall<0.001 P-non-linear=0.0157). This study indicates a linear correlation between MetS and HbEO, with MetS risk escalating as HbEO levels increase. The prevalence of MetS varies depending on BMI, age and gender, and these factors can also influence MetS prevalence when exposed to EO.

Proteomic Analysis of Salivary Extracellular Vesicles from COVID-19 Patients Reveals a Specific Anti-COVID-19 Response Protein Signature.

Despite the understanding of the coronavirus disease-19 (COVID-19), the role of salivary extracellular vesicles (sEVs) in COVID-19 remains unclear. Exploring the proteomic cargo of sEVs could prove valuable for diagnostic and prognostic purposes in assessing COVID-19. The proteomic cargo of sEVs from COVID-19(+) subjects and their healthy close contacts (HCC) was explored. sEVs were isolated by ultracentrifugation from unstimulated saliva samples, and subsequently characterized through nanoparticle tracking, transmission electron microscopy, and Western blot analyses. The proteomic cargo of sEVs was processed by LC-MS/MS. sEVs were morphologically compatible with EVs, with the presence of Syntenin-1 and CD81 EV markers. The sEV pellet showed 1417 proteins: 1288 in COVID-19(+) cases and 1382 in HCC. In total, 124 proteins were differentially expressed in sEVs from COVID-19(+) subjects. "Coronavirus-disease response", "complement and coagulation cascades", and "PMN extracellular trap formation" were the most enriched KEGG pathways in COVID-19(+) cases. The most represented biological processes were "Hemoglobin and haptoglobin binding" and "oxygen carrier activity", and the best-denoted molecular functions were "regulated exocytosis and secretion" and "leucocyte and PMN mediated immunity". sEV proteomic cargo in COVID-19(+) suggests activity related to immune response processes, oxygen transport, and antioxidant mechanisms. In contrast, in HCC, sEV signature profiles are mainly associated with epithelial homeostasis.

Bioassembly of hemoglobin-loaded photopolymerizable spheroids alleviates hypoxia-induced cell death

The delivery of oxygen within tissue engineered constructs is essential for cell survivability; however, achieving this within larger biofabricated constructs poses a significant challenge. Efforts to overcome this limitation often involve the delivery of synthetic oxygen generating compounds. The application of some of these compounds is problematic for the biofabrication of living tissues due to inherent issues such as cytotoxicity, hyperoxia and limited structural stability due to oxygen inhibition of radical-based crosslinking processes. This study aims to develop an oxygen delivering system relying on natural-derived components which are cytocompatible, allow for photopolymerization and advanced biofabrication processes, and improve cell survivability under hypoxia (1% O2). We explore the binding of human hemoglobin (Hb) as a natural oxygen deposit within photopolymerizable allylated gelatin (GelAGE) hydrogels through the spontaneous complex formation of Hb with negatively charged biomolecules (heparin, hyaluronic acid, and bovine serum albumin). We systematically study the effect of biomolecule inclusion on cytotoxicity, hydrogel network properties, Hb incorporation efficiency, oxygen carrying capacity, cell viability, and compatibility with 3D-bioassembly processes within melt electrowritten (MEW) scaffolds. All biomolecules were successfully incorporated within GelAGE hydrogels, displaying controllable mechanical properties and cytocompatibility. Results demonstrated efficient and tailorable Hb incorporation within GelAGE-Heparin hydrogels. The developed system was compatible with microfluidics and photopolymerization processes, allowing for the production of GelAGE-Heparin-Hb spheres. Hb-loaded spheres were assembled into MEW polycaprolactone scaffolds, significantly increasing the local oxygen levels. Ultimately, cells within Hb-loaded constructs demonstrated good cell survivability under hypoxia. Taken together, we successfully developed a hydrogel system that retains Hb as a natural oxygen deposit post-photopolymerization, protecting Hb from free-radical oxidation while remaining compatible with biofabrication of large constructs. The developed GelAGE-Heparin-Hb system allows for physoxic oxygen delivery and thus possesses a vast potential for use across broad tissue engineering and biofabrication strategies to help eliminate cell death due to hypoxia.

Transcriptome in Liver of Periparturient Dairy Cows Differs between Supplementation of Rumen-Protected Niacin and Rumen-Protected Nicotinamide.

To investigate the difference between rumen-protected niacin (RPN) and rumen-protected nicotinamide (RPM) in the transcriptome of genes relating to the lipid metabolism of the liver of periparturient dairy cows, 10 healthy Chinese Holstein cows were randomly divided into two groups and fed diets supplemented with 18.4 g/d RPN or 18.7 g/d RPM, respectively. The experiment lasted from 14 days before to 21 days after parturition. Liver biopsies were taken 21 days postpartum for transcriptomic sequencing. In addition, human LO2 cells were cultured in a medium containing 1.6 mmol/L of non-esterified fatty acids and 1 mmol/L niacin (NA) or 2 mmol/L nicotinamide (NAM) to verify the expression of the 10 genes selected from the transcriptomic analysis of the liver biopsies. The expression of a total of 9837 genes was detected in the liver biopsies, among which 1210 differentially expressed genes (DEGs) were identified, with 579 upregulated and 631 downregulated genes. These DEGs were associated mainly with lipid metabolism, oxidative stress, and some inflammatory pathways. Gene ontology (GO) enrichment analysis showed that 355 DEGs were enriched in 38 GO terms. The differences in the expression of these DEGs between RPN and RPM were predominantly related to the processes of steroid catabolism, steroid hydroxylase, monooxygenase activity, oxidoreductase activity, hemoglobin binding, and ferric iron binding, which are involved mainly in lipid anabolism and redox processes. The expressions of FADS2, SLC27A6, ARHGAP24, and THRSP in LO2 cells were significantly higher (p < 0.05) while the expressions of BCO2, MARS1, GARS1, S100A12, AGMO, and OSBPL11 were significantly lower (p < 0.05) on the NA treatment compared to the NAM treatment, indicating that NA played a role in liver metabolism by directly regulating fatty acid anabolism and transport, inflammatory factor expression, and oxidative stress; and NAM functioned more as a precursor of nicotinamide adenine dinucleotide (NAD, coenzyme I) and nicotinamide adenine dinucleotide phosphate (NADP, coenzyme II) to participate indirectly in biological processes such as ether lipid metabolism, cholesterol metabolism, energy metabolism, and other processes.

Blood gene expression biomarkers of response to anti-TNF drugs in pediatric inflammatory bowel diseases before initiation of treatment

Background/aimsChanges in gene expression profiles among individuals with inflammatory bowel diseases (IBDs) could potentially influence the responsiveness to anti-TNF treatment. The aim of this study was to identify genes that could serve as predictors of early response to anti-TNF therapies in pediatric IBD patients prior to the initiation of treatment. MethodsWe conducted a prospective, longitudinal, and multicenter study, enrolling 24 pediatric IBD patients aged less than 18 years who were initiating treatment with either infliximab or adalimumab. RNA-seq from blood samples was analyzed using the DESeq2 library by comparing responders and non-responders to anti-TNF drugs. ResultsBioinformatic analyses unveiled 102 differentially expressed genes, with 99 genes exhibiting higher expression in responders compared to non-responders prior to the initiation of anti-TNF therapy. Functional enrichment analyses highlighted defense response to Gram-negative bacteria (FDR = 2.3 ×10–7) as the most significant biological processes, and hemoglobin binding (FDR = 0.002), as the most significant molecular function. Gene Set Enrichment Analysis (GSEA) revealed notable enrichment in transcriptional misregulation in cancer (FDR = 0.016). Notably, 13 genes (CEACAM8, CEACAM6, CILP2, COL17A1, OLFM4, INHBA, LCN2, LTF, MMP8, DEFA4, PRTN3, AZU1, and ELANE) were selected for validation, and a consistent trend of increased expression in responders prior to drug administration was observed for most of these genes, with findings for 4 of them being statistically significant (CEACAM8, LCN2, LTF2, and PRTN3). ConclusionsWe identified 102 differentially expressed genes involved in the response to anti-TNF drugs in children with IBDs and validated CEACAM8, LCN2, LTF2, and PRTN3. Genes participating in defense response to Gram-negative bacterium, serine-type endopeptidase activity, and transcriptional misregulation in cancer are good candidates for anticipating the response to anti-TNF drugs in children with IBDs.

LCN2 and ELANE overexpression induces sepsis.

Sepsis is a syndrome characterized by a systemic inflammatory response due to the invasion of pathogenic microorganisms. The relationship between Lipocalin-2 (LCN2), elastase, neutrophil expressed (ELANE) and sepsis remains unclear. The sepsis datasets GSE137340 and GSE154918 profiles were downloaded from gene expression omnibus generated from GPL10558. Batch normalization, differentially expressed Genes (DEGs) screening, weighted gene co-expression network analysis (WGCNA), functional enrichment analysis, Gene Set Enrichment Analysis (GSEA), immune infiltration analysis, construction and analysis of protein-protein interaction (PPI) networks, Comparative Toxicogenomics Database (CTD) analysis were performed. Gene expression heatmaps were generated. TargetScan was used to screen miRNAs of DEGs. 328 DEGs were identified. According to Gene Ontology (GO), in the Biological Process analysis, they were mainly enriched in immune response, apoptosis, inflammatory response, and immune response regulation signaling pathways. In cellular component analysis, they were mainly enriched in vesicles, cytoplasmic vesicles, and secretory granules. In Molecular Function analysis, they were mainly concentrated in hemoglobin binding, Toll-like receptor binding, immunoglobulin binding, and RAGE receptor binding. In Kyoto Encyclopedia of Genes and Genomes (KEGG), they were mainly enriched in NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, P53 signaling pathway, and legionellosis. Seventeen modules were generated. The PPI network identified 4 core genes (MPO, ELANE, CTSG, LCN2). Gene expression heatmaps revealed that core genes (MPO, ELANE, CTSG, LCN2) were highly expressed in sepsis samples. CTD analysis found that MPO, ELANE, CTSG and LCN2 were associated with sepsis, peritonitis, meningitis, pneumonia, infection, and inflammation. LCN2 and ELANE are highly expressed in sepsis and may serve as molecular targets.

In Vitro Hemoglobin Binding and Molecular Docking of Synthesized Chitosan-Based Drug-Carrying Nanocomposite for Ciprofloxacin-HCl Drug Delivery System.

Understanding the intermolecular interactions between antibiotic drugs and hemoglobin is crucial in biological systems. The current study aimed to investigate the preparation of chitosan/polysorbate-80/tripolyphosphate (CS-PS/TPP) nanocomposite as a potential drug carrier for Ciprofloxacin-HCl drug (CFX), intended for controlled release formulation and further used to interact with bovine hemoglobin. Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis-differential thermal analysis (TGA-DTA), scanning electron microscopy (SEM), dynamic light scattering (DLS), and X-ray diffraction analyses were used to characterize the CS-PS/TPP nanocomposite and its CFX-loaded nanocomposite. The second series of biophysical properties were performed on the Ciprofloxacin-loaded CS-PS/TPP (NCFX) for interaction with bovine hemoglobin (BHb). The interactions of (CFX and NCFX) with redox protein hemoglobin were investigated for the first time through a series of in vitro experimental techniques to provide comprehensive knowledge of the drug-protein binding interactions. Additionally, the effect of inclusion of PS-80 on the CFX-BHb interaction was also studied at different concentrations using fluorescence spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, and circular dichroism (CD) under physiological conditions. The binding process of CFX and NCFX was spontaneous, and the fluorescence of BHb was quenched due to the static mechanism formation of the (CFX/BHb) and (NCFX/BHb) complexes. Thermodynamic parameters ΔG, ΔH, and ΔS at various temperatures indicate that the hydrogen bonding and van der Waals forces play a major role in the CFX-BHb association.

Detection of Developmental Asexual Stage-Specific RNA Editing Events in Plasmodium falciparum 3D7 Malaria Parasite.

Transcriptional variation has been studied but post-transcriptional modification due to RNA editing has not been investigated in Plasmodium. We investigated developmental stage-specific RNA editing in selected genes in Plasmodium falciparum 3D7. We detected extensive amination- and deamination-type RNA editing at 8, 16, 24, 32, 40, and 46 h in tightly synchronized Plasmodium. Most of the editing events were observed in 8 and 16 h ring-stage parasites. Extensive A-to-G deamination-type editing was detected more during the 16 h ring stage (25%) than the 8 h ring stage (20%). Extensive U-to-C amination-type editing was detected more during the 16 h ring stage (31%) than the 8 h ring stage (22%). In 28S, rRNA editing converted the loop structure to the stem structure. The hemoglobin binding activity of PF3D7_0216900 was also altered due to RNA editing. Among the expressed 28S rRNA genes, PF3D7_0532000 and PF3D7_0726000 expression was higher. Increased amounts of the transcripts of these two genes were found, particularly PF3D7_0726000 in the ring stage and PF3D7_0532000 in the trophozoite and schizont stages. Adenosine deaminase (ADA) expression did not correlate with the editing level. This first experimental report of RNA editing will help to identify the editing machinery that might be useful for antimalarial drug discovery and malaria control.

The oral pathogen Porphyromonas gingivalis gains tolerance to the antimicrobial peptide DGL13K by synonymous mutations in hagA.

Porphyromonas gingivalis is a keystone pathogen for periodontal disease. The bacteria are black-pigmented and require heme for growth. P. gingivalis exhibit resistance to many antimicrobial peptides, which contributes to their success in the oral cavity. P. gingivalis W50 was resistant to the antimicrobial peptide LGL13K but susceptible to the all-D-amino acid stereoisomer, DGL13K. Upon prolonged exposure to DGL13K, a novel non-pigmented mutant was isolated. Exposure to the L-isomer, LGL13K, did not produce a non-pigmented mutant. The goal of this study was to characterize the genomic and cellular changes that led to the non-pigmented phenotype upon treatment with DGL13K. The non-pigmented mutant showed a low minimum inhibitory concentration and two-fold extended minimum duration for killing by DGL13K, consistent with tolerance to this peptide. The DGL13K-tolerant bacteria exhibited synonymous mutations in the hagA gene. The mutations did not prevent mRNA expression but were predicted to alter mRNA structure. The non-pigmented bacteria were deficient in hemagglutination and hemoglobin binding, suggesting that the HagA protein was not expressed. This was supported by whole cell enzyme-linked immunosorbent assay and gingipain activity assays, which suggested the absence of HagA but not of two closely related gingipains. In vivo virulence was similar for wild type and non-pigmented bacteria in the Galleria mellonella model. The results suggest that, unlike LGL13K, DGL13K can defeat multiple bacterial resistance mechanisms but bacteria can gain tolerance to DGL13K through mutations in the hagA gene.

An innovative Schiff-base colorimetric chemosensor for the selective detection of Cu2+ ions and its applications.

A novel Schiff base moiety, (E)-4-(1-hydrazonoethyl)benzene-1,3-diol (2), and 2,4-dihydroxybenzaldehyde were condensed in a 1 : 1 molar ratio to generate 4-((E)-1-(((Z)-2,4dihydroxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (L), which was then characterized using high-resolution mass spectrometry (HRMS), 1H-NMR, 13C NMR, and single-crystal XRD techniques. UV-vis absorbance measurements were used to determine whether the Schiff base could detect the cupric ions more effectively than the other transition metal ions. When Cu2+ ions were involved, a new band was observed at 462 nm. From the Job plot, the binding stoichiometry for the anticipated L : Cu2+ partnership is determined to be 1 : 1. For the purpose of validating structural correlations and absorption data, DFT simulations were performed. Further, docking studies for L indicated high binding affinity for human hemoglobin, providing vital information about the ligand's favorable binding locations inside hemoglobin binding sites and the consequent interactions with HHb. The binding coefficient and limit of detection were found to be 3.02 × 104 M-1 and 42.09 nM, respectively. Reversibility of the complex was seen upon the addition of EDTA to the L-Cu2+ solution, and a colorimetric variation simulating the "INHIBIT" molecular logic gate was seen upon the addition of Cu2+ and EDTA to L. Furthermore, the chemosensor's potential application in the detection of Cu2+ in the solid state by chemosensor L also confirms its usefulness in real-world applications emphasizing its versatility and practical utility.

Temporal proteomic and phosphoproteomic profiling of the left ventricle in a novel rat model of ischemia/reperfusion with or without ischemic preconditioning

Abstract Background The relationship between ischemic preconditioning (IPC) and myocardial stunning is incompletely understood. Preconditioning has been shown to protect against ischemic injury, whereas myocardial stunning, i.e. transient contractile dysfunction in the setting of ischemia, can lead to severe heart failure and has been considered a deleterious process. To understand this relationship, we recently developed a novel rat model of ischemia-reperfusion (I/R) injury in the presence or absence of IPC. In the presence of IPC (2 cycles of 5 min of I/R followed by 13.5 min of ischemia), we observe a profound myocardial stunning at 4h of reperfusion which completely resolves at 48h. In the absence of IPC, we observe necrosis at 4h of reperfusion and sustained injury at 48h. Despite extensive studies on IPC and myocardial stunning in the setting of I/R, the molecular basis of the relationship between these phenomena are still unknown. Purpose To investigate alterations in protein expression after IPC and myocardial stunning in a novel rat model of I/R injury. Methods We utilized tandem mass tags and mass spectrometry to identify temporal changes in the proteomic- and phosphoproteomic-profiles in left ventricular (LV) tissue from rat hearts following 13.5 min of ischemia and 4h of reperfusion in the presence of absence of IPC. Results Proteomics analysis of the LV tissues collected at baseline showed that 115 proteins were differentially expressed between IPC and non-IPC groups. We observed abundant changes in sarcoplasmic proteins, sarcoplasmic reticulum-related proteins, and actin-based cell projection proteins. Most of these proteins are implicated in arylesterase activity, haemoglobin binding, and nitrogen metabolism. At 4h, differentially expressed proteins were predominantly related to wound healing, coagulation, and lipase activity. Phosphoproteomic analysis at baseline showed that 686 phosphosites were differentially regulated between IPC and non-IPC groups. Aberrant phosphorylation patterns mostly occurred in sarcomere proteins, Z-disc, I-band, contractile fiber associated with actin binding, and the thyroid hormone signalling pathway. At 4h, 520 phosphosites differed between IPC and non-IPC rats; these phosphosites were abundant in contractile fiber proteins, myofibril proteins, and sarcomere proteins associated with calcium signalling and cardiomyopathies. Conclusions This study demonstrates marked changes in proteins and phosphosites related to lipase activity, haemoglobin binding, calcium signalling, and the cardiac contractile apparatus in IPC versus non-IPC groups. This extensive catalogue of novel proteins and phosphosites associated with IPC and myocardial stunning in the setting of I/R injury sets the stage for future manipulation of myocardial stunning and preconditioning in both experimental and clinical settings.

Quantitative Model-Based Assessment of Multiple Sickle Cell Disease Therapeutic Approaches Alone and in Combination

In sickle cell disease (SCD) red blood cell sickling is driven by concentration dependent polymerization of mutation-carrying deoxygenated hemoglobin (deoxHbS). Multiple treatment strategies aim to reduce the concentration of deoxHbS to prevent sickling. Evidence for the utility of this approach is seen in patients endogenously expressing &amp;gt;30% HbF who are largely asymptomatic. In addition to inducing HbF, two other strategies, stabilizing oxygenated hemoglobin (oxyHbS) and lowering 2,3-BPG similarly aim to reduce deoxHbS. However, the degree of oxyHbS stabilization or 2,3-BPG lowering needed to achieve a similar reduction in deoxHbS as 30% HbF is not known. Furthermore, it may not be possible to reach the desired efficacy by a single therapeutic agent making it important to understand how these mechanisms would perform in combination. To address these questions, we extended the Monod-Wymann-Changeux (MWC) oxygen saturation model to include hemoglobin binding to 2,3-BPG and oxyHbS stabilizers and added an additional species to represent HbF. It was assumed that oxyHbS stabilizer occupancy provided equivalent polymerization protection as HbF, which may not be true for all molecules in this class. The model allows simulation of varying levels of oxyHbS stabilizer occupancy, 2,3-BPG lowering, or HbF intervention alone or in combination to calculate the deoxHbS concentration at oxygen tensions found in peripheral tissue and assess polymerization protection. By this approach, the model predicts 51% occupancy by an oxyHbS stabilizer or reduction of 2,3-BPG to 1.8 mM will give equivalent polymerization protection as 30% HbF. Simulations indicate combining any two mechanisms results in a further decrease in deoxHbS compared to either mechanism alone, suggesting combination therapy will have added benefit in the case of dose limitations. By including the main strategies aimed at reducing deoxHbS, this model represents a unified framework that is useful in supporting decisions across the R&amp;D continuum and can be adapted as new clinical data becomes available to gain new and increasingly confident insights into treatment strategies for SCD patients.