Apo-transferrin (apo-TRF) is a vital protein for maintaining iron balance in the body, which is produced by the liver. Indisulam (IDM) has been extensively used to treat cancer in clinical study and has been identified as a molecular glue. Iron imbalances in the body are believed to encourage the growth and spread of cancer cells. Thus, understanding the interactions between apo-TRF and IDM may serve as a foundation for identifying novel therapeutic strategies for cancer associated with iron imbalances. In this study, multi-spectroscopic methods and computer simulations were employed to explore the binding mode between apo-TRF and IDM, as well as to investigate IDM's impact on the biological functions of apo-TRF. Multi-spectroscopic studies indicated that IDM and apo-TRF formed binary complexes with Ka of 1.274 × 104 M-1 at 298 K. The H-bonds and van der Waals forces were the dominant interaction forces based on an analysis of the thermodynamic parameters (ΔHθ = -37.565 kJ/mol, ΔSθ = -46.665 J mol-1 K-1). Three-dimensional (3D) and circular dichroism (CD) spectra revealed the conformational of apo-TRF changed by IDM, resulting in a looser and more unfolded structure. With escalating concentrations of IDM, a notable reduction in the binding affinity between apo-TRF and Fe3+ was observed, indicating that IDM could potentially alter iron transfer mediated by apo-TRF. Molecular docking analysis indicated that IDM docked in the apo-TRF iron-binding pocket. After in-depth analysis of the molecular dynamic results, it was found that Asp392 played an important role in this interaction. In addition, accessible surface area (ASA) values of key residues (Tyrosine, Aspartate, and Histidine) for iron transfer were altered, which could be a possible reason for the change in iron transport.

Plasma exchange is an effective treatment for Kawasaki disease (KD), suggesting that plasma from patients with KD bears its causative agents. The aim of this study was to use mass spectrometry to identify candidate agents in patient sera. Serum samples were obtained from 17 KD patients. In six patients, samples were collected in each of three phases: the acute phase prior to acetylsalicylic acid (ASA) and intravenous immunoglobulin administration (Phase A1), the remission phase with ASA (Phase A2), and the remission phase without any medication (Phase A3). Sera from the remaining 11 patients were collected during Phases A1 and A2. The study also included two age- and gender-matched control groups, one with eight afebrile children and one with eight febrile children diagnosed with infectious disease. Patients in Phase A1 and febrile controls did not differ in body temperature, white blood cell counts, or C-reactive protein levels. Mass spectrometry analysis revealed that the intensity levels of m/z 9416, identified as apolipoprotein CIII (Apo CIII), were lower in Phase A1 samples compared with samples from patients in Phases A2 and A3, and from febrile controls (all comparisons, p < 0.01). Serum Apo CIII levels were also lower in Phase A1 samples compared with samples from Phase A2 patients and afebrile controls (both p < 0.01), but samples from patients in Phase A2 did not differ significantly from those of the afebrile controls (p = 0.55). This study demonstrated that serum Apo CIII level was decreased in the acute phase of KD.

Different groups of synthetic dyes might lead to environmental pollution. The binding affinity among hazardous materials with biomolecules necessitates a detailed understanding of their binding properties. Malachite Green might induce a change in the iron transfer by Apo-transferrin. Spectroscopic studies showed malachite green oxalate (MGO) could form the apo-transferrin-MGO complex and change the Accessible Surface Area (ASA) of the key amino acids for iron transfer. According to the ASA results the accessible surface area of Tyrosine, Aspartate, and Histidine of apo-transferrin significantly were changed, which can be considered as a convincing reason for changing the iron transfer. Moreover, based on the fluorescence data MGO could quench the fluorescence intensity of apo-transferrin in a static quenching mechanism. The experimental and Molecular Dynamic simulation results represented that the binding process led to micro environmental changes, around tryptophan residues and altered the tertiary structure of apo-transferrin. The Circular Dichroism (CD) spectra result represented a decrease in the amount of the α-Helix, as well as, increase in the β-sheet volumes of the apo-transferrin structure. Moreover, FTIR spectroscopy results showed a hypochromic shift in the peaks of amide I and II. Molecular docking and MD simulation confirmed all the computational findings.

Soil pH is a key controller of denitrification. We analysed the metagenomics/transcriptomics and phenomics of two soils from a long-term liming experiment, SoilN (pH 6.8) and un-limed SoilA (pH 3.8). SoilA had severely delayed N2O reduction despite early transcription of nosZ (mainly clade I), encoding N2O reductase, by diverse denitrifiers. This shows that post-transcriptionally hampered maturation of the NosZ apo-protein at low pH is a generic phenomenon. Identification of transcript reads of several accessory genes in the nos cluster indicated that enzymes for NosZ maturation were present across a range of organisms, eliminating their absence as an explanation for the failure to produce a functional enzyme. nir transcript abundances (for NO2[-] reductase) in SoilA suggest that low NO2[-] concentrations in acidic soils, often ascribed to abiotic degradation, are primarily due to biological activity. The accumulation of NO2[-] in neutral soil was ascribed to high nar expression (nitrate reductase). The -omics results revealed dominance of nirK over nirS in both soils while qPCR showed the opposite, demonstrating that standard primer pairs only capture a fraction of the nirK pool. qnor encoding NO reductase was strongly expressed in SoilA, implying an important role in controlling NO. Production of HONO, for which some studies claim higher, others lower, emissions from NO2[-] accumulating soil, was estimated to be ten times higher from SoilA than from SoilN. The study extends our understanding of denitrification-driven gas emissions and the diversity of bacteria involved and demonstrates that gene and transcript quantifications cannot always reliably predict community phenotypes.

To search immune defense proteins in skin mucus of Japanese flounder fed with a diet containing high concentration of ascorbic acid, we carried out 2D-PAGE and compared the resolved pattern of proteins between control group that fed commercial diet and ascorbic acid supplemented group (AsA group) fed a diet supplemented with high concentration of ascorbic acid (2,000 mg/kg) for 7 days. The results revealed that there were many proteins exhibited distinct increase in AsA group. Among them, 6 regions that showed a dramatic elevation were chosen for protein identification using LC-MS/MS analysis and Mascot database search. Six proteins were identified, i.e. serotransferrin (Sero), transferrin (Trans), warm temperature acclimation-related 65 kDa protein (Wap65), complement component c3 (C3), hemoglobin beta-A chain (Hbβ) and apolipoprotein A-1 (Apo). Quantitative RT-PCR analysis showed that the mRNA level of Hbβ in epidermis of AsA group gave much higher increase (11.6 folds) than control group; the levels of Sero/Trans, Wap65, C3 and Apo showed no apparent difference between the two groups. The mRNA levels of wap65 and c3 in the liver and Apo in the kidney of AsA group exhibited significant increase in comparison to control group. In the case of secreted immunoglobulin M (IgM) and lysozyme (lyz), no difference of the mRNA levels of IgM in epidermis, gill, kidney, spleen and intestine, and lyz in epidermis, gill, spleen and intestine, was observed. The results of in situ hybridization confirmed the elevation of Hbβ mRNA level in the epidermis tissue of AsA group. Our present study provided additional evidence showing the effectiveness of AsA in activating innate immune defense system in skin mucosal tissue of fish.

Dehydroascorbate reductase (DHAR) is a key enzyme for glutathione (GSH)-dependent reduction of dehydroascorbate (DHA) to recycled ascorbate (AsA) in plants, and plays a major role against the toxicity of reactive oxygen species (ROS). Previously, we proposed that the increase of AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity against photooxidative stress in Chlamydomonas reinhardtii. In the present work, we use site-directed mutagenesis and crystal structure analysis to elucidate the molecular basis of how C. reinhardtii DHAR (CrDHAR1) is involved in the detoxification mechanisms. Mutagenesis data show that the D21A, D21N and C22A mutations result in severe loss of the enzyme's function, suggesting crucial roles of Asp-21 and Cys-22 in substrate binding and catalysis. The mutant K11A also exhibits reduced redox activity (∼50%). The crystal structure of apo CrDHAR1 further provides insights into the proposed mechanism centering on the strictly conserved Cys-22, which is suggested to initiate the redox reactions of DHA and GSH. Furthermore, in vitro oxidation of the recombinant CrDHAR1 in the presence of 1 mM H2O2 has minor effects on the Km for the substrates but significantly reduces the kcat. The enzyme's activity and its mRNA abundance in the C. reinhardtii cells are increased by treatment with 0.2-1 mM H2O2 but decreased when H2O2 is ≥ 1.5 mM. The latter decrease is accompanied by oxidative damage and lower AsA concentrations. These biochemical and physiological data provide new insights into the catalytic mechanism of CrDHAR1, which protects the C. reinhardtii cells from oxidative stress-induced toxicity.