Autophagy Is Involved in the Viability of Overexpressing Thioredoxin o1 Tobacco BY-2 Cells under Oxidative Conditions

Autophagy is an essential process for the degradation of non-useful components, although the mechanism involved in its regulation is less known in plants than in animal systems. Redox regulation of autophagy components is emerging as a possible key mechanism with thioredoxins (TRXs) proposed as involved candidates. In this work, using overexpressing PsTRXo1 tobacco cells (OEX), which present higher viability than non-overexpressing cells after H2O2 treatment, we examine the functional interaction of autophagy and PsTRXo1 in a collaborative response. OEX cells present higher gene expression of the ATG (Autophagy related) marker ATG4 and higher protein content of ATG4, ATG8, and lipidated ATG8 as well as higher ATG4 activity than control cells, supporting the involvement of autophagy in their response to H2O2. In this oxidative situation, autophagy occurs in OEX cells as is evident from an accumulation of autolysosomes and ATG8 immunolocalization when the E-64d autophagy inhibitor is used. Interestingly, cell viability decreases in the presence of the inhibitor, pointing to autophagy as being involved in cell survival. The in vitro interaction of ATG4 and PsTRXo1 proteins is confirmed by dot-blot and co-immunoprecipitation assays as well as the redox regulation of ATG4 activity by PsTRXo1. These findings extend the role of TRXs in mediating the redox regulation of the autophagy process in plant cells.

Interaction of Carrier Protein with Potential Metallic Drug Candidate N-Glycoside ‘GATPT’: Validation by Multi-Spectroscopic and Molecular Docking Approaches

Lysozyme is often used as a model protein to study interaction with drug molecules and to understand biological processes which help in illuminating the therapeutic effectiveness of the drug. In the present work, in vitro interaction studies of 1-{(2-hydroxyethyl)amino}-2-amino-1,2-dideoxy-d-glucose triphenyl tin (IV) (GATPT) complex with lysozyme were carried out by employing various biophysical methods such as absorption, fluorescence, and circular dichroism (CD) spectroscopies. The experimental results revealed efficient binding affinity of GATPT with lysozyme with intrinsic binding (Kb) and binding constant (K) values in the order of 105 M−1. The number of binding sites and thermodynamic parameters ΔG, ΔH, and ΔS at four different temperatures were also calculated and the interaction of GATPT with lysozyme was found to be enthalpy and entropy driven. The CD spectra revealed alterations in the population of α–helical content within the secondary structure of lysozyme in presence of GATPT complex. The morphological analysis of the complex with lysozyme and lysozyme-DNA condensates was carried out by employing confocal and SEM studies. Furthermore, the molecular docking studies confirmed the interaction of GATPT within the larger hydrophobic pocket of the lysozyme via several non-covalent interactions.

Pharmacological modulation of the cAMP signaling of two isoforms of melanocortin-3 receptor by melanocortin receptor accessory proteins in the tetrapod Xenopus laevis

As a member of the seven-transmembrane rhodopsin-like G protein-coupled receptor superfamily, the melanocortin-3 receptor is vital for the regulation of energy homeostasis and rhythms synchronizing in mammals and its pharmacological effect could be directly influenced by the presence of melanocortin accessory proteins, MRAP1 and MRAP2. The tetrapod amphibian Xenopus laevis (xl) retains higher duplicated genome than extant teleosts and serves as an ideal model system for embryonic development and physiological studies. However, the melanocortin system of the Xenopus laevis has not been thoroughly evaluated yet. In this work, we performed sequence alignment, phylogenetic and synteny analysis of two xlMC3Rs. Co-immunoprecipitation and immunofluorescence assay further confirmed the co-localization and in vitro interaction of xlMC3Rs with xlMRAPs on the plasma membrane. Our results demonstrated that xlMRAP2.L/S could improve α-MSH stimulated xlMC3Rs signaling and suppress their surface expression. Moreover, xlMC3R.L showed a similar profile on the ligands and surface expression in the presence of xlMRAP1.L. Overall, the distinct pharmacological modulation of xlMC3R.L and xlMC3R.S by dual MRAP2 proteins elucidated the functional consistency of melanocortin system during genomic duplication of tetrapod vertebrates.

Molecular basis for PICS-mediated piRNA biogenesis and cell division

By incorporating two mutually exclusive factors, PID-1 and TOST-1, C. elegans PICS complex plays important roles in piRNA biogenesis, chromosome segregation and cell division. We firstly map the interaction network between PICS subunits, then uncover the mechanisms underlying the interactions between PICS subunits by solving several complex structures, including those of TOFU-6/PICS-1, ERH-2/PICS-1, and ERH-2/TOST-1. Our biochemical experiment also demonstrates that PICS exists as an octamer consisting of two copies of each subunit. Combining structural analyses with mutagenesis experiments, we identify interfacial residues of PICS subunits that are critical for maintaining intact PICS complex in vitro. Furthermore, using genetics, cell biology and imaging experiments, we find that those mutants impairing the in vitro interaction network within PICS, also lead to dysfunction of PICS in vivo, including mislocalization of PICS, and reduced levels of piRNAs or aberrant chromosome segregation and cell division. Therefore, our work provides structural insights into understanding the PICS-mediated piRNA biogenesis and cell division.

In Vitro Interaction and Killing-Kinetics of Amphotericin B Combined with Anidulafungin or Caspofungin against Candida auris

Treatment of invasive infections caused by Candida auris is challenging due to the limited therapeutic options. The combination of antifungal drugs may be an interesting and feasible approach to be investigated. The aim of this study was to examine the in vitro activity of amphotericin B in combination with anidulafungin or caspofungin against C. auris. In vitro static time–kill curve experiments were conducted for 48 h with different combinations of amphotericin B with anidulafungin or caspofungin against six blood isolates of C. auris. The antifungal activity of 0.5 mg/L of amphotericin B was limited against the six isolates of C. auris. Similarly, echinocandins alone had a negligible effect, even at the highest tested concentrations. By contrast, 1 mg/L of amphotericin B showed fungistatic activity. Synergy was rapidly achieved (8 h) with 0.5 mg/L of amphotericin B plus 2 mg/L of anidulafungin or caspofungin. These combinations lead to a sustained fungistatic effect, and the fungicidal endpoint was reached against some C. auris isolates. Additionally, ≥0.5 mg/L of either of the two echinocandins with 1 mg/L of amphotericin B resulted in fungicidal effect against all C. auris isolates. In conclusion, combinations of amphotericin B with anidulafungin or caspofungin provided greater killing with a lower dose requirement for amphotericin B compared to monotherapy, with synergistic and/or fungicidal outcomes.

Aminopolycarboxylate Chelators Are Effective for Whole-body Clearance of Free 225ac: A Method to Reduce Unexpected Radiation Exposure During Targeted Alpha Therapy

Purpose: Actinium-225 (225Ac) is a promising radionuclide used in targeted alpha therapy (TAT). Although 225Ac labelling of bifunctional chelating ligands is effective, previous in vivo studies have reported that free 225Ac can be released from the drugs. Notably, such free 225Ac predominantly accumulates in the liver and can cause unexpected toxicity. To accelerate the clinical development of 225Ac TAT, methods for addressing unexpected toxicity are therefore needed. In this study, we evaluated various chelators in vitro and in vivo with regard to reducing and excreting free 225Ac and compared their chemical structures. Methods: Nine candidate chelators (D-penicillamine, dimercaprol, Ca-DTPA, Ca-EDTA, CyDTA, GEDTA TTHA, Ca-TTHA, and DO3A) were tested. In vitro interaction of 225Ac and chelators was investigated. Biodistribution and dosimetry of free 225Ac were examined in mice prior to the in vivo chelating study. For in vivo chelation, nine candidate chelators were administered 1 h after free 225Ac injection, and biodistribution was compared 4 h after 225Ac injection in mice. Two favourable chelators were then investigated intensively for biodistribution 24 h after the 225Ac injection.Results: The liver exhibited pronounced 225Ac uptake corresponding to an estimated human absorbed dose of 4.76 SvRBE5/MBq. Aminopolycarboxylate chelators with five and six carboxylic groups, Ca-DTPA and Ca-TTHA, significantly reduced 225Ac retention in the liver (22% and 30%, respectively). Significant 225Ac reductions were observed in the heart and the remainder of the body with both Ca-DTPA and Ca-TTHA, and in the lung, kidney, and spleen for Ca-TTHA. In vitro interaction analysis supported the in vivo reduction ability of Ca-DTPA and Ca-TTHA.Conclusions. Aminopolycarboxylate chelators with five and six carboxylic groups, Ca-DTPA and Ca-TTHA, were effective for whole-body clearance of free 225Ac, with a significant reduction in the liver. This method could reduce undesirable radiation exposure from free 225Ac during 225Ac TAT.

A Complex Network of Sigma Factors and sRNA StsR Regulates Stress Responses in R. sphaeroides

Adaptation of bacteria to a changing environment is often accompanied by remodeling of the transcriptome. In the facultative phototroph Rhodobacter sphaeroides the alternative sigma factors RpoE, RpoHI and RpoHII play an important role in a variety of stress responses, including heat, oxidative stress and nutrient limitation. Photooxidative stress caused by the simultaneous presence of chlorophylls, light and oxygen is a special challenge for phototrophic organisms. Like alternative sigma factors, several non-coding sRNAs have important roles in the defense against photooxidative stress. RNAseq-based transcriptome data pointed to an influence of the stationary phase-induced StsR sRNA on levels of mRNAs and sRNAs with a role in the photooxidative stress response. Furthermore, StsR also affects expression of photosynthesis genes and of genes for regulators of photosynthesis genes. In vivo and in vitro interaction studies revealed that StsR, that is under control of the RpoHI and RpoHII sigma factors, targets rpoE mRNA and affects its abundance by altering its stability. RpoE regulates expression of the rpoHII gene and, consequently, expression of stsR. These data provide new insights into a complex regulatory network of protein regulators and sRNAs involved in defense against photooxidative stress and the regulation of photosynthesis genes.