Balasubramanian Moovarkumudalvan

Enterobacteriaceae represent a clinically challenging bacterial family owing to the global emergence of multidrug resistance. Bacteriophages, the natural predators of these bacteria, have emerged as powerful tools for detecting, targeting, and inhibiting these pathogens. This review provides a comprehensive overview of bacteriophages targeting Enterobacteriaceae hosts, focusing on their diversity, virion architecture, and the molecular determinants of host specificity. The dynamic co-evolution between bacterial defence strategies and phage counter-defences is discussed to illustrate the complexity of the phage-Enterobacteriaceae host interactions. Translational applications of Enterobacteriaceae phages, including phage therapy, engineered phages, phage-derived lysins as antimicrobials, and phage-based diagnostics for rapid and specific pathogen detection is also explored alongside their limitations including limited large-scale clinical trials, regulatory and manufacturing hurdles, and the need for robust host-range prediction and standardization. Overall, this review elaborates implications of bacteriophage-Enterobacteriaceae interactions to combat antimicrobial resistance and support the development of next-generation antibacterial therapies and diagnostics.

Introduction: The tryptophan (TRP)-serotonin pathway is disrupted and redirected towards kynurenine by the action of indoleamine 2,3-dioxygenase 2 (IDO2) and kynureninase (KYNU). In ovarian cancer, concurrent kynurenine activation encourages tumor growth and immune evasion. The investigation of phytochemicals has focused on identifying therapeutic targets in ovarian cancer. Methods: Gene expression, survival plots, and comparisons of correlated genes were computed to obtain holistic insights into the identified targets. A Kaplan-Meier analysis was performed to examine the survival patterns of IDO1 and KYNU targets. Molecular docking was performed to determine the binding affinity of ligands, and the Desmond module of Schrodinger was used to confirm the docking scores through simulation. Physiochemical and ADMET properties were assessed using SwissADME and pkCSM. Electronic effects and stability were further confirmed using Gaussian software. Results: Protein-protein interactions of TRP metabolism revealed IDO2 and KYNU as potential targets involved in pathway diversion. The comparison of mutated and non-mutated targets showed no significant difference, reflecting the tumor’s heterogeneity and the limited number of mutated samples. Molecular docking studies of 368 phytochemicals from six sources, reported to have anti-ovarian cancer properties, revealed 11 phytochemicals with higher binding affinities of -9.7 kcal/mol for IDO2 and -9.4 kcal/mol for KYNU. The interaction between the target and ligand was confirmed by molecular dynamics simulation using the Desmond module V3 of Schrödinger software. The stability and reactivity of phytochemicals were assessed using HOMO-LUMO and energy gap calculations. The highest electron stability was shown in 24MC (6.95 eV) and C24β (6.93 eV) compared with controls. The identified phytochemicals were further evaluated for their ADMET properties to refine therapeutic hit compounds. Compounds, such as Hemanthidine (HAT), Ergometrine (ERG), Tazettine (TZT), Tenatoprazole (TU-199), Rucaparib (CO-338), and Nicotinylalanine (NAL), have been reported to improve drug absorption. Discussion: By altering the ovarian cancer microenvironment, targeting immunometabolism with a clinically relevant approach improves therapeutic efficacy. Target expression clarifies the immune-rich tumor microenvironment rather than the actual tumor growth. Higher-binding- affinity compounds demonstrate high electronic stability in energy estimates as well as improved drug availability in pharmacokinetic investigations. However, this finding remains a computational approach and requires experimental validation. Discussion: By altering the ovarian cancer microenvironment, targeting immunometabolism with a clinically relevant approach improves therapeutic efficacy. Target expression clarifies the immune-rich tumor microenvironment rather than the actual tumor growth. Higher-binding-affinity compounds demonstrate high electronic stability in energy estimates as well as improved drug availability in pharmacokinetic investigations. However, this finding remains a computational approach and requires experimental validation. Conclusion: This computational study identifies suitable hit compounds for targeting TRP depletion, expanding the scope for subsequent in vitro and in vivo exploration to expedite drug discovery.

Dysregulation of tryptophan metabolism activates Kynurenine (KYN), which facilitates tumor immune escape and ovarian cancer progression frequently results in high mortality due to delayed diagnosis. Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) enzymes are primarily responsible for dysregulation in tryptophan metabolism. It was well understood that KYN became life-threatening, causing a cancerous condition. Recently, the urge has been satisfied with potential phytochemicals. Phytochemicals of Taxus baccata, Nelumbo nucifera, Saraca asoca, Trichosanthes cucumerina, Aracauria columnaris and Hymenocallis littoralis were chosen to target kynurenine activation. The molecular docking analysis of IDO1 and TDO2 with 368 phytochemicals reveals higher binding energy for Hinokiflavone, Hippeastrine, Campesterol, Clionasterol, [ ]-sitosterol, Violaxanthin, [ ]-amyrin and [ ]-carotene compounds, which were further analyzed to determine the pharmacokinetic properties and toxicity of hit compounds. Density functional theory (DFT) analysis was performed to determine the electronic effect of hit compounds, resulting in sturdy stability for Campesterol, Clionasterol and [ ]-sitosterol. Molecular dynamic simulation of protein–ligand complexes was further confirmed by the Desmond module V3 of Schrödinger software investigating phytochemicals possessing pharmacological effects with higher binding stability, promising a strategic approach to synthesizing phytochemicals encapsulated nanocarriers for drug delivery to combat tryptophan loss. This in silico approach portrays the interaction of beneficiary phytochemicals with IDO1 and TDO2 in ovarian cancer, restoring the tryptophan homeostasis, thus expanding the scope for in vitro and in vivo studies and hastening drug discovery and delivery.

Hemoglobin (Hb) is an important oxygen carrying protein inherent in erythrocytes facilitates exchange of oxygen and carbon dioxide from body tissues. Bioengineered synthetic erythrocytes with enhanced Hb proteins have been developed as efficient blood transfusion substitutes to supply oxygen to patients in extreme situations. This has driven research towards the molecular characterization and structural elucidation of Hb in organisms thriving in extreme conditions. Camel is recognized as a typical organism to investigate molecular level adaptations to surpass extreme ecological conditions in the arid zone. The molecular architecture of Hb is linked to the oxygen requirements of an organism to sustain at extreme living conditions. Molecular structure of camel Hb can be determined by X-ray crystallography. In this study, camel Hb is purified and crystallized in the monoclinic space group P2₁ with cell dimensions of a = 52.75 Å, b = 116.78 Å, c = 52.80 Å and β = 120.07 Å. The crystal packing parameters reveal that a whole biological molecule is present in the asymmetric unit with the final R-factor and R_free value 23.1 and 29.5%, respectively. The Quaternary structural analysis of the camel Hb [PDB ID: 3GDJ] suggests its state to be identical to human R-state Hb.

The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene.

Hemoglobin (Hb) is the key metalloprotein within red blood cells involved in oxygen transportation from lungs to body cells. The heme-iron atom inherent within Hb effectuates the mechanism of oxygen transportation and carbon dioxide removal. Structural investigations on avian Hb are limited when compared with the enormous work has been carried out on mammalian Hb. Here, the crystal structure of T-state methemoglobin (T-metHb) from domestic duck (Anas platyrhynchos), a low oxygen affinity avian species, determined to 2.1Å resolution is presented. Duck T-metHb crystallized in the orthorhombic space group C222₁ with unit cell parameters a = 59.89, b = 109.42 and c = 92.07Å. The final refined model with R-factor: 19.5% and R_free: 25.2% was obtained. The structural analysis reveals that duck T-metHb adopts a unique quaternary structure that is distinct from any of the avian liganded Hb structures. Moreover, it closely resembles the deoxy Hb of bar-headed goose, a high oxygen-affinity species. Besides the amino acid αPro119 located in the α1β1 interface, a unique quaternary structure with a constrained heme environment is attributed for the intrinsic low oxygen-affinity of duck Hb. This study reports the first protein crystal structure of low oxygen-affinity avian T-metHb from Anas platyrhynchos.

The Saccharomyces cerevisiae Agp2 is a plasma membrane protein initially reported to be an uptake transporter for L-carnitine. Agp2 was later rediscovered, together with three additional proteins, Sky1, Ptk2, and Brp1, to be involved in the uptake of the polyamine analogue bleomycin-A5, an anticancer drug. Mutants lacking either Agp2, Sky1, Ptk2, or Brp1 are extremely resistant to polyamines and bleomycin-A5, suggesting that these four proteins act in the same transport pathway. We previously demonstrated that pretreating cells with the protein synthesis inhibitor cycloheximide (CHX) blocked the uptake of fluorescently labelled bleomycin (F-BLM), raising the possibility that CHX could either compete for F-BLM uptake or alter the transport function of Agp2. Herein, we showed that the agp2Δ mutant displayed striking resistance to CHX as compared to the parent, suggesting that Agp2 is required to mediate the physiological effect of CHX. We examined the fate of Agp2 as a GFP tag protein in response to CHX and observed that the drug triggered the disappearance of Agp2 in a concentration- and time-dependent manner. Immunoprecipitation analysis revealed that Agp2-GFP exists in higher molecular weight forms that were ubiquitinylated, which rapidly disappeared within 10 min of treatment with CHX. CHX did not trigger any significant loss of Agp2-GFP in the absence of the Brp1 protein; however, the role of Brp1 in this process remains elusive. We propose that Agp2 is degraded upon sensing CHX to downregulate further uptake of the drug and discuss the potential function of Brp1 in the degradation process.