Archives of Biochemistry and Biophysics

Artemisinin is an effective antimalarial drug sourced from Artemisia annua, but its low and variable yields require enhancement either semi-synthetically or in-planta to meet the global demand for treatment. Though essential enzymes have been identified in the artemisinin biosynthetic pathway, including an essential Cytochrome P450 monooxygenase (CYP71AV1), there are still many unknowns. Cytochrome P450 reductase 1 (herein, AaCPR1), has been experimentally confirmed as an electron transfer partner for CYP71AV1 in its three step oxygenation of key artemisinin precursors. However, the recent discovery of a highly related CPR, herein AaCPR2, introduces the possibility that another, potentially more catalytically favourable interaction, could exist for CYP71AV1. Therefore, enzyme kinetics and differential scanning fluorimetry (DSF) were used in the characterisation of both AaCPR1 and AaCPR2 to determine the existence and source of their catalytic differences. Tested enzyme activity under cytochrome c and NADPH concentrations revealed that AaCPR1 had lower Km and higher kcat/Km values, while AaCPR2 had higher Vmax and kcat values. This suggests that AaCPR1 is more effective at reducing cytochrome c when substrate conditions are limiting, whereas AaCPR2 is more effective than AaCPR1 at reducing cytochrome c when substrate conditions are saturating. This implies a functional partitioning of the two enzymes on the basis of substrate availability. The DSF results provided deeper insight into the different protein-ligand interactions between the two enzymes. AaCPR2 reached lower maximum melting temperatures across all tested conditions, whereas AaCPR1 had higher maximum melting temperatures. Thus, AaCPR1 exhibits higher thermal stability and has a higher temperature threshold than AaCPR2. This contributes to the notion that the AaCPRs are functionally divergent also on the basis of temperature. The cumulative differences in melting behaviour between the two enzymes led to the hypothesis that AaCPR1 and AaCPR2 exhibit different domain motions that may lead to preferential catalysis for one redox partner over another. This was further supported by the prediction of a highly variable loop region between the two enzymes at the connecting domain just after the flexible hinge. If such loops are highly mobile, as predicted, then the residue differences therein could provide a bio-structural basis for the kinetic and thermal/biophysical differences observed between AaCPR1 and AaCPR2. These data support that AaCPR1 and AaCPR2 possess fundamental biophysical differences despite their high degree of relatedness. Ultimately, these differences suggest differential metabolic functions of the two enzyme in artemisinin biosynthesis and/or other important secondary metabolic processes.

The human mitochondrial amidoxime reducing component 1 (mARC1) is a molybdenum-dependent enzyme whose protein-coding variants confer protection against common metabolic liver diseases. Whereas the frequent A165T variant acts largely through accelerated cellular degradation, the basis of protection by the rarer M187K variant remains obscure, as it has been reported that there are no differences between the "wild-type" and M187K variant protein in terms of cellular protein levels and localisation. Here, the crystal structure of the human mARC1 M187K variant, crystallised as a T4 lysozyme fusion after iterative micro-seeding, was determined at 1.63 [A] resolution. The variant structure is essentially superimposable with the previously reported "wild-type" and A165T structures, with pairwise root-mean-square deviations of 0.3-0.4 [A], and the pentacoordinated molybdenum cofactor is fully intact. Differential scanning fluorimetry across a broad pH range revealed only a modest, pH-dependent decrease in thermal stability associated with the exchange, most pronounced at alkaline pH. These data suggest that the disease-protective effect of M187K is unlikely to originate from gross structural rearrangement or active-site perturbation. SynopsisThe structure of the disease-relevant human mARC1 M187K variant was determined at 1.63 [A] resolution after iterative micro-seeding. The variant does not display relevant perturbations of the overall protein fold or active site structure, but differential scanning fluorimetry detects a pH-dependent decrease in overall protein stability associated with the M187K amino acid exchange.

The mucosal chemoattractant C-X-C motif chemokine ligand 17 (CXCL17) was recently identified as a ligand for the orphan G protein-coupled receptor 25 (GPR25). Although CXCL17 orthologs have been identified in fishes, amphibians, and mammals, their presence in reptiles and birds remains unclear. In this study, we employed bioinformatic searches based on gene synteny and sequence features to identify CXCL17 orthologs in public databases. We identified functional CXCL17 orthologs in 46 reptilian species, including lizards, snakes, turtles, and alligators. In contrast, we found only non-functional gene relics in 22 bird species, suggesting the avian lineage lost functional CXCL17 during evolution. A recombinant reptilian CXCL17 from the loggerhead turtle (Caretta caretta), termed Cc-CXCL17, directly bound to and efficiently activated its corresponding receptor, Cc-GPR25, in a C-terminal fragment-dependent manner. Activation of Cc-GPR25 by Cc-CXCL17 also induced chemotactic movement of transfected human embryonic kidney (HEK) 293T cells. In cross-species activity assays, CXCL17s from human and tropical clawed frog could activate Cc-GPR25 albeit with lower potency, but fish orthologs lacked this activity; all tested CXCL17s had no detectable activity towards chicken GPR25, but Cc-CXCL17 had low activity towards mallard GPR25. Our findings demonstrate the presence of functional CXCL17 orthologs in extant reptiles and provide evidence for their evolutionary loss in birds, offering new insights into the phylogenetic distribution of the newly identified CXCL17-GPR25 signaling system.

Antifungal drug resistance in Aspergillus fumigatus is a major global health concern. Due to the increasing frequency of drug-resistant strains, there is a growing need for novel antifungal drugs. Inhibition of the cysteine biosynthesis pathway in A. fumigatus represents a promising strategy for the development of antifungal adjuvants. Within this pathway, the previously uncharacterised enzyme AfuCys1a fulfils several desirable characteristics as an adjuvant target. To enable structure-based inhibitor design, AfuCys1a was structurally and functionally characterised using X-ray crystallography, biochemical assays, and comparative sequence and structural analysis. In the search for effective and viable adjuvants, this research characterises the catalytic mechanism and substrate recognition of AfuCys1a and identifies 2-picolinic acid and related compounds as promising starting points for inhibitor development, working towards the larger goal of combating rising deaths from infection by A. fumigatus.

[FeFe]-hydrogenases are very active biocatalysts for H2 conversion. However, their active site is vulnerable to irreversible degradation initiated by O2 binding at the catalytic iron ion (Fed) of the active center. CbA5H, the [FeFe]-hydrogenases from Clostridium beijerinckii exhibits stability towards oxygen (O2) due to its ability to reversibly enter an inactive state termed Hinact upon contact with O2. We previously proposed that the close distance of approximately 3.1 [A] between the thiol of a nearby cysteine (C367) and the Fed, based on a 2.9 [A] crystal structure of CbA5H in the Hinact state, enables their binding to each other. This binding therefore was suggested to shield the Fed from O2 damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of the Fed over the thiol of C367. In this study, we present the crystal structure of CbA5H in the Hinact state at an improved resolution of 2.15 [A]. The structure reveals a direct binding between the thiol of C367 and the Fed with a distance of approximated 2.77 [A] which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 [A] bond distance is strikingly long when compared with other iron-sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the Hinact state.

AAA proteases are hexameric ATP-dependent metallopeptidases that perform crucial proteolytic activities within prokaryotic and eukaryotic membranes. Structurally, protomers are comprised of catalytically active C-terminal domains that are anchored to the membrane by an N-terminal autonomous folding unit. In this study, we determined the fold, stability, and oligomeric state of the N-terminal intermembrane domains of human spastic paraplegia type 7 (SPG7)/ paraplegin protein and its bacterial orthologue FtsH using circular dichroism (CD), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS) and X-ray crystallography. Solution-state analysis revealed that the N-terminal domain of paraplegin is a monomer in solution whereas FtsH forms a dimer. Unexpectedly, the N-terminal domain of paraplegin presents as a domain-swapped homodimer in our crystal structure that involves the first helix and first two beta-strands from one monomer and beta-strand 3, helix 2 and beta-strand 4 from another symmetry-related molecule. However, together they form an assembly which is similar to protomers observed for the N-terminal regions of FtsH and AfG3L2. Drawing from our structural data, we postulate that domain-swapping interactions of the N-terminal regions contribute to stability of the AAA protease hexamer containing paraplegin, demonstrating the extensive flexibility of the N-terminal portion of this protein and its role in achieving the appropriate molecular architecture required for function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/720153v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f4b9b5org.highwire.dtl.DTLVardef@1cc2242org.highwire.dtl.DTLVardef@dd211borg.highwire.dtl.DTLVardef@1a87722_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFtsH-IMS forms a homo-dimer in solution, whereas paraplegin-IMS presents as a well-folded monomer in solution C_LIO_LIparaplegin-IMS crystallises as a domain-swapped homo-dimer but its domain-swapped monomers are structurally similar to other IMS-regions C_LIO_LIAfG3L2/paraplegin hexamer formation may be supported by domain swapping in paraplegin-IMS C_LIO_LIdomain-swapping in paraplegin could be a Bonafide feature under certain cellular conditions and may be related to disease in spastic paraplegia C_LI

Catechinopyranocyanidins (Cpcs) which consist of diastereomers A and B are pigments derived from adzuki beans and are compounds in which the catechin and cyanidin skeletons are condensed to a pyrano ring. While catechins and anthocyanidins possess high antioxidant capacity, the physiological functions of Cpcs remains unclear. In this study, the antioxidant capacity of Cpcs was evaluated by in vitro antioxidant assays and by assessing their cytoprotective activity against oxidative stress in normal human dermal fibroblasts (NHDFs). Antioxidant capacity based on the hydrogen atom transfer (HAT) mechanism, as assessed by the ORAC assay revealed that Cpcs exhibit 14.1 mol TE/mol (Trolox equivalent antioxidant capacity: TEAC). Meanwhile, capacity based on the single electron transfer (SET) mechanism, as assessed by the DPPH, ABTS and CUPRAC assays revealed, they exhibit 2.1-3.6 mol TE/mol. Since TEAC value of Cpcs demonstrated by the HAT based mechanism higher than its SET based oxidative capacity suggesting that the antioxidant capacity of Cpcs is driven by the HAT mechanism. In cell culture experiments, Cpcs ameliorate cell toxicity in rotenone-induced injury model, suggesting to cytoprotective activity against mitochondrial dysfunction-dependent apoptosis. These results reveal novel physiological functions of Cpcs which may serve as a design guideline for elucidating in vivo dynamics based on antioxidant mechanisms.

Statins, widely used cholesterol-lowering drugs, inhibit the mevalonate pathway and reduce coenzyme Q (CoQ) biosynthesis, potentially impairing mitochondrial function. Because astrocytes are essential for maintaining brain redox homeostasis, statin-induced mitochondrial dysfunction in these cells may contribute to CNS pathology. We examined the effects of a six-day statin exposure on mitochondrial bioenergetics in rat astrocytes, focusing on mitochondrial CoQ (mtCoQ) deficiency. Treatment with 200 nM atorvastatin or simvastatin decreased the total mtCoQ pool (mtCoQ9 + mtCoQ10) by 30-35% and decreased the antioxidant pool mtCoQH2 by 40%, whereas the levels of mitochondrial antioxidant proteins, including superoxide dismutase 2 and uncoupling proteins, remained unchanged. Mitochondria of statin-treated astrocytes showed decreased respiratory activity, membrane potential, and ATP synthesis, and increased mtCoQ reduction leading to increased H2O2 production during the oxidation of complex I (CI) and CII substrates. Statin treatment also altered the organization of the respiratory chain, leading to a downregulation of the CI+CIII2+CIV and CIII2+CIV supercomplexes and decreased protein levels and activity of all respiratory chain complexes. Furthermore, a decrease in cytochrome a + a3 content was accompanied by a reduction in the maximum activity of CIV. CoQ10 supplementation elevated mtCoQ levels, restored respiratory function, and decreased H2O2 production in the mitochondria of statin-treated astrocytes. Prolonged statin exposure alters mtCoQ redox homeostasis and impairs mitochondrial bioenergetic function in astrocytes. CoQ10 supplementation attenuates these changes, supporting its potential role in protecting astrocyte mitochondria from statin-induced dysfunction.

Punicalagin, an ellagic acid polyphenol from pomegranate, has been proposed as an antagonist of protein disulfide isomerase (PDI) and endoplasmic reticulum resident protein 57 (ERp57), thiol oxidoreductases that regulate protein folding and extracellular thrombotic signaling. Here, biochemical oxidase and reductase assays on PDI show that punicalagin inhibits both activities with micromolar potency, thereby extending earlier work that described only disulfide reductase inhibition. In parallel, thiol labeling of catalytic cysteines revealed no change in the redox state, supporting a noncovalent, allosteric of inhibition. Molecular docking and molecular dynamics simulations showed that punicalagin binds stably and preferentially to defined sites on the Nterminal domains of PDI through extensive hydrogen bonding and van der Waals contacts, which is an alternative binding mode to previously reported C-terminal binding. Finally, artificial intelligence-driven network analysis identified PDI as a high-confidence target of punicalagin and related galloylated polyphenols, alongside additional signaling proteins. Together, these findings provide further mechanistic framework for punicalagin-mediated antagonism of PDI and highlight galloylated polyphenols as promising scaffolds for protein disulfide isomerase-targeted therapeutics. HighlightsO_LIPunicalagin, a galloylated polyphenol, antagonizes not only the reductase activity but also the oxidase activity of protein disulfide isomerase C_LIO_LIProtein disulfide isomerase inhibition by punicalagin is through N-terminal binding C_LIO_LIPunicalagin inhibits conformationally rather than catalytic cysteine modification C_LIO_LIArtificial intelligence network analysis reveals pathway inhibition by punicalagin C_LI

Heat shock protein 90 (HSP90) assists protein folding and maturation of many important signaling proteins. In various diseases, HSP90 clients contribute to aberrant signaling, and HSP90 inhibition is being explored as a potential therapeutic approach. Commonly researched HSP90 inhibitors target the ATP-binding pocket, thereby disrupting the ATP-induced closure of HSP90. Drugs disrupting the HSP90 ATPase cycle by targeting the closed state of the chaperone have not been developed. Here, we present de novo design and selection of protein binders interacting exclusively with the closed state HSP90. Two such binders, H2 and H4, were identified that feature a similar fold and comparable affinities for HSP90 but display different binding kinetics. The structures of the HSP90 complexes with H2 and H4 were determined by cryo-EM single particle analysis, and they revealed high accuracy of the BindCraft predictions. H2 and H4 compete with p23 at one but not both symmetrical p23 binding sites on HSP90. H2 expressed in HEK293T cells moderately elevated expression of HSP70 and had no effect on the HSP90 level, suggesting muted heat shock response. Overall, this study demonstrates that the de novo binders represent novel and promising tools to probe the potential utility of HSP90 inhibition by targeting its closed state.

The chemokine-like peptide GPR15LG is a known agonist of G protein-coupled receptor 15 (GPR15), a ligand-receptor pair primarily implicated in mammalian mucosal immunity and lymphocyte homing. However, the evolutionary origin and phylogenetic distribution of this signaling system remain poorly understood due to the extreme sequence diversity of GPR15LG orthologs. In this study, we identified GPR15LG orthologs in several fish species for the first time according to their conserved gene synteny, genomic organization, and amino acid sequence features. A representative ortholog from the spotted gar (Lepisosteus oculatus), termed Lo-GPR15LG, was recombinantly prepared and functionally characterized using NanoLuc Binary Technology (NanoBiT)-based {beta}-arrestin recruitment assay and homogenous ligand-receptor binding assay. Our results demonstrated that Lo-GPR15LG directly binds to and efficiently activates its cognate receptor, Lo-GPR15, with a dissociation constant (Kd) of approximately 60 nM and an EC50 value of approximately 10 nM. Functional assays further revealed that receptor activation is critically dependent on the conserved C-terminal residues. Notably, human and fish orthologs exhibited no cross-species activity, consistent with their high sequence divergence. These findings reveal that the GPR15LG-GPR15 signaling system originated in ancient fish ancestors and has remained a conserved signaling axis throughout vertebrate evolution, suggesting a fundamental role in immunity across all vertebrate lineages.

Rhodiola rosea is a traditional medicinal plant often classified as an adaptogen, with reported effects in supporting the bodys response to physical, environmental, and emotional stressors. The present study investigated the antioxidant properties of Rhodiola rosea extract and its major chemical constituents to provide insight into their potential mechanisms of action. Through in vitro biochemical assays, we demonstrated that Rhodiola rosea extract has the capacity to reduce hydrogen peroxide (H2O2) levels. Among its primary chemical components, rosavin significantly decreased H2O2, whereas salidroside had no effect. Neither compound affected superoxide levels. Structural analysis revealed that the intact phenylpropanoid glycoside architecture of rosavin is required for activity, as its individual components, arabinose and rosin, showed no inhibitory effect. Further investigation demonstrated that rosavin attenuates H2O2-mediated oxidation of thiol groups, supporting a role in cellular redox regulation. In cultured human cells, rosavin mitigated reductions in cell viability induced by exposure to H2O2, indicating cytoprotective effects under oxidative stress conditions. Finally, in an in vivo model, administration of SARS-CoV-2 spike protein increased circulating levels of H2O2, which were subsequently reduced following rosavin treatment. Collectively, these findings identify rosavin as a structurally dependent antioxidant component of Rhodiola rosea that modulates H2O2-associated oxidative stress and supports further investigation of phenylpropanoid glycosides as adaptogens.

Thymosin {beta}4 (T{beta}4) is a conserved acidic polypeptide with 43-amino acids participating in multiple pathophysiological processes. In this study in vivo effects of T{beta}4 on liver regeneration are investigated in carbon-tetrachloride (CCL4) induced rodent animal liver jury models. Results illustrate that exogenous T{beta}4 treatment significantly reduced CCL4-rendered liver necrosis around central vein. At 48 hours after CCL4 insults hepatocytes proliferation occur mainly around the periportal area, while hepatocytes proliferation around the necrosis area is prominently increased by exogenous T{beta}4 treatment. The holistic proliferation level of liver tissues are also enhanced by exogenous T{beta}4. Hepatocyte proliferation activities negatively correlate with the necrosis extent of the liver tissue. These results suggested firstly exogenous T{beta}4 treatment could enhance liver regeneration and exhibit prosperous potential for application in clinical conditions such as liver transplantation.

EIF2alpha kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2alpha-ATF4 pathway. We report a protocol for producing the minimal kinase domain of full-length human HRI, termed HRIKD-delta-KI, where the unstructured 140 amino acid (aa) kinase insert (KI) within HRI kinase domain (HRIKD) is replaced with a 2aa glycine/serine (GS) linker. X-ray crystal structures were determined of apo-HRIKD-delta-KI and of its complex with ATP at 2.1 & 2.5 Angstrom resolution respectively. Both structures display a canonical bi-lobal kinase fold. However, they remain in a non-productive state with a displaced C-helix, disassembled R-spine, and a disordered activation segment hindering the substrate site. Biophysical assays (fluorescence based thermal shift & Synchrotron Radiation Circular Dichroism) demonstrate HRIKD-delta-KI retains its functional ligand-binding conformation. All together, these findings define structural and ligand-binding features of HRI to support ongoing drug discovery efforts in blood cancer.

Mitochondrial morphology and function are critical determinants of neuronal function and survival, with disruptions in mitochondrial dynamics often preceding the overt neuronal dysfunction seen in neurodegenerative diseases such as Alzheimers disease, Huntingtons disease and Parkinsons disease. The kynurenine pathway accounts for 95% of dietary tryptophan catabolism and many of the metabolites are neuroactive, including redox-active 3-hydroxykynurenine (3-HK). 3-HK is present under normal physiological conditions in the central nervous system (CNS) and is elevated during inflammation. While supraphysiological levels of 3-HK have been associated with neurotoxicity, the effects of physiological concentrations on neuronal cells, and specifically their mitochondria, remain poorly understood. Here we assessed viability, ATP levels and redox status to determine cellular health and function in neuronal cells exposed to physiological levels of 3-HK, alongside confocal imaging and transcriptomic profiling, finding significant alterations in mitochondrial function and morphology. Interestingly, a biphasic influence of 3-HK on mitochondrial morphology was observed, with an elongated network as well as decreased surface area and volume being observed only at the lowest concentration of 3-HK, reflecting normal physiological levels. At the highest 3-HK concentration tested, reflecting an inflammatory situation, an increased number of mitochondria were present, accompanied by increased activation of caspase-3/7 and enhanced production of mitochondrial superoxide. These results highlight a previously unknown role for 3-HK in regulating mitochondrial function and structure, possibly through altered fission and fusion events, suggesting that subtle changes in kynurenine pathway metabolism may contribute to early mitochondrial dysfunction in neurological disease.

The chemical constituents and cytoprotective potential of Cyathea podophylla, a Vietnamese fern, remain poorly investigated. This study aimed to isolate its compounds and evaluate their in vitro cytoprotective activity against 6-hydroxydopamine (6-OHDA)-induced toxicity in F11 cells. Compounds were chromatographically isolated and structurally characterized using NMR and HR-ESI-MS. Seven compounds were identified: five phenolics (trans-cinnamic acid, (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one, p-coumaric acid, 3,4-dihydroxybenzoic acid, 4-O-acetyl-caffeic acid), 5-hydroxymethylfurfural, and butyl-{beta}-D-fructofuranoside. Six of these are newly reported for the Cyathea genus. In MTT assays, butyl-{beta}-D-fructofuranoside exhibited the strongest cytoprotective effect (69.6% cell protection at 10 {micro}M, p < 0.001), followed by (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one (39.2% at 10 {micro}M). The remaining compounds lacked significant activity. These findings expand the phytochemical profile of Cyathea podophylla and provide preliminary evidence of its cytoprotective properties against 6-OHDA-induced injury, warranting further mechanistic and in vivo validation.

BackgroundHuntington disease (HD) is a neurological disorder caused by an aberrant CAG expansion in the HTT gene, producing a mutant protein (mHTT). Although HD is classically characterized by adult-onset cortical and striatal degeneration, accumulating evidence suggests that altered cortical development may also contribute to disease pathogenesis. ObjectiveWe sought to investigate the impact of mHTT on neocortical patterning, which is a largely unexplored aspect of HD. MethodsUsing the HdhQ140 HD knock-in mouse model, we performed immunofluorescence and in situ hybridization to analyze the patterning of the cortex from embryonic day 10 to postnatal day 7. ResultsDuring embryogenesis, HTT expression exhibited a high medial-to-low lateral gradient in the neocortex, like that observed for key transcription factors involved in cortical patterning. Notably, HTT expression was absent from the cortical hem, a critical patterning center. In HD, the protein gradient remained unchanged whereas the expression in medial pallium seemed increased. During the early development of the cerebral hemispheres, the expression of morphogens and signaling pathways, including Shh, Fgf8, and Wnt/BMP genes, were disrupted in organizing centers, leading to altered expression of major neocortical transcription factors. At postnatal stages, the motor and somatosensory cortical areas were misplaced. These developmental alterations were associated with postnatal sensorimotor deficits relevant to HD. ConclusionsOur findings demonstrate that HD-related neurodevelopmental alterations arise as early as embryonic day 10 in mice. This supports previous work suggesting that defects in brain development contribute to HD pathogenesis prior to clinical onset.

Emerging drug resistance against the malaria parasite is worrisome and necessitates the development of novel antimalarials. Himalayan Buransh (Rhododendron arboreum) is a well-known medicinal plant found in the northern states of India. In this study, we observed the pleiotropic antimalarial activities and immunomodulation exhibited by the aqueous extract of Buransh flower (AEBF). AEBF demonstrated significant IC50 values (16-29 {micro}g/ml) against the asexual stages of various P. falciparum strains (3D7, Dd2-chloroquine-resistant and C580Y-artemisinin resistant). The oral administration of AEBF (200 mg/kg) in mice, suppressed [~]80% P. berghei parasitemia, improved mean survival time (MST-23.5 days) and prevented splenomegaly. Notably, the combination of AEBF and artesunate not only cleared primary infection, but also conferred sustained immunity. This immunomodulatory effect, driven by protective IFN-{gamma} resulted in reduced parasitemia during a homologous challenge without the need for further treatment. It is important to highlight the malaria transmission blocking activity of AEBF, resulting in reduced sexual stage male gametocyte exflagellation. Furthermore, the virtual drug screening of selected bioactive constituents from Buransh flower demonstrated potent binding against multiple P. falciparum proteins, suggested a pleiotropic mode of action. Altogether, our results corroborated the first ever evidence of the multistage antimalarial potential of Buransh flower, supported by in vitro cell studies, in vivo rodent malaria model and in silico docking analyses. Based on our studys findings and the traditional use of Buransh juice as a medicinal beverage in Uttarakhand, India, we propose exploring it as an adjunct therapy for drug-resistant malaria, subject to further clinical validation.

The Rho family of small GTPases plays a critical role in regulating actin cytoskeleton dynamics during endocytic processes in E. histolytica, including phagocytosis, pinocytosis, and trogocytosis. These proteins act as molecular switches, transitioning between inactive GDP-bound and active GTP-bound states, with guanine nucleotide exchange factors (GEFs) catalyzing this transition. Among the GEFs, EhFP10--a FYVE-domain-containing protein harbouring Dbl homology (DH) and pleckstrin homology (PH) domain was observed in phagocytosis along with seven functionally characterized Rho GTPases (EhRho1, EhRho2, EhRho4, EhRho5, EhRho6, EhRho8, and EhRho13). To study the specificity of FP10, a combination of GEF activity, binding affinity, and molecular dynamics simulations was used to characterize the interactions between EhFP10 and seven Rho GTPases systematically. The results revealed EhRho2 as the most specific and high-affinity interactor of EhFP10, with the highest nucleotide exchange rate and lowest dissociation constant (KD = 0.58 {micro}M). Structural modeling, sequence alignment, and interaction mapping further demonstrated that EhRho2 retains critical contact residues--such as Glu33, Arg4, and Leu69--that are variably absent in other isoforms, correlating with decreased GEF responsiveness. Molecular dynamics simulations and cross-correlation analyses supported the presence of a stable and coordinated interaction interface in the EhFP10-EhRho2 complex, distinguishing it from less active complexes. These findings indicate a highly selective GEF-GTPase module in E. histolytica, analogous to those in higher eukaryotes. The results uncover a potential regulatory mechanism specific to pathogenic amoebae and present EhFP10-EhRho2 as a novel therapeutic target for disrupting cytoskeleton-mediated processes crucial to virulence.

Polyphenols are compounds synthesized by plants as part of their chemical defense system to counteract biotic and abiotic stressors. These compounds share two key chemical characteristics: their aromatic groups make them insoluble in water, while hydroxy groups provide redox properties. These characteristics may explain how polyphenols interact with mitochondrial membranes (which are lipophilic) and participate in redox (electron scavenging) reactions of the electron transport chain, ultimately affecting ATP synthesis via oxidative phosphorylation. This interaction accounts for both the beneficial and adverse effects of polyphenols. However, no research has examined how hydroxyl groups or a lipophilic environment influence the biological activity of polyphenols. Therefore, this study aimed to explore the impact of hydroxy groups and a lipophilic environment on the biological activity of polyphenols. We tested four polyphenols (quercetin, naringenin, resveratrol, and gallic acid) with varying numbers of hydroxyl and other functional groups to determine how hydroxyl groups affect their biological activity (toxicity) in Saccharomyces cerevisiae. Additionally, we evaluated different fatty acids to understand how a lipophilic environment influences polyphenol biological activity. The results of this study support the two main ideas of our hypothesis: 1) a lipid solvent increases the toxicity of polyphenols, and 2) the molecule with the most hydroxyl groups is the most toxic (as seen with quercetin, which has five hydroxyl groups). Consequently, the increased toxicity of polyphenols in lipid solvents, along with their association with oxidizable groups, opens the door to the development of new technologies based on polyphenols.