Low Affinity Research Articles

Palmitoleic acid sensitizes vancomycin-resistant Staphylococcus aureus to vancomycin by outpacing the expression of resistance genes.

The rise in antibiotic resistance limits the availability of antibiotics to treat bacterial infections. Despite this, antibiotic development pipelines remain sparse which makes using adjuvants to reverse antibiotic resistance a promising therapeutic strategy. The use of vancomycin, a frontline antibiotic used to treat hospitalized patients with methicillin-resistant Staphylococcus aureus (MRSA) infections, is complicated by high rates of treatment failure. Vancomycin binds to the D-ala-D-ala terminus of the nascent peptidoglycan precursor lipid II, preventing cell wall biosynthesis. Vancomycin-resistant strains of S. aureus and Enterococci typically express a van gene cluster that is induced in response to vancomycin and results in the synthesis of an alternative lipid precursor with a peptide chain ending in D-ala-D-lac. Vancomycin has low affinity for the D-ala-D-lac terminus, and the bacteria can resume growth even in the presence of an otherwise lethal dose of vancomycin. We previously showed that palmitoleic acid, a host-produced monounsaturated fatty acid, combined with vancomycin led to an accumulation of large fluid patches in the bacterial membrane, resulting in membrane destabilization and cell death. In this study, we observed that palmitoleic acid increases the rate of vancomycin killing by more than 50-fold, compared to vancomycin alone. This rapid bactericidal activity by the combined treatment sensitized vancomycin-resistant S. aureus (VRSA) and vancomycin-resistant Enterococcus (VRE) to vancomycin, likely by outpacing the expression of vancomycin resistance genes. This study represents an important step in the ongoing effort to mitigate antibiotic resistance.IMPORTANCEThe development of antibiotics has transformed medicine, reducing the incidence and severity of bacterial infections and allowing for advancements in healthcare, including invasive surgeries and organ transplants. However, the rise of antibiotic resistance poses a significant threat to these medical advancements, leading to treatment failures that result in increased patient morbidity and mortality, as well as substantial healthcare costs. Vancomycin-resistant Enterococcus (VRE) species are prevalent in hospital settings and chronic infections. Although high-level vancomycin resistance in S. aureus is rare, S. aureus can acquire plasmids expressing vancomycin resistance genes from resistant Enterococcal species during infection, further complicating treatment. In this study, we find that palmitoleic acid increases the rate of vancomycin killing and restores sensitivity to vancomycin-resistant S. aureus (VRSA) and VRE isolates.

Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling

Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.

Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling.

Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.

Abstract A014: OICR41103: a chemical probe for investigating DCAF1 function and therapeutic potential

Abstract DCAF1 is a multifunctional protein that plays an important role in cellular processes, including protein degradation, cell cycle regulation, and immune function, by serving as a substrate receptor for RING-type CRL4 and the HECT family EDVP E3 ubiquitin ligases. Due to its involvement in various biological pathways and diseases, such as cancer and viral infections, there is a growing need for tools to study and regulate DCAF1 functions. Building upon our previous discovery of OICR-38268, a small-molecule binder of the DCAF1 WD40 domain, we have developed an improved chemical probe, OICR-41103. This compound exhibits enhanced binding affinity and selectivity for the WD40 domain of DCAF1. We successfully crystallized OICR-41103 with the WD40 domain, revealing its binding mode. Our probe binds to the WD40 domain of DCAF1 with a low nanomolar affinity in biophysical assays, and engages this domain in cells at nanomolar concentrations. This work presents a valuable chemical probe for studying DCAF1 function and provides a foundation for future drug discovery efforts, either by directly targeting DCAF1 or leveraging it for PROTAC-based strategies Citation Format: Mahmoud Noureldin, Serah Kimani, Brian Wilson, Levon Halabelian, Rima Al-awar. OICR41103: a chemical probe for investigating DCAF1 function and therapeutic potential. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A014

An MST-based assay reveals new binding preferences of IFIT1 for canonically and non-canonically capped RNAs.

IFIT proteins (interferon-induced proteins with tetratricopeptide repeats) are key components of the innate immune response that bind to viral and cellular RNA targets to inhibit viral translation and replication. The RNA target recognition is guided by molecular patterns, particularly at the RNA 5' ends. IFIT1 preferably binds RNAs modified with the 7-methylguanosine (m7G) cap-0 structure, while RNAs with cap-1 structure are recognized with lower affinity. Less is known about the propensity of IFIT1 to recognize non-canonical RNA 5' ends, including hypermethylated and non-canonical RNA caps. Deciphering the structure-function relationship for IFIT1-RNA interaction may improve the understanding of cellular selection of IFIT targets and guide the design of exogenously delivered therapeutic RNAs, but requires high-throughput and robust analytical methods. Here, we report a biophysical assay for quick, direct, in-solution affinity assessment of differently capped RNAs with IFIT1. The procedure, which relies on measuring microscale thermophoresis (MST) of fluorescently labelled protein as a function of increasing ligand concentration, is applicable to RNAs of various lengths and sequences without the need for their labelling or affinity tagging. Using the assay, we examined thirteen canonically and non-canonically 5'-capped RNAs, revealing new binding preferences of IFIT1. The 5' terminal m6A mark in the m7G cap had a protective function against IFIT1, which was additive with the effect observed for the 2'-O position (m6Am cap-1). In contrast, an increased affinity for IFIT1 was observed for several non-canonical caps, including trimethylguanosine (TMG), unmethylated (G), and flavin-adenine dinucleotide (FAD) caps. The results suggest new potential cellular targets of IFIT1 and may contribute to broadening the knowledge on the mechanisms of the innate immune response as well as the more effective design of chemically modified mRNAs.

Target-conditioned diffusion generates potent TNFR superfamily antagonists and agonists.

Despite progress in designing protein-binding proteins, the shape matching of designs to targets is lower than in many native protein complexes, and design efforts have failed for the tumor necrosis factor receptor 1 (TNFR1) and other protein targets with relatively flat and polar surfaces. We hypothesized that free diffusion from random noise could generate shape-matched binders for challenging targets and tested this approach on TNFR1. We obtain designs with low picomolar affinity whose specificity can be completely switched to other family members using partial diffusion. Designs function as antagonists or as superagonists when presented at higher valency for OX40 and 4-1BB. The ability to design high-affinity and high-specificity antagonists and agonists for pharmacologically important targets in silico presages a coming era in protein design in which binders are made by computation rather than immunization or random screening approaches.

Analysis of the Monophyletic Lineage of Avian Influenza H5N1 Which Circulated in Venezuelan Birds During the 2022–2023 Outbreak

Avian influenza subtype H5N1 has caused outbreaks worldwide since 1996, with the emergence of the Guandong lineage in China. The current clade 2.3.4.4b has evolved from this lineage, with increased virulence and mass mortality events in birds and mammals. The objective of this study was the analysis of 17 viral genomes of H5N1 avian influenza isolated in Venezuela during the 2022–2023 outbreak. The eight viral genomic segments were amplified using universal primers and sequenced via next-generation sequencing. The sequences were analyzed to confirm the H5 hemagglutinin clade, identify possible genetic reassortments, and perform a phylogenetic and docking analysis of the viral isolates. The viruses found in Venezuela belonged, as expected, to clade 2.3.4.4b and formed a monophyletic clade with North American influenza viruses, with no evidence of further reassortment. The introduction of the virus in South America is associated with bird migration through the Atlantic (Venezuela), Atlantic/Mississippi (Choco, Colombia), and Pacific migratory flyways, with the emergence of several viral lineages. Several mutations were found in all segments of the genome, although none of the key mutations was involved in mammalian adaptation. Moreover, in silico structural analysis suggests, as expected, that the viral hemagglutinin maintained a predilection for avian α2,3-linked sialic acid. The unprecedented pathogenic outbreak of avian influenza disease in South America was associated with the circulation of three different lineages, which maintain a lower affinity for the mammalian receptor.

Correlation between Insulin antibodyand HbA1c in Diabetes Mellitus

Insulin antibodies (INAs) are found in both types of diabetic patients and have low binding capacity and high affinity leading to hypoglycemia or hyperglycemia in type II patients taking insulin, which differs from the insulin antibodies found in type I that contain a high capacity of insulin binding and low affinity. This work aims to detect the levels of insulin antibodies and HbA1c in diabetic patients, and find the correlation between them and its potential role in glucose control. The study involved 60 patients with diabetes, divided into two groups type I and type II, each group containing 30 patients, in addition to 30 healthy individuals as a control group. High-performance liquid chromatography in a D-10 analyzer was used to measure HbA1c while ELISA was used to measure INAs in control and patients .Results: The current finding showed significant an elevated mean levels of HbA1c and INAs in both of patients groups compare to healthy, and there was non-significant weak positive correlation between them (R= 0.107 , P= 0.4 ).Together, elevated levels of HbA1c and INA, and the positive correlation between their levels, provide evidence of the effect of insulin antibodies on glycemic control and the effects of HbA1c levels in patients with diabetes.

A High-Affinity Monoclonal Antibody Against the Pancreatic Ductal Adenocarcinoma Target, Anterior Gradient-2 (AGR2/PDIA17)

Background/Objectives: Anterior Gradient-2 (AGR2/PDIA17) is a member of the protein disulfide isomerase (PDI) family of oxidoreductases. AGR2 is up-regulated in several solid tumors, including pancreatic ductal adenocarcinoma (PDAC). Given the dire need for new therapeutic options for PDAC patients, we investigated the expression and function of AGR2 in PDAC and developed a novel series of affinity-matured AGR2-specific single-chain variable fragments (scFvs) and monoclonal antibodies. Results: We found that AGR2 was expressed in approximately 90% of PDAC but not normal pancreas biopsies, and the level of AGR2 expression correlated with increasing disease stage. AGR2 expression was inversely related to SMAD4 status in PDAC and colorectal cancer cell models and was secreted from cells into their media. In normal tissues, a high density of AGR2 was detected in the epithelium of cells in the digestive tract but was lacking in most other normal tissue systems. The addition of recombinant AGR2 to cell culture and genetic overexpression of AGR2 increased the adhesion, motility, and invasiveness of both human and mouse PDAC cells. Human phage display library screening led to the discovery of multiple AGR2-specific scFv clones that were affinity-matured to produce monoclonal antibody (MAb) clones with low picomolar binding affinity (S31R/A53F/Y). These high-affinity MAbs inhibited AGR2-mediated cell adhesion, migration, and binding to LYPD3, which is a putative cell surface binding partner of AGR2. Conclusions: Our study provides novel, high-affinity, fully human, anti-AGR2 MAbs that neutralize the pro-tumor effects of extracellular AGR2 in PDAC.

Central dopamine receptors: Radiotracers unveiling the Role of dopaminergic tone in obesity.

Brain dopamine type 2 and 3 receptors (D2/3R) have been postulated to play a role in obesity. However, results from molecular neuroimaging studies exploring these receptors in obesity are not consensual. These inconsistencies may be due to the distinct characteristics of radiotracers that confound the interpretation of D2/3R assessment. Only three meta-analyses reported their results across radiotracers. Although all agree that obesity severity influences D2/3R availability, results vary for [11C]raclopride. Further, D2/3R assessment has been commonly interpreted as reflecting receptor density or availability. An alternative interpretation could be related to changes in endogenous central dopaminergic tone. The main question is whether the hypothesis of a quadratic relationship between dopaminergic tone and degree of obesity is suitable for the distinct characteristics of radiotracers. To answer this question and clarify the role of dopaminergic tone in obesity, we systematically reviewed this issue across radiotracers. Out of 514 articles, 15 articles were selected for review. Besides obesity severity, this study highlights the influence of radiotracer characteristics when assessing D2/3R. The tested hypothesis proved to be more suitable for radiotracers more susceptible to endogenous dopamine or with a lower affinity to D2/3R, supporting the quadratic relationship between dopaminergic tone and degree of obesity. While the role of D2/3R density in obesity may be relevant, dopaminergic tone seems to have a greater impact on the obesity-related differences found in these receptors. Finally, neuropsychological factors should be tested in addition to body mass index, as they may better reflect altered brain dopaminergic function.

Polyphosphate Kinase from Burkholderia cenocepacia, One Enzyme Catalyzing a Two-Step Cascade Reaction to Synthesize ATP from AMP

This study characterizes a novel polyphosphate kinase from Burkholderia cenocepacia (BcPPK2-III), an enzyme with potential applications in ATP regeneration processes. Bioinformatic and structural analyses confirmed the presence of conserved motifs characteristic of PPK2 enzymes, including Walker A and B motifs, and the subclass-specific residue E137. Molecular docking simulations showed AMP had the highest binding affinity (−7.0 kcal/mol), followed by ADP (−6.5 kcal/mol), with ATP having the lowest affinity (−6.3 kcal/mol). It was overexpressed in Escherichia coli, after purification enzymatic activity assays revealed that BcPPK2-III needed divalent cations (Mg2⁺, Mn2⁺, Co2⁺) as cofactors to be active. Functional assays revealed its ability to synthesize ATP from AMP through a stepwise phosphorylation mechanism, forming ADP as an intermediate, achieving 70% ATP conversion (TTN 4354.7) after 24 h. Kinetic studies indicated cooperative behavior and substrate preference, with AMP phosphorylation to ADP being the most efficient step. The enzyme demonstrated high thermostability (T50 = 62 °C) and a broad pH stability range (pH 6.0–9.0), making it suitable for diverse biocatalytic applications. The study highlights BcPPK2-III as a robust and versatile candidate for cost-effective ATP regeneration, offering advantages in industrial processes requiring stoichiometric amounts of ATP.

In situ self-reassembling nanosystem enhances PD-L1 blockade for cancer immunotherapy

Although immune checkpoint inhibitors (ICIs) have made great progress in cancer treatment, their off-tumor distribution, low affinity of traditional ICIs and insufficient T cells infiltration at tumor site limit immunotherapeutic efficacy. Herein, we engineer a highly specific and effective PD-L1 inhibitor (PEC) that modulates the level of binding sites with PD-L1. Specifically, PEC is a hybrid system composed of E. coli membrane expressing PD-L1 binding protein and cancer cell membrane. Notably, PEC can target the tumor site, produce oxygen in response to H2O2, rupture into membrane fragments, and reassemble to form vesicles retaining the PD-L1 binding protein. Through in situ fracture and reassembly, PEC transforms from a hybrid membrane to a single E. coli membrane, leading to the increased density of PD-L1 binding protein. Consequently, the reassembled vesicles can bind to more PD-L1 on tumor cells and induce its degradation in lysosomes. Furthermore, the cGAS-STING signaling activators HZD is encapsulated into PEC to promote T cells infiltration. We demonstrate that PEC@HZD achieves sequential T cells recruitment and functional enhancement, thus stimulating a powerful antitumor immune response. This work provides a new perspective on tailoring ICIs to improve cancer immunotherapy.

Overexpression of ace2 compensating for the acetylcholinesterase activity loss from ace1 mutations accelerated the development of eggs and early nymphs in Nilaparvata lugens.

Acetylcholinesterase (AChE) guarantees the acetylcholine signal in insect central nervous system, and is the target of organophosphorus and carbamate insecticides, towards which resistance was often reported due to AChE mutations. In Nilaparvata lugens, a major pest on rice, two mutations (G119S and F331C) were detected in AChE1 in a chlorpyrifos-resistant (CHL) strain. The double mutations in AChE1 reduced total AChE activity in metabolizing acetylcholine, such as low protein stability, substrate affinity and catalytic efficiency, which needed compensation in a special way. In CHL strain, the transcriptional level of ace2 encoding AChE2 was systematically elevated, such as over 30-fold overexpression in brain. The ace2 overexpression not only compensated for AChE activity loss in brain due to AChE1 mutations, but also accelerated the development of eggs and early nymphs in CHL strain. When performing ace2 RNAi in CHL eggs, the egg and early nymph duration were recovered. In CHL eggs, the transcriptional levels of three basic helix-loop-helix transcription factors (Ase2, Ato1 and SCL), which were closely related to neural development, were significantly upregulated. Their respective RNAi in CHL strain also significantly recovered the egg duration, as RNAi towards ace2, which partially explained the reason for the accelerated development of eggs and early nymphs. The results revealed AChE2 non-canonical function in insect embryonic development, and uncovered a physiological effect caused by ace2 overexpression as a compensation action for resistance mechanism due to AChE1 mutations, providing a base for insecticide resistance management in insects.

Crystal structures of Cystathionine β-lyase and Cystathionine β-lyase like protein from Bacillus cereus ATCC 14579

Cystathionine β-lyase (CBL) and cystathionine β-lyase-like protein (CBLP) are key PLP-dependent enzymes involved in methionine biosynthesis. In Bacillus cereus ATCC 14579 CBL (BcCBL) and CBLP (BcCBLP) catalyze the conversion of cystathionine to homocysteine and pyruvate. In this study, we found that both BcCBL and BcCBLP effectively catalyze cystathionine cleavage, with BcCBLP exhibiting a higher catalytic efficiency (kcat) and low substrate affinity (Km). We determined their crystal structures in complex with pyridoxal phosphate (PLP). BcCBL, forming a tetramer, aligns with typical CBLs in sulfur amino acid metabolism, while BcCBLP, forming a dimer, resembles the bifunctional MalY enzyme from Escherichia coli, indicating potential additional regulatory roles. These structural and functional insights highlight the distinct roles of BcCBL and BcCBLP in cellular metabolism. This study provides valuable insights into the structural diversity and potential functions of these enzymes, contributing to the broader knowledge of PLP-dependent enzymatic mechanisms.

In-silico based Designing of benzo[d]thiazol-2-amine Derivatives as Analgesic and Anti-inflammatory Agents.

Benzo[d]thiazoles represent a significant class of heterocyclic compounds renowned for their diverse pharmacological activities, including analgesic and antiinflammatory properties. This molecular scaffold holds substantial interest among medicinal chemists owing to its structural versatility and therapeutic potential. Incorporating the benzo[d]thiazole moiety into drug molecules has been extensively investigated as a strategy to craft novel therapeutics with heightened efficacy and minimized adverse effects. The aim of the present research work was to design, synthesize and characterize the new benzo[d]thiazol-2-amine derivatives as potent analgesic and anti-inflammatory agents. The synthesis of the presented benzo[d]thiazol-2-amine derivatives was performed by condensing-(4-chlorobenzylidene) benzo[d]thiazol-2-amine with a number of substituted phenols in the presence of potassium iodide and anhydrous potassium carbonate in dry acetone. IR spectroscopy, 1HNMR spectroscopy, 13CNMR spectroscopy and Mass spectroscopy methods were used to characterize the structural properties of all 13 newly synthesized derivatives. The molecular properties of these newly synthesized derivatives were estimated to study the attributes of drug-like candidates. Benzo[d]thiazol-2-amine derivatives were molecularly docked with selective enzymes COX-1 and COX-2. Analgesic and anti-inflammatory activities of synthesized compounds were evaluated by using albino rats. Findings of the research suggested that compounds G3, G4, G6, G8 and G11 possess higher binding affinity than diclofenac sodium, when docking was performed with enzyme COX-1. Compounds G1, G3, G6, G8 and G10 showed lower binding affinity than Indomethacin when docking was performed with enzyme COX-2. In vitro evaluation of the COX-1 and COX-2 enzyme inhibitory activities was performed for synthesized compounds. Compounds G10 and G11 exhibited significant COX-1 and COX-2 enzyme inhibitory action with an IC50 value of 5.0 and 10 μM, respectively. Using the hot plate method and the carrageenan-induced rat paw edema model, the synthesized compounds were screened for their biological activities, including analgesic and anti-inflammatory activities. Highest analgesic action was exhibited by derivative G11 and the compound G10 showed the highest anti-inflammatory response. Inhibition of COX may be considered as a mechanism of action of these compounds. It was concluded that synthesized derivatives G10 and G11 exhibited significant analgesic and anti-inflammatory effect; therefore, the said compounds may be subjected to further clinical investigation for establishing these as future compounds for the treatment of pain and inflammation.

Discovery of a novel KV7.2/7.3 channels agonist for the treatment of neuropathic pain

Here, we designed, synthesized and evaluated a series of compounds as KV7.2/7.3 channels (or KCNQ2/3) agonists. The new compounds were assayed in vitro for KCNQ2/3 and other receptors binding affinity. The desired compound 16 showed high activity for KCNQ2/3 (EC50 = 1.03 ± 0.07 μM) without acute liver injury compared to flupirtine. It demonstrated powerful dose-dependent effects in multiple analgesic models, such as chronic constriction injury (CCI, ED50 = 12.02 mg/kg) and streptozotocin-induced diabetic peripheral neuropathic pain (DPNP, ED50 = 9.63 mg/kg) models. Additionally, compound 16 showed low affinity for human ether-a-go-go-related gene (hERG), high thresholds for acute toxicity, good motor performance in the rotarod test and acceptable pharmacokinetic properties. These results suggest the potentiality of compound 16 for the treatment of neuropathic pain.

Green synthesis and anticancer activity of titanium dioxide nanoparticles using the endophytic fungus Aspergillus sp.

Titanium dioxide nanoparticles (TiO2 NPs) have attracted significant attention for their unique physicochemical features and various applications. This study demonstrated the biosynthesis of TiO2 NPs using Aspergillus fungal extract that served as a green and eco-friendly reducing and stabilizing agent. The biosynthesized nanoparticles were analyzed using SEM and TEM to determine their morphology, size, and distribution, FTIR to determine functional groups, and Zeta potential to assess their surface charge and stability. An extensive review of the Protein Data Bank (PDB) and literature indicated that TiO2 could target various cancer-relevant matrix metalloproteinases. In vitro screening indicated promising anticancer effects against the MCF7 breast cancer cell line. To investigate the possible mode of action of TiO2 NPs as an anticancer agent, human matrix metalloproteinase-3 was highlighted as a protein inhibited by metallic ions like PtCl2. Therefore, we investigated whether TiO2 could similarly interact with the active site of MMP-3. We hypothesized that TiO2 could interact with the MMP-3 active site and replace PtCl2 with modelled TiO2 in its co-crystallized binding site. A 100 ns-long MDS, binding free energy (ΔGBinding) of PtCl2 and TiO2 within MMP-3 binding site indicated that TiO2's enhanced binding affinity and stability, as evidenced by a ΔGBinding of −7.23 kcal/mol and average RMSD of 0.89 Å, compared to PtCl2's lower affinity. In conclusion, endophytic fungi can be used efficiently in the biosynthesis of nanoparticles. Our study indicated TiO2 NPs have a potential anticancer effect, suggesting TiO2 binds to MMP3, potentially offering comparable inhibitory effects on the enzyme's activity.

Investigation of Cr2SiC Ceramic MAX Phase Coated Metallic Bipolar Plates in Ex-situ Conditions for Proton Exchange Membrane Fuel Cells

Essential for compact and lightweight proton exchange membrane fuel cell (PEMFC) stacks, metallic bipolar plates (MBPs) suffer from durability and conductivity issues due to surface corrosion and passivation. This study conducts ex-situ characterization of Cr2SiC ceramic MAX phase coatings on stainless steel (SS) 316 L substrates to evaluate their suitability for MBP application. The investigation encompasses coating structure, surface morphology, corrosion resistance, surface wettability, in-plane electrical conductivity, and interfacial contact resistance. X-ray diffraction and X-ray photoelectron spectroscopy confirm the presence of Cr2SiC coatings on SS316L, while energy-dispersive X-ray spectroscopy verifies uniform coverage and elemental weight percentage. Corrosion resistance is evaluated using potentiostatic and potentiodynamic polarization tests, showing excellent resistance with low corrosion current density (Icorr) at both 25 °C (3.29E-03 μA/cm2) and 80 °C (4.32E-02 μA/cm2), meeting US Department of Energy (DOE) technical targets. Potentiodynamic polarization reveals large corrosion potential and small Icorr values at both temperatures, outperforming uncoated samples. Electrochemical impedance spectroscopy post-accelerated corrosion tests show high charge transfer resistance at 25 °C (3.68E+05 Ω cm2) and 80 °C (3.02E+05 Ω cm2), indicating stability in acidic environments. Surface wettability analysis indicates low water affinity with a large contact angle (75°) and low surface free energy (28.92 mJ/m2) for the coated samples as compared to the uncoated samples. Electrical conductivity meets DOE targets with an in-plane conductivity of 4.59E+05 S/m and interfacial contact resistance of 8.04 mΩcm2 after 5-h accelerated corrosion tests at 80 °C. These results suggest that Cr2SiC coated SS316L exhibits excellent corrosion resistance, surface wettability, and electrical characteristics, making them viable for PEMFC applications.

Cryo-EM structure of a photosystem I variant containing an unusual plastoquinone derivative in its electron transfer chain.

Photosystem I (PS I) is a light-driven oxidoreductase responsible for converting photons into chemical bond energy. Its application for renewable energy was revolutionized by the creation of the MenB deletion (ΔmenB) variant in the cyanobacterium Synechocystis sp. PCC 6803, in which phylloquinone is replaced by plastoquinone-9 with a low binding affinity. This permits its exchange with exogenous quinones covalently coupled to dihydrogen catalysts that bind with high affinity, thereby converting PS I into a stable solar fuel catalyst. Here, we reveal the 2.03-Å-resolution cryo-EM structure of a recent MenB variant of PS I. The quinones and their binding environment are analyzed in the context of previous biophysical data, thereby enabling a protocol to solve future PS I hybrids and constructs from this genetically tractable cyanobacterium.

Exploration of urease-aided calcium carbonate mineralization by enzyme analyses of Neobacillus mesonae strain NS-6.

Urease containing nickel cofactor is crucial for urea-hydrolytic induced calcium carbonate (CaCO3) precipitation (UICP). However, limited information exists regarding the influence of amino acid residues interacting with nickel ions in its structure on induced CaCO3 mineralization. Herein, RT-qPCR was used to demonstrate that the addition of NiCl2 dramatically upregulated the expression of urease structural gene ureC that was correlated with nickel binding in Neobacillus mesonae strain NS-6. Homology modeling and molecular docking were employed to construct the three-dimensional structure of urease and seek the key residues involved in nickel binding process, and virtual mutation technology was adopted to inform three key residues coordinated with nickel ions and urea, His249, His275, and Asp363. Four metrics, including root mean square deviation values for mutations of those key residues in urease-urea complexes severally and wild-type, were calculated by molecular dynamics simulations when they were mutated into alanine, respectively. Subsequently, the mutations of H249A, H275A, and D363A were characterized using western blotting to reveal a decrease in the relative expression and activity of urease, along with a corresponding reduction in CaCO3 precipitation. Ultimately, the mutations also exhibited that they had lower substrate affinity and catalytic efficiency for urea through enzymatic properties analysis. The findings suggested that those residues played a pivotal role in UICP of strain NS-6, which would expand the theoretical basis for modulating urease activity.IMPORTANCEUrease-producing bacterium is of great importance in diverse application fields, such as environmental remediation, due to its key driving characteristics in catalyzing urea hydrolysis via urea-hydrolytic induced CaCO3 precipitation (UICP). As essential cofactors of urease, nickel ions play a crucial role in regulating urease catalysis and maintaining structural stability. Numerous investigations have emphasized the impact of nickel ions on urease activity in recent years, to our best knowledge, only a few literatures have studied the molecular-level regulation of nickel-ligand residues. This study focused on the highly urease-producing bacterial Neobacillus mesonae NS-6 to explore the effects of specific nickel-ligand residues on the urease-aided CaCO3 mineralization process using molecular simulation predictions and targeted mutation experiments. The aim was to provide a molecular-level understanding of the interactive effects between urea and critical residues associated with the urease active center, as well as propose an effective modification strategy to enhance the application of UICP in future environmental areas.