Low Affinity Research Articles

Characterization of a cytochrome P450 that catalyzes the O-demethylation of lignin-derived benzoates

Cytochromes P450 (P450s) are a superfamily of heme-containing enzymes possessing a broad range of monooxygenase activities. One such activity is O-demethylation, an essential and rate-determining step in emerging strategies to valorize lignin that employ carbon-carbon bond cleavage. We recently identified PbdA, a P450 from Rhodococcus jostii RHA1, and PbdB, its cognate reductase, which catalyze the O-demethylation of para-methoxylated benzoates (p-MBAs) to initiate growth of RHA1 on these compounds. PbdA had the highest affinity (Kd = 3.8 ± 0.6 μM) and apparent specificity (kcat/KM = 20 000 ± 3 000 M-1 s-1) for p-MBA. The enzyme also O-demethylated two related lignin-derived aromatic compounds with remarkable efficiency: veratrate and isovanillate. PbdA also catalyzed the hydroxylation and dehydrogenation of p-EB even though RHA1 did not grow on this compound. Atomic-resolution structures of PbdA in complex with p-MBA, p-EB and veratrate revealed a cluster of three residues that form hydrogen bonds with the substrates’ carboxylate: Ser87, Ser237 and Arg84. Substitution of these residues resulted in lower affinity and O-demethylation activity on p-MBA as well as increased affinity for the acetyl analogue, p-methoxyacetophenone. The S87A and S237A variants of PbdA also catalyzed the O-demethylation of an aldehyde analogue of p-MBA, p-methoxy-benzaldehyde, while the R84M variant did not, despite binding this compound with high affinity. These results suggest that Ser87, Ser237 and Arg84 are not only important determinants of specificity but also help to orientate that substrate correctly in the active site. This study facilitates the design of biocatalysts for lignin valorization.

T-Cell Receptors Cross-Reactive to Coronaviral Epitopes Homologous to the SPR Peptide.

The COVID-19 pandemic caused by the rapid spread of the novel coronavirus SARS-CoV-2, has promoted an interest in studying the T-cell immune response. It was found that the polyclonal and cross-reactive T-cell response against seasonal coronaviruses and other SARS-CoV-2 strains reduced disease severity. We investigated the immunodominant T-cell epitope SPRWYFYYYL from the nucleocapsid protein of SARS-CoV-2. The immune response to this epitope is characterized by the formation of highly homologous (convergent) receptors that have been found in the T-cell receptor (TCR) repertoires of different individuals. This epitope belongs to a group of highly conserved peptides that are rarely mutated in novel SARS-CoV-2 strains and are homologous to the epitopes of seasonal coronaviruses. It has been suggested that the cross-reactive response to homologous peptides contributes to the reduction of COVID-19 severity. However, some investigators have questioned this hypothesis, suggesting that the low affinity of the cross-reactive receptors reduces the strength of the immune response. The aim of this study was to evaluate the effect of amino acid substitutions in the SPR epitope on its binding affinity to specific TCRs. For this, we performed antigen-dependent cellular expansions were performed using samples from four COVID-19-transfected donors and sequenced their TCR repertoires. The resulting SPR-specific repertoire of β-chains in TCRs had a greater sequence diversity than the repertoire of α-chains. However, the TCR repertoires of all four donors contained public receptors, three of which were cloned and used to generate the Jurkat E6-1 TPR cell line. Only one of these receptors was activated by the SPR peptide and recognized with the same affinity by its mutant homologue LPRWYFYYY from seasonal coronaviruses. This indicates that the presence of the mutation did not affect the strength of the immune response, which may explain why the cross-reactive response to the SPR epitope is so frequent and contributes positively to COVID-19 infection.

Effectiveness of supercooled freezing in suppressing ice recrystallization during frozen storage

Ice recrystallization often occurs during frozen food storage, an undesirable occurrence that can cause further damage to food cells and tissues. One factor that triggers its occurrence is the initial structure of the ice crystals formed during the freezing phase. During frozen storage, small ice crystals may recrystallize due to their high surface-to-volume ratio and excess free energy. In this study, the ice recrystallization of supercooled frozen tofu was evaluated during a 12-week frozen storage period and compared with that of slowly and rapidly frozen tofu. The storage temperature was at − 10 ± 2 ℃. Slow freezing at − 10 ℃ static air has produced large crystals up to 0.6 mm before storage and tended not to grow further. Rapid freezing at − 80 ℃ static air with elongated ice crystals exhibits the highest recrystallization rate, especially on the surface of the sample, where the maximum ice crystals grew from 0.23 mm before storage to 0.45 mm after 12 weeks. Supercooled freezing has produced small and homogeneous ice crystals, the largest being 0.15 mm, but their spherical shape tends to have a low affinity for recrystallization, which has allowed the crystals to maintain their size and structure during the frozen storage.

Human NGF "Painless" Ocular Delivery for Retinitis Pigmentosa: An In Vivo Study.

Retinitis pigmentosa (RP) is a family of genetically heterogeneous diseases still without a cure. Despite the causative genetic mutation typically not expressed in cone photoreceptors, these cells inevitably degenerate following the primary death of rods, causing blindness. The reasons for the "bystander" degeneration of cones are presently unknown but decrement of survival factors, oxidative stress, and inflammation all play a role. Targeting these generalized biological processes represents a strategy to develop mutation-agnostic therapies for saving vision in large populations of RP individuals. A classical method to support neuronal survival is by employing neurotrophic factors, such as NGF. This study uses painless human NGF (hNGFp), a TrkA receptor-biased variant of the native molecule with lower affinity for nociceptors and limited activity as a pain inducer; the molecule has identical neurotrophic power of the native form but a reduced affinity for the p75NTR receptors, known to trigger apoptosis. hNGFp has a recognized activity on brain microglial cells, which are induced to a phenotype switch from a highly activated to a more homeostatic configuration. hNGFp was administered to RP-like mice in vivo with the aim of decreasing retinal inflammation and also providing retinal neuroprotection. However, the ability of this treatment to counteract the bystander degeneration of cones remained limited.

Interaction Between SARS-CoV-2 Spike Protein S1 Subunit and Oyster Heat Shock Protein 70.

There is growing evidence that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contaminates the marine environment and is bioaccumulated in filter-feeding shellfish. Previous study shows the Pacific oyster tissues can bioaccumulate the SARS-CoV-2, and the oyster heat shock protein 70 (oHSP70) may play as the primary attachment receptor to bind SARS-CoV-2's recombinant spike protein S1 subunit (rS1). However, detailed information about the interaction between rS1 and oHSP70 is still unknown. In this study, we confirmed that the affinity of recombinant oHSP70 (roHSP70) for rS1 (KD = 20.4nM) is comparable to the receptor-binding affinity of rACE2 for rS1 (KD = 16.7nM) by surface plasmon resonance (SPR)-based Biacore and further validated by enzyme-linked immunosorbent assay (ELISA). Three truncated proteins (roHSP70-N/C/M) and five mutated proteins (p.I229del, p.D457del, p.V491_K495del, p.K556I, and p.ΣroHSP70) were constructed according to the molecular docking results. All three truncated proteins have significantly lower affinity for rS1 than the full-length roHSP70, indicating that all three segments of roHSP70 are involved in binding to rS1. Further, the results of SPR and ELISA showed that all five mutant proteins had significantly lower affinity for rS1 than roHSP70, suggesting that amino acids at these sites are involved in binding to rS1. This study provides a preliminary theoretical basis for the bioaccumulation of SARS-CoV-2 in oyster tissues or using roHSP70 as the capture unit to selectively enrich virus particles for detection.

Photophysical properties of donor (D)-acceptor (A)-donor (D) diketopyrrolopyrrole (A) systems as donors for applications to organic electronic devices.

Fourteen substituted diketopyrrolopyrrole (DPP) molecules in a donor (D)-acceptor (DPP)-donor (D) arrangement were designed. We employed density functional theory, time-dependent DFT, DFT-MRCI and the ab initio wave function second-order algebraic diagrammatic construction (ADC(2)) methods to investigate theoretically these systems. The examined aromatic substituents have one, two, or three hetero- and non-hetero rings. We comprehensively investigated their optical, electronic, and charge transport properties to evaluate potential applications in organic electronic devices. We found that the donor substituents based on one, two, or three aromatic rings bonded to the DPP core can improve the efficiency of an organic solar cell by fine-tuning the highest occupied molecular orbital/lowest unoccupied molecular orbital levels to match acceptors in typical bulk heterojunctions acceptors. Several properties of interest for organic photovoltaic devices were computed. We show that the investigated molecules are promising for applications as donor materials when combined with typical acceptors in bulk heterojunctions because they have appreciable energy conversion efficiencies resulting from their low ionization potentials and high electron affinities. This scenario allows a more effective charge separation and reduces the recombination rates. A comprehensive charge transfer analysis shows that D-A (DDP)-D systems have significant intramolecular charge transfer, further confirming their promise as candidates for donor materials in solar cells. The significant photophysical properties of DPP derivatives, including the high fluorescence emission, also allow these materials to be used in organic light-emitting diodes.

Development of ketobenzothiazole-based peptidomimetic TMPRSS13 inhibitors with low nanomolar potency.

TMPRSS13, a member of the Type II Transmembrane Serine Proteases (TTSP) family, is involved in cancer progression and in cell entry of respiratory viruses. To date, no inhibitors have been specifically developed toward this protease. In this study, a chemical library of 65 ketobenzothiazole-based peptidomimetic molecules was screened against a proteolytically active form of recombinant TMPRSS13 to identify novel inhibitors. Following an initial round of screening, subsequent synthesis of additional derivatives supported by molecular modelling, uncovered important molecular determinants involved in TMPRSS13 inhibition. One inhibitor, N-0430, achieved low nanomolar affinity towards TMPRSS13 activity in a cellular context. Using a SARS-CoV-2 pseudovirus cell entry model, we further show the ability of N-0430 to block TMPRSS13-dependent entry of the pseudovirus. The identified peptidomimetic inhibitors and the molecular insights of their potency gained from this study will aid in the development of specific TMPRSS13 inhibitors.

In-silico Identification of citrus phytochemicals for chemical interaction of CotH of Mucor and human GRP78 receptor and its experimental verification through FTIR spectroscopy

After suffering from serious critical disease such as recent COVID-19 pandemic, secondary fungal infection (Specially Mucormycosis) has become a serious concern throughout the world. Glucose regulated protein 78 (GRP78) is a multifunctional protein which acts as a receptor in human body for spore Coat homolog (CotH) cell surface protein of Mucorales. The development of immunity with easily available plant products is vital and essential due to having less effect on other biological activities. Here, In-silico method has been employed to screen antifungal citrus phyto-chemicals through molecular docking with CotH and its ADMET study due its rapid and cost effective quality compared to the trial and error methods using experimental studies. Limonin and naringin have been selected as potential ligands due to having its low binding affinity and no risk factor as per Lipinski's rule of five and medicinal chemistry. Molecular interaction of naringin and limonin with CotH obtained by molecular docking shows better binding affinity of −8.7 and – 8.9 kcal/mol respectively. The binding of CotH-GRP78 complex (developed through protein-protein molecular docking) with limonin resists more in the spreading of Mucormycosis in comparison to naringin. The FTIR spectra of limonin-CotH and Naringin-CotH sample exhibits the evidences of hydrogen bond for the formation of both strong limonin-CotH and naringin-CotH complex assuring the development of immunity with citrus fruit containing limonin and naringin.

Preparing and Assessment of Biocidal La Nano-complex Treated Filter Capacity against Isolated Microbes from Air Conditioning Systems in COVID-19 Rehabilitation Rooms

Mucormycosis is a severe fungal infection which mainly caused by filamentous fungi of the Absidia sp., Rhizopus sp., Cunninghamella sp, Mucor sp., and Rhizomucor sp. Moreover, the pandemic of the SARS-CoV-2 virus expands the need to interfere with spread of the airborne respiratory infections. Accordingly, developing cutting-edge solutions to restrict and/or prevent air contamination by infectious microbes are very warranted. The current work aims to prepare biocidal La-nano complex treated filters and assess their anti-fungal capacity against 20 Rhizopus oryzae, 10 Candida albicans, and 11 Aspergillus fumigatus. These fungi were isolated from the inside parts of the air conditioning systems in the rehabilitation rooms for COVID-19 patients. The obtained results demonstrated that the prepared were able to significantly decrease the invading microbes and eradicate Rhizopus, Aspergillus, Mucor, Candida albicans isolates at 0.64 mg/ml concentration. DFT study compares the electronic properties and reactivity of a ligand in its uncoordinated form with its lanthanum complex. The ligand exhibits lower binding energy, ionization potential, electron affinity, absolute electronegativity, and chemical potential when coordinated with lanthanum. In contrast, the lanthanum complex has a smaller energy gap, absolute hardness, and global softness.

Rational Design of High Affinity Interaction Between CC Chemokine Binding Protein vCCI and CCL17/TARC.

The poxvirus-derived protein vCCI (viral CC chemokine inhibitor) binds almost all members of the CC chemokine family with nanomolar affinity, inhibiting their pro-inflammatory actions. Understanding the affinity and specificity of vCCI could lead to new anti-inflammatory therapeutics. CCL17, also known as TARC, is unusual among CC chemokines by having only micromolar binding to vCCI. We have used sequence analysis and molecular simulations to determine the cause of this weak binding, which identified several locations in CCL17 where mutations seemed likely to improve binding to vCCI. Based on the aforementioned analysis, we expressed and tested multiple mutants of CCL17. We found two single point mutants V44K and Q45R that increased binding affinity to vCCI by 2-3-fold and, in combination, further improved affinity by 7-fold. The CCL17 triple mutant G17R/V44K/Q45R yielded a Kd of 0.25 ± 0.13 μM, a 68-fold improvement in affinity compared to the complex with wild-type CCL17. A quadruple mutant G17R/V44K/Q45R/R57W showed high affinity (0.59 ± 0.09 μM) compared to the wild type but lower affinity than the triple mutant. This work demonstrates that sequence comparisons and molecular simulations can predict chemokine mutations that increase the level of binding to vCCI, an important first step in developing engineered chemokine inhibitors useful for anti-inflammatory therapy.

Long-lasting antistatic hydrophilic polyethylene terephthalate fabric using greener reagents

AbstractPolyethylene terephthalate (PET) is the most common synthetic polymer used in the textile sector by virtue of its unique superior performance attributes. However, the comfort characteristics of PET fabrics, such as their inadequate sweat-absorbing capability, low affinity for most dyestuff classes, and susceptibility to the accumulation of electrostatic charge, make them unfavorable compared to natural fibers. Rendering PET fabrics with hydrophilic and antistatic properties is difficult due to the lack of reactive functional groups. Herein, a long-lasting eco-friendly strategy to impart some desirable properties to PET fabrics was developed. The PET fabric was saponified in an aqueous caustic soda solution, followed by treatment with an amino acid, namely lysine, aspartic acid, serine, tyrosine, or cysteine, as a coupling agent, and eventually, the fabric was finished with the protein biopolymer gelatin using the pad-dry-cure method. The effects of treatment of PET fabric with the aforementioned reagents on its surface hydrophilicity, roughness, antistatic, thermal stability, ultraviolet protection, air permeability, yellowness, bending stiffness, and tensile properties were monitored. The discrepancy between the chemical structures of the untreated and finished fabrics was determined using Fourier transform infrared spectroscopy as well as by determining the carboxylic and nitrogen contents. The morphological and crystal structures of the treated fabrics were examined using scanning electron microscopy and X-ray diffraction pattern, respectively. The results indicate that a maximum add-on was obtained upon treatment of the saponified PET fabrics with 0.5 M lysine followed by 7.5% (on the weight of the fiber) gelatin using the pad-dry-cure method.The finished PET fabrics exhibited improved hydrophilic and antistatic properties with adequate protection against UV rays.Ingeneral, treatment of PET fabrics with gelatin, using a bi-functional amino acid as a coupling agent, is an effective and durable method to improve some performance and comfort features of PET fabric without having a remarkable negative effect on the fabric’s mechanical properties.

Origin of biogeographically distinct ecotypes during laboratory evolution

Resource partitioning is central to the incredible productivity of microbial communities, including gigatons in annual methane emissions through syntrophic interactions. Previous work revealed how a sulfate reducer (Desulfovibrio vulgaris, Dv) and a methanogen (Methanococcus maripaludis, Mm) underwent evolutionary diversification in a planktonic context, improving stability, cooperativity, and productivity within 300–1000 generations. Here, we show that mutations in just 15 Dv and 7 Mm genes within a minimal assemblage of this evolved community gave rise to co-existing ecotypes that were spatially enriched within a few days of culturing in a fluidized bed reactor. The spatially segregated communities partitioned resources in the simulated subsurface environment, with greater lactate utilization by attached Dv but partial utilization of resulting H2 by low affinity hydrogenases of Mm in the same phase. The unutilized H2 was scavenged by high affinity hydrogenases of planktonic Mm, producing copious amounts of methane. Our findings show how a few mutations can drive resource partitioning amongst niche-differentiated ecotypes, whose interplay synergistically improves productivity of the entire mutualistic community.

Investigation on the machining characteristics of AZ91 magnesium alloy using uncoated and CVD-diamond coated WC-Co inserts

An investigation has been performed to study the compatibility of machining AZ91 magnesium alloy in dry conditions using uncoated and CVD diamond-coated WC-Co inserts. The machining characteristics such as built-up edge (BUE) formation, surface roughness, cutting forces and chip morphology were studied at the cutting speeds, 350, 650 and 950 m/min. The results revealed severe adhesion of the AZ91 Mg alloy on the uncoated WC-Co inserts with increasing cutting speed and contributed to an increase in surface roughness and cutting forces. The CVD diamond-coated inserts have shown negligible built-up edges at various cutting speeds due to their unique properties like low coefficient of friction, low chemical affinity and high thermal conductivity. Snarled types of long continuous chips were observed while machining with CVD diamond-coated WC-Co inserts, whereas short continuous chips were observed with uncoated WC-Co inserts.

Multiple strategies approach: A novel crosslinked hydrogel forming chitosan-based microneedles chemowrap patch loaded with 5-fluorouracil liposomes for chronic wound cancer treatment

Untreated or poorly managed chronic wounds can progress to skin cancer. Topically applied 5-fluorouracil (5-FU), a nonspecific cytostatic agent, can cause various side effects. Its high polarity also results in low cell membrane affinity and bioavailability. Hydrogel, used for its occlusive effect, is one platform for treating chronic wounds combined with PEGylated liposomes (LPs), developed to increase drug-skin affinity. This research aimed to develop a novel hydrogel forming chitosan-based microneedles (HFM) chemowrap patch containing 5-FU PEGylated LPs, improving 5-FU efficiency for pre-carcinogenic and carcinogenic skin lesions. The results indicated that the 5-FU-PEGylated LPs-loaded HFM chemowrap patch exhibited desirable physical and mechanical characteristics with complete penetration ability. Furthermore, in vivo skin permeation studies demonstrated the highest percentage of 5-FU permeated the skin (42.06 ± 11.82 %) and skin deposition (75.90 ± 1.13 %) compared to the other treatments, with demonstrated superior percentages of complete wound healing in in vivo (47.00 ± 5.77 % wound healing at day 7) and in NHF cells (92.79 ± 7.15 % at 48 h). Furthermore, 5-FU-PEGylated LPs-loaded HFM chemowrap patches exhibit efficient anticancer activity while maintaining safety for normal cells. The results also show that the developed formulation of a 5-FU-PEGylated LPs-loaded HFM chemowrap patch could enhance apoptosis higher than that of the 5-FU solution. Consequently, 5-FU PEGylated LPs-loaded HFM chemowrap patch represented a promising drug delivery approach for treating pre-carcinogenic and carcinogenic skin lesions.

A broadly cross-reactive i-body to AMA1 potently inhibits blood and liver stages of Plasmodium parasites

Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium.

Simultaneous capture of trace benzene and SO2 in a robust Ni(II)-pyrazolate framework

Benzene and SO2, coexisting as hazardous air pollutants in some cases, such as in coke oven emissions, have led to detrimental health and environmental effects. Physisorbents offer promise in capturing benzene and SO2, while their performance compromises at low concentration. Particularly, the simultaneous capture of trace benzene and SO2 under humid conditions is attractive but challenging. Here, we address this issue by constructing a robust pyrazolate metal-organic framework (MOF) sorbent featuring rich accessible Ni(II) sites with low affinity to water and good stability. This material achieves a high benzene uptake of 5.08 mmol g–1 at 10 Pa, surpassing previous benchmarks. More importantly, it exhibits an adsorption capacity of ~0.51 mmol g–1 for 10 ppm benzene and ~1.21 mmol g–1 for 250 ppm SO2 under 30% relative humidity. This work demonstrates that a pioneering MOF enables simultaneous capture of trace benzene and SO2, highlighting the potential of physisorbents for industrial effluent remediation, even in the presence of moisture.

A comprehensive insight from molecular docking and dynamics with clinical investigation on the impact of direct oral anticoagulants on atheroprotective protein in atrial fibrillation

BackgroundDirect oral anticoagulants (DOACs) have high potency against their therapeutic target and are widely used in the treatment of atrial fibrillation (AF). Most DOACs are often claimed to have adverse effects due to off-target inhibition of essential proteins. Human serum paraoxonase 1 (PON1), one of the essential proteins, known for its anti-inflammatory and antioxidant properties, could be affected by DOACs. Thus, a comparative evaluation of DOACs and their effect on PON1 protein will aid in recommending the most effective DOACs for AF treatment. This study aimed to assess the impact of DOACs on PON1 through a combination of computational and experimental analyses.MethodsWe focus on apixaban, dabigatran, and rivaroxaban, the most recommended DOACs in AF treatment, for their impact on PON1 through molecular docking and molecular dynamics (MD) simulation to elucidate the binding affinity and drug-protein structural stability. This investigation revealed the most influential DOACs on the PON1 protein. Then experimental validation was performed in DOAC-treated AF participants (n = 42; 19 treated with dabigatran and 23 treated with rivaroxaban) compared to a healthy control group (n = 22) through gene expression analysis in peripheral blood mononuclear cells (PBMC) and serum enzyme concentration.ResultsOur computational investigation showed rivaroxaban (−4.24 kcal/mol) exhibited a lower affinity against the PON1 protein compared to apixaban (−5.97 kcal/mol) and dabigatran (−9.03 kcal/mol) through molecular docking. Dabigatran holds complex interactions with PON1 at GLU53, TYR197, SER193, and ASP269 by forming hydrogen bonds. Additionally, MD simulation revealed that dabigatran disrupts PON1 stability, which may contribute functional changes. Further experimental validation revealed a significant down-regulation (p < 0.05) of PON1 gene expression in PBMC and decreased serum PON1 enzyme concentration on DOAC treatment. Rivaroxaban as about 48% has inhibitory percentage and dabigatran as about 75% of inhibitory percentage compared to healthy control.ConclusionOverall, our computational and experimental results clearly show the higher inhibitory effect of dabigatran than rivaroxaban. Hence, rivaroxaban will be a better drug candidate for improving the outcome of AF.

A Novel Confocal Scanning Protein-Protein Interaction Assay (PPI-CONA) Reveals Exceptional Selectivity and Specificity of CC0651, a Small Molecule Binding Enhancer of the Weak Interaction between the E2 Ubiquitin-Conjugating Enzyme CDC34A and Ubiquitin.

Protein-protein interactions (PPIs) are some of the most challenging target classes in drug discovery. Highly sensitive detection techniques are required for the identification of chemical modulators of PPIs. Here, we introduce PPI confocal nanoscanning (PPI-CONA), a miniaturized, microbead based high-resolution fluorescence imaging assay. We demonstrate the capabilities of PPI-CONA by detecting low affinity ternary complex formation between the human CDC34A ubiquitin-conjugating (E2) enzyme, ubiquitin, and CC0651, a small molecule enhancer of the CDC34A-ubiquitin interaction. We further exemplify PPI-CONA with an E2 enzyme binding study on CC0651 and a CDC34A binding specificity study of a series of CC0651 analogues. Our results indicate that CC0651 is highly selective toward CDC34A. We further demonstrate how PPI-CONA can be applied to screening very low affinity interactions. PPI-CONA holds potential for high-throughput screening for modulators of PPI targets and characterization of their affinity, specificity, and selectivity.

Confinement Effect in Multilayer Films Made from Semicrystalline and Bio-Based Polyamide and Polylactic Acid.

Bio-based multilayer films were prepared by using the innovative nanolayer coextrusion process to produce films with a number of alternating layers varying from 3 to 2049. For the first time, a semicrystalline polymer was confined by another semicrystalline polymer by nanolayering in order to develop high barrier polyamide (PA11)/polylactic acid (PLA) films without compromising thermal stability and mechanical behavior. This process allows the preparation of nanostratified films with thin layers (down to nanometric thicknesses) in which a confinement effect can be induced. The stratified structure has been investigated, and the layer thicknesses have been measured. Barrier properties were successfully correlated to the microstructure, as well as the thermal behavior, and mechanical properties. The layer continuity was fully achieved for most of the films, but some layer breakups have been observed on the film with the thinnest PLA layer (2049-layers film). Coextruding PLA with PA11 has induced an increase in PLA crystallinity (from 4 to 16%) along with an increase in thermal stability of the multilayer films without impacting PA11 properties. Gas barrier properties were driven by the PLA confined layers due to the microstructural rearrangement by increasing crystallinity, whereas water barrier properties were governed by the PA11 confining layers due to its lower water affinity. As a consequence, a decrease of water permeability (up to 11 times less permeable for the 6M film) but an increase of gas barrier properties (barrier improvement factor (BIF) of 66% for the 0M film for N2 and BIF of 36% for the 6M film for CO2 for instance) were evidenced as the layer number was increased. This study paves the way for the development of ecofriendly materials with outstanding barrier performances and highlights the importance of nonmiscible polymers adhesion at melt state and additives presence.

Thiostrepton induces spindle abnormalities and enhances Taxol cytotoxicity in MDA-MB-231 cells

BackgroundThiostrepton (TST) is a known inhibitor of the transcription factor Forkhead box M1 (FoxM1) and inducer of heat shock response (HSR) and autophagy. TST thus may be one potential candidate of anticancer drugs for combination chemotherapy.Methods and ResultsImmunofluorescence staining of mitotic spindles and flow cytometry analysis revealed that TST induces mitotic spindle abnormalities, mitotic arrest, and apoptotic cell death in the MDA-MB-231 triple-negative breast cancer cell line. Interestingly, overexpression or depletion of FoxM1 in MDA-MB-231 cells did not affect TST induction of spindle abnormalities; however, TST-induced spindle defects were enhanced by inhibition of HSP70 or autophagy. Moreover, TST exhibited low affinity for tubulin and only slightly inhibited in vitro tubulin polymerization, but it severely impeded tubulin polymerization and destabilized microtubules in arrested mitotic MDA-MB-231 cells. Additionally, TST significantly enhanced Taxol cytotoxicity. TST also caused cytotoxicity and spindle abnormalities in a Taxol-resistant cell line, MDA-MB-231-T4R.ConclusionsThese results suggest that, in addition to inhibiting FoxM1, TST may induce proteotoxicity and autophagy to disrupt cellular tubulin polymerization, and this mechanism might account for its antimitotic effects, enhancement of Taxol anticancer effects, and ability to overcome Taxol resistance in MDA-MB-231 cells. These data further imply that TST may be useful to improve the therapeutic efficacy of Taxol.