Structure-Based Optimization of Carbendazim-Derived Tubulin Polymerization Inhibitors through Alchemical Free Energy Calculations.

Carbendazim derivatives, commonly used as antiparasitic drugs, have shown potential as anticancer agents due to their ability to induce cell cycle arrest and apoptosis in human cancer cells by inhibiting tubulin polymerization. Crystallographic structures of α/ß-tubulin multimers complexed with nocodazole and mebendazole, two carbendazim derivatives with potent anticancer activity, highlighted the possibility of designing compounds that occupy both benzimidazole- and colchicine-binding sites. In addition, previous studies have demonstrated that the incorporation of a phenoxy group at position 5/6 of carbendazim increases the antiproliferative activity in cancer cell lines. Despite the significant progress made in identifying new tubulin-targeting anticancer compounds, further modifications are needed to enhance their potency and safety. In this study, we explored the impact of modifying the phenoxy substitution pattern on antiproliferative activity. Alchemical free energy calculations were used to predict the binding free energy difference upon ligand modification and define the most viable path for structure optimization. Based on these calculations, seven compounds were synthesized and evaluated against lung and colon cancer cell lines. Our results showed that compound 5a, which incorporates an α-naphthyloxy substitution, exhibits the highest antiproliferative activity against both cancer lines (SK-LU-1 and SW620, IC50 < 100 nM) and induces morphological changes in the cells associated with mitotic arrest and mitotic catastrophe. Nevertheless, the tubulin polymerization assay showed that 5a has a lower inhibitory potency than nocodazole. Molecular dynamics simulations suggested that this low antitubulin activity could be associated with the loss of the key H-bond interaction with V236. This study provides insights into the design of novel carbendazim derivatives with anticancer activity.

Improvement of Laccase Production by Thielavia terrestris Co3Bag1. Enhancing the Bio-Catalytic Performance of the Native Thermophilic TtLacA via Immobilization in Copper Alginate Gel Beads.

A 32-fold increase in laccase activity production by the thermophilic biomass-degrading fungus T. terrestris Co3Bag1 was achieved when the microorganism was grown on a modified medium containing fructose, sodium nitrate, and copper. A 70 kDa laccase (TtLacA), produced under the above conditions, was purified, immobilized in copper alginate gel beads, and characterized. TtLacA, both free and immobilized enzymes, exhibited optimal activity at pH 3.0, at a temperature of 65 and 70 °C, respectively, although both displayed 70% of activity from 40 to 70 °C. Free and immobilized enzymes retained at least 80% of relative activity in the pH range from 3 to 4.6. Immobilized TtLacA manifested a 2.3-fold higher thermal stability than the free form of the enzyme at 60 and 70 °C. Immobilized TtLacA retained 95% initial activity for six consecutive reuse cycles at 60 °C, and also retained 86% of initial activity after 12 days of storage at 4 °C. Based on the biochemical features, thermophilic TtLacA may be an efficient enzyme for dye decolorization and other industrial applications at high temperatures or acidic conditions. This work represents the first report about the immobilization and biochemical characterization of a thermophilic laccase from a member of the genus Thielavia.

First Report of Erysiphe betae Causing Powdery Mildew on Chard (Beta vulgaris var. cicla) in Mexico.

Chard (Beta vulgaris var. cicla; Chenopodiaceae) is a vegetable native to the Mediterranean, widely cultivated for its nutritional properties. In June 2020, an outbreak of powdery mildew was detected in a commercial crop of chard in San Martín Texmelucan, Puebla (19°14'37.1"N; 98°27'12.5"W), Mexico. The disease was present in 86% of the plants (n=400) and the pathogen was found to cover up to 95% of the surface of the leaves. Initially, small whitish patches were observed on both sides of the leaves. Subsequently, the patches grew rapidly to cover most of the leaf surface and premature senescence of infected leaves was observed. The signs of the pathogen were observed as abundant whitish masses of conidia. Microscopic analysis of the fungus showed amphigenous mycelia with lobed hyphal appressoria. Conidiophores (n=30) were simple and erect, 93133 × 7.58.5 µm. Foot cells (n=30) were cylindrical, predominately straight, and rarely somewhat curved at the base, 30.036.5 µm, followed by a longer cell and two shorter cells, and the conidium. Conidia (n=100) were hyaline, ellipsoid-ovoid, 3745 × 1416 µm. Germ tubes (n =30) were terminal, short (0.52.0 times the conidial width) and stout. Conidial appressoria (n=30) were mostly lobed, showing from 2-6 lobes. Chasmothecia were not found. The morphological characteristics observed correspond to previous descriptions of Erysiphe betae by Braun and Cook et al. (2012). A voucher specimen (accession no. UACH450) was deposited in the Department of Agricultural Parasitology Herbarium at the Chapingo Autonomous University. To confirm identification, DNA was extracted from the fungus, and the internal transcribed spacer (ITS) and the 28S gene region of rDNA from one sample were amplified by PCR, using the primers ITS1/ITS4 (White et al. 1990) and PM3 (Takamatsu and Kano 2001)/TW14 (Mori et al. 2000). The sequences obtained from our specimen were registered to the GenBank under the accession numbers ON157053 and ON157047 for ITS and LSU, respectively. Our sequences shared 100% identity for ITS (KX574674) and 99.8% for LSU (OM033348 and OM368494) with sequences of E. betae in BLAST'n search. Based on phylogenetic analysis using the Maximum Likelihood method including a published ITS + 28S dataset for Erysiphe species, the isolate UACH450 was grouped into a clade with E. betae. Takamatsu et al. (2015) found that E. betae, E. malvae and E. heraclei are phylogenetically indistinguishable (they form the E. heraclei species complex), nevertheless, E. malavae infects Lavatera and Malva (Malvaceae), E. heraclei predominately forms on hosts of Apiaceae and E. betae is commonly found on Beta and Chenopodium (Chenopodiaceae) (Braun and Cook 2012). Pathogenicity was verified by spraying a suspension of conidia (1107 conidia/ml) onto the leaves of six healthy chard plants and six plants were sprayed with sterile distilled water to serve as controls. All plants were maintained at temperatures from 28 2 °C and relative humidity of 802 %. All inoculated leaves developed powdery mildew symptoms after 14 days, whereas the control plants remained symptomless. The pathogenicity test was performed twice, observing the same results. The recovered pathogen showed the same morphological characteristics as the inoculated pathogen, thus fulfilling Koch's postulates. To our knowledge, this is the first report of Erysiphe betae causing powdery mildew on Beta vulgaris var. cicla in Mexico. This pathogen has been previously reported in Iraq (Amano, 1986) and Greece (Vakalounakis and Kavroulakis, 2017) on Beta vulgaris var. cicla. Also, Erysiphe betae has been reported in Mexico on Chenopodium and throughout the world on sugar beet (Farr and Rossman, 2022). This pathogen is a major issue as it can completely cover the leaves of the diseased plants, making them difficult to market.

Calidad del Sistema de Gestión de los Servicios de Salud en la red asistencial amazónica Tarapoto / Quality of the Health Services Management System in the Tarapoto Amazonian healthcare network

La atención de salud de óptima calidad es fundamental para atender los requerimientos sanitarios de zonas vulnerables, incluyendo la prevención y el control de las enfermedades desatendidas, tropicales y transmitidas por vectores, presentes en las regiones amazónicas como la provincia San Martín y su principal centro poblacional, Tarapoto. La investigación se realizó en un diseño no experimental, con enfoque cuantitativo, de tipo descriptivo y corte transversal, entre mayo de 2019 y enero de 2020. El universo que se estableció para dicha investigación fue el sector de asegurados de la red Asistencial de Tarapoto EsSalud, que en el año 2019 estaba conformado por 118.672 personas. Se consideró una población de 1.722 pacientes del Hospital II Tarapoto, adscrito a la red Asistencial EsSalud, cuya atención se relacionó con eventos de vigilancia epidemiológica, según los reportes de la Oficina de Inteligencia Sanitaria de esta entidad de ese mismo año. El análisis del instrumento aplicado a los 318 voluntarios dentro de este grupo, permitió establecer la percepción de la calidad del servicio de la red. Tomando en cuenta las 5 dimensiones de la calidad en la atención a los pacientes, se evidenciaron fallas en la capacidad de respuesta (50,8%) y fiabilidad (57,3%) de la red asistencial Tarapoto. La empatía obtuvo una puntuación equivalente al 65,1%, cercano a los 67,6% de la dimensión seguridad. Los elementos tangibles fueron los mejor calificados por los usuarios, con una proporción de 68,1%(AU)

Optimal quality health care is essential to meet the health requirements of vulnerable areas, including the prevention and control of neglected, tropical and vector-borne diseases present in Amazonian regions such as San Martín province and its main population center, Tarapoto. The research was carried out in a non-experimental design, with a quantitative, descriptive and cross-sectional approach, between May 2019 and January 2020. The universe that was established for said research was the insured sector of the Tarapoto EsSalud Healthcare network, which in 2019 was made up of 118,672 people. A population of 1,722 patients from Hospital II Tarapoto, assigned to the EsSalud Assistance network, was considered, whose care was related to epidemiological surveillance events, according to reports from the Health Intelligence Office of this entity that same year. The analysis of the instrument applied to the 318 volunteers within this group, allowed to establish the perception of the quality of the network service. Taking into account the 5 dimensions of quality in patient care, failures in response capacity (50.8%) and reliability (57.3%) of the Tarapoto healthcare network were evidenced. Empathy obtained a score equivalent to 65.1%, close to 67.6% for the security dimension. Tangible items were the best rated by users, with a proportion of 68.1%(AU)

Strategies used by nature to fix the red, purple and blue colours in plants: a physical chemistry approach.

While identified by the respective flavylium cation, anthocyanins are much more than this molecule. The flavylium cation (generally appearing only at very acidic pH values) is one of the molecules of a complex sequence of pH dependent molecular species reversibly interconnected by different chemical reactions. These species include the red flavylium cation, purple quinoidal base and blue or bluish anionic quinoidal bases. At the common pH of the vacuoles of simpler anthocyanins, the red flavylium cation is present only at very acidic pH values and at moderately acidic pHs there is no significant colour of the purple quinoidal base. Moreover, the blue or bluish anionic quinoidal base appearing around neutral pH values is not stable. Intermolecular (copigmentation) and intramolecular (in acylated anthocyanins) interactions increase the colour hue and yield bathochromic shifts in the absorption bands, permitting to extend the pH domain of the flavylium cation and increase the mole fraction of the quinoidal bases. Metal complexation is another strategy. In particular, the Al3+ cation plays an essential role in the blue colour of hydrangea. The most sophisticated structures are however the metaloanthocyanins, such as the one that gives the blue colour of commelina communis, constituted of six anthocyanins, six flavanones and two metals. In this work we discuss how physical chemical tools are indispensable to account for the chemical behaviour of these complex systems. The experimental procedures and the equations needed to calculate all equilibrium constants of anthocyanins and the consequent pH dependent mole fraction distributions in the absence or presence of copigments are described in detail. Reverse pH jumps monitored by stopped flow have been shown to be an indispensable tool to calculate these parameters.

Anthocyanin Color Stabilization by Host-Guest Complexation with p-Sulfonatocalix[n]arenes.

Flavylium-based compounds in their acidic and cationic form bring color to aqueous solutions, while under slightly acidic or neutral conditions they commonly bring discoloration. Selective host-guest complexation between water-soluble p-sulfonatocalix[n]arenes (SCn) macrocycles and the flavylium cationic species can increase the stability of the colored form, expanding its domain over the pH scale. The association constants between SCn and the cationic (acid) and neutral basic forms of flavylium-based compounds were determined through UV-Vis host-guest titrations at different pH values. The affinity of the hosts for synthetic chromophore was found to be higher than for a natural anthocyanin (Oenin). The higher affinity of SC4 for the synthetic flavylium was confirmed by 1H NMR showing a preferential interaction of the flavylium phenyl ring with the host cavity. In contrast with its synthetic counterpart, the flavylium substitution pattern in the anthocyanin seems to limit the inclusion of the guest in the host's binding pocket. In this case, the higher affinity was observed for the octamer (SC8) likely due to its larger cavity and higher number of negatively charged sulfonate groups.

Evolution of Flavylium-Based Color Systems in Plants: What Physical Chemistry Can Tell Us.

Anthocyanins are the basis of the color of angiosperms, 3-deoxyanthocyanins and sphagnorubin play the same role in mosses and ferns, and auronidins are responsible for the color in liverworts. In this study, the color system of cyanidin-3-O-glucoside (kuromanin) as a representative compound of simpler anthocyanins was fully characterized by stopped flow. This type of anthocyanin cannot confer significant color to plants without intra- or intermolecular interactions, complexation with metals or supramolecular structures as in Commelina communis. The anthocyanin's color system was compared with those of 3-deoxyanthocyanins and riccionidin A, the aglycone of auronidins. The three systems follow the same sequence of chemical reactions, but the respective thermodynamics and kinetics are dramatically different.

Correction to "New Procedure to Calculate All Equilibrium Constants in Flavylium Compounds: Application to the Copigmentation of Anthocyanins".

[This corrects the article DOI: 10.1021/acsomega.9b01066.].

Chemical evolution of the colour systems generated by riccionidin A, 3-deoxyanthocyanidins and anthocyanins.

The kinetics and thermodynamics (in acidic solutions) of the five chemical species reversibly interconnected by external stimuli (a multistate), such as pH and light, generated by the liverworts colorant riccionidin A were investigated. The degradation products of the multistate formed after 10 days at neutral pH were identified. The behaviour of riccionidin A multistate was compared with previous results reported for the equivalent systems based on 3-deoxyanthocyanidins (found in mosses and ferns) and anthocyanins (ubiquitous in angiosperms). The five chemical species have mutatis mutandis similar structures in the three multistates. The most dramatic difference is the extremely slow interconversion rate between flavylium cation and trans-chalcone in riccionidin A and related compounds multistates (tens of days) when compared with deoxyanthocyanins (a few days) and anthocyanins (several hours), at room temperature. The mole fraction distribution of the five species that constitute the multistate as a function of pH is also different in the three families of compounds. Some considerations regarding the chemical evolution of the three systems are given.

Unveiling the formation 1 : 2 supramolecular complexes between cucurbit[7]uril and a cationic calix[4]arene derivative.

The formation of host-guest complexes between cucurbit[7]uril (CB7) and a tetracationic calix[4]arene derivative in the so-called cone conformation was investigated by 1H NMR, DOSY NMR, isothermal titration calorimetry and ESI-MS. The results point to the formation of 1 : 1, 1 : 2 and 2 : 1 CB7 : calixarene complexes with binding constants of 3 × 106 M-1, ≈2 × 102 M-1, and 9 × 104 M-1 respectively. The study demonstrates, on one hand, that despite having four potential recognition sites, the calixarene only binds two CB7 molecules and, on the other, that for sterically crowded binding motifs that prevent CB7 inclusion with optimized hydrophobic and ion-dipole interactions, the formation of 1 : 2 complexes can be observed most likely due to formation of external binding of the cationic moieties to the CB7 carbonyl portals.

Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

New Procedure To Calculate All Equilibrium Constants in Flavylium Compounds: Application to the Copigmentation of Anthocyanins.

A new experimental procedure to calculate all equilibrium constants of the multistate of species of anthocyanins and related compounds, including those in basic medium, is reported. The procedure is based on a series of pH jumps monitored by stopped flow from an extended pH range of solutions at pseudo-equilibrium (when there is no significant formation of trans-chalcones) or at equilibrium to pH = 1.0. The experimental procedure is described for the anthocyanin model compound 4'-hydroxyflavylium, which exhibits a peculiar behavior in moderately acidic medium, because the quinoidal base, hemiketal, and cis-chalcone have similar mole fractions at pseudo-equilibrium, permitting good discrimination among these species. The experimental procedure can be extended to the copigmentation phenomenon and allow the calculation of the 1:1 copigmentation constants of the flavylium cation, quinoidal base, hemiketal, and cis- and trans-chalcones (this last from the equilibrium) and their respective ionized forms. The method was applied to calculate the copigmentation constants of the model compound 4'-hydroxyflavylium as well as malvidin-3-glucoside with caffeine. In the last compound, the strongest interaction takes place with the quinoidal base (K = 303 M-1) and flavylium cation (K = 134 M-1) and, to a lesser extent, with the ionized quinoidal base (K = 43 M-1) and cis-chalcone (K = 17 M-1). The caffeine interaction with the hemiketal and the other ionized species is negligible.

Impact of a Water-Soluble Gallic Acid-Based Dendrimer on the Color-Stabilizing Mechanisms of Anthocyanins.

The interaction of two anthocyanins with a water-soluble polyanionic dendrimer was studied through UV/Vis, stopped-flow, and NMR spectroscopy. Cyanidin-3-glucoside (cy3glc) revealed a stronger interaction than malvidin-3-glucoside (mv3glc) at pH 1 according to their apparent association constants. A higher color increased was also obtained for cy3glc at pH 3.5 as a result of this stronger interaction. A high-frequency chemical shift of the cy3glc aromatic protons suggest the formation of ionic pairs. The interaction parameters (K≈700 m-1 , n≈295) indicated the binding of approximately two anthocyanin molecules by each sulfate group. The equilibrium and rate constants of cy3glc in the presence of dendrimer showed an increased stability of the flavylium cation and a higher protection of this species from hydration (pK'a and pKh increased almost one pH unit). The tuning and color stabilization of anthocyanins by using this dendrimer allow novel applications as colorimetric sensors for food packaging.

Rationalizing the Color in Heavenly Blue Anthocyanin: A Complete Kinetic and Thermodynamic Study.

All equilibrium and rate constants of heavenly blue anthocyanin (HBA 1) as well as the derivatives with two (HBA 2) or none (HBA 3) acylated units were determined. The three acylated units of the sugar in position 3 of the peonidin chromophore of HBA 1 are essential to confer the peculiar stability of its purple and blue colors. The sugars generate an efficient protective environment around position 2 (and 4) of the flavylium cation, through an intramolecular sandwich-type stacking that retards 35-fold the hydration reaction ( kh) and increases 8.8-fold the dehydration reaction ( k-h), when compared with the peonidin chromophore HBA 3. The conjugation of these two rates lowers 308-fold the hydration equilibrium constant ( Kh), corresponding to a raise of the energy level of the hemiketal by 14.2 kJ mol-1. Conversely, the p Ka of the quinoidal base in HBA 1 is only slightly stabilized in comparison with that of HBA 2 and HBA 3. The energy level of hemiketal increases with the number of acylated units, but the inversion of energies between hemiketal and quinoidal base takes place exclusively for HBA 1 (three acylated units), permitting in moderately acidic solutions the stabilization of the purple quinoidal base. Identical inversion of energy was observed for the corresponding ionized species, allowing the stabilization of the blue ionized quinoidal base in slightly basic solutions. At pH values higher than 8, the hydroxyl groups of the hydroxycinnamic acid units start to deprotonate disrupting the intramolecular sandwich-type stacking and the more or less slow degradation of the anthocyanin is observed.

Taxonomic identification of the thermotolerant and fast-growing fungus Lichtheimia ramosa H71D and biochemical characterization of the thermophilic xylanase LrXynA.

The zygomycete fungus Lichtheimia ramosa H71D, isolated from sugarcane bagasse compost, was identified by applying phylogenetic analysis based on the DNA sequence of the Internal Transcribed Spacer (ITS), and subsequent secondary structure analysis of ITS2. L. ramosa H71D was able to grow over a wide range of temperatures (25-45 °C), manifesting optimal growth at 37 °C. A 64 kDa xylanase (named LrXynA) was purified from the culture supernatant of L. ramosa H71D grown on 2% carboxymethylcellulose (CMC), as the only carbon source. LrXynA displayed optimal activity at pH 6 and temperature of 65 °C. The enzyme retained more than 50% of its maximal activity over a broad range of pH values (4.5-7.5). Enzyme half-life (t½) times at 55, 65 and 75 °C were 80, 25, and 8 min, respectively. LrXynA showed higher affinity (k M of 2.87 mg/mL) and catalytic efficiency (k cat /k M of 0.651 mg s/mL) towards Beechwood xylan in comparison to other substrates such as Birchwood xylan, Oat-spelt xylan, CMC, Avicel and Solka floc. The predominant final products from LrXynA-mediated hydrolysis of Beechwood xylan were xylobiose and xylotriose, suggesting that the enzyme is an endo-ß-1,4 xylanase. Scanning electron microscopy (SEM) imaging of sugar cane bagasse (SCB) treated with LrXynA, alone or in combination with commercial cellulases, showed a positive effect on the hydrolysis of SCB. To our knowledge, this is the first report focusing on the biochemical and functional characterization of an endo-ß-1,4 xylanase from the thermotolerant and fast-growing fungus Lichtheimia ramosa.

pH-Driven self-sorting in a four component host-guest system.

A four component host-guest system comprising ß-cyclodextrin (ß-CD), cucurbit[7]uril (CB7) and two chalcone guests was demonstrated to undergo pH-triggered self-sorting in aqueous solution. This phenomenon arises from the contrasting complexation induced pKa shifts displayed by these guests after complexation with CB7.