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The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.

In this work, we present a comprehensive investigation of the entrapment of laccase, a biotechnologically relevant enzyme, into levan-based nanoparticles (LNPs). The entrapment of laccase was achieved concomitantly with the synthesis of LNP, catalyzed by a truncated version of a levansucrase from Leuconostoc mesenteroides . The study aimed to obtain a biocompatible nanomaterial, able to entrap functional laccase, and characterize its physicochemical, kinetic and thermal stability properties. The experimental findings demonstrated that a colloidal stable solution of spherically shaped LNP, with an average diameter of 68 nm, was obtained. An uniform particle size distribution was observed, according to the polydispersity index determined by DLS. When the LNPs synthesis was performed in the presence of laccase, biocatalytically active nanoparticles with a 1.25-fold larger diameter (85 nm) were obtained, and a maximum load of 243 μg laccase per g of nanoparticle was achieved. The catalytic efficiency was 972 and 103 (μM·min) -1 , respectively, for free and entrapped laccase. A decrease in k cat values (from 7050 min -1 to 1823 min - 1) and an increase in apparent Km (from 7.25 μM to 17.73 μM) was observed for entrapped laccase, compared to the free enzyme. The entrapped laccase exhibited improved thermal stability, retaining 40% activity after 1 h-incubation at 70°C, compared to complete inactivation of free laccase under the same conditions, thereby highlighting the potential of LNPs in preserving enzyme activity under elevated temperatures. The outcomes of this investigation significantly contribute to the field of nanobiotechnology by expanding the applications of laccase and presenting an innovative strategy for enhancing enzyme stability through the utilization of fructan-based nanoparticle entrapments.

Purpose The aim of this study was to assess the risk factors for colistin-resistant carbapenemase-producing Enterobacterales (CR-CPE), and describe the mortality associated with this organism, in a low-income country. Methods A descriptive, observational, and prospective multicenter study was carried out in Guayaquil, Ecuador. All patients with carbapenem-resistant Enterobacterales admitted between December 2021 and May 2022 were enrolled. Infection definitions were established according to the Centers for Disease Control and Prevention (CDC) protocols. The presence of carbapenemase-producing Enterobacterales was confirmed with a multiplex PCR for bla KPC, bla NDM, bla OXA-48, bla VIM, and bla IMP genes. MCR-1 production was studied molecularly, and MLST assays were carried out. Results Out of 114 patients enrolled in the study, 32 (28.07%) had at least one positive sample for CR-CPE. Klebsiella pneumoniae ST512-KPC-3 was the most frequent microorganism isolated. Parenteral feeding, β-lactamase inhibitor use, recent hemodialysis, and renal failure were all considered independent risk factors for carrying CR-CPE. A mortality of 41.22% was detected, but we could not find any difference between colistin-resistant and colistin-susceptible CPE. MCR-1 production was not detected in any of the isolates studied. Conclusion A significant burden for CR-CPE was found in a South American country that was mainly caused by the high-risk clone K. pneumoniae ST512-KPC-3 and not mediated by mcr-1 production. Its acquisition involved parenteral feeding, β-lactamase inhibitor use, recent hemodialysis, and renal failure as independent risk factors, demonstrating the critical need for infection prevention and stewardship programs to avoid dissemination to other countries in the region.

Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus) sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc). To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor) from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation.

KEY MESSAGE : Two novel Wx - B1 null alleles that enlarge the genetic variability for this wheat gene were characterized, whose effects on wheat quality could be different to those of the Wx - B1b allele. The starch composition of wheat grain has a primary influence on flour quality. Wheat starch consists of two types of glucose polymers: amylose (22–35 % of the total) and amylopectin (68–75 % of the total). Amylose is synthesized by waxy proteins. Several studies have contributed to the catalogue of waxy alleles available for breeders, and the search for novel alleles of these and other proteins related to flour quality continues. In this report, we describe the characterization of two novel Wx-B1 alleles (Wx-B1k and Wx-B1m) in a collection of macha, Indian dwarf and club wheat. Several accessions lacking Wx-B1 protein were detected, and some were caused by the common Wx-B1b null allele. Of the other accessions, four from Indian dwarf wheat showed the insertion of 4 bp within the seventh exon, and one from club wheat had a deletion of four nucleotides in the second exon. These mutations were novel and provisionally catalogued as Wx-B1k and Wx-B1m, respectively, and could be used to enlarge the genetic variability for this gene.

Ethers can be found in the environment as structural, active or even pollutant molecules, although their degradation is not efficient under environmental conditions. Fungal unspecific heme-peroxygenases (UPO were reported to degrade low-molecular-weight ethers through an H2O2-dependent oxidative cleavage mechanism. Here, we report the oxidation of a series of structurally related aromatic ethers, catalyzed by a laboratory-evolved UPO (PaDa-I) aimed at elucidating the factors influencing this unusual biochemical reaction. Although some of the studied ethers were substrates of the enzyme, they were not efficiently transformed and, as a consequence, secondary reactions (such as the dismutation of H2O2 through catalase-like activity and suicide enzyme inactivation) became significant, affecting the oxidation efficiency. The set of reactions that compete during UPO-catalyzed ether oxidation were identified and quantified, in order to find favorable conditions that promote ether oxidation over the secondary reactions.

In this work, a commercial peroxidase was immobilized onto porous silicon (PS) support functionalized with 3-aminopropyldiethoxysilane (APDES) and the performance of the obtained catalytic microreactor was studied. The immobilization steps were monitored and the activity of the immobilized enzyme in the PS pores was spectrophotometrically determined. The enzyme immobilization in porous silicon has demonstrated its potential as highly efficient enzymatic reactor. The effect of a polar organic solvent (acetonitrile) and the temperature (up to 50°C) on the activity and stability of the biocatalytic microreactor were studied. After 2-h incubation in organic solvent, the microreactor retained 80% of its initial activity in contrast to the system with free soluble peroxidase that lost 95% of its activity in the same period of time. Peroxidase immobilized into the spaces of the porous silicon support would be perspective for applications in treatments for environmental security such as removal of leached dye in textile industry or in treatment of different industrial effluents. The system can be also applied in the field of biomedicine.

Stem and progenitor cells undergo a global elevation of nascent transcription, or hypertranscription, during key developmental transitions involving rapid cell proliferation. The chromatin remodeler Chd1 mediates hypertranscription in pluripotent cells but its mechanism of action remains poorly understood. Here we report a novel role for Chd1 in protecting genome integrity at promoter regions by preventing DNA double-stranded break (DSB) accumulation in ES cells. Chd1 interacts with several DNA repair factors including Atm, Parp1, Kap1 and Topoisomerase 2β and its absence leads to an accumulation of DSBs at Chd1-bound Pol II-transcribed genes and rDNA. Genes prone to DNA breaks in Chd1 KO ES cells are longer genes with GC-rich promoters, a more labile nucleosomal structure and roles in chromatin regulation, transcription and signaling. These results reveal a vulnerability of hypertranscribing stem cells to accumulation of endogenous DNA breaks, with important implications for developmental and cancer biology. The chromatin remodeler Chd1 mediates hypertranscription in embryonic stem (ES) cells and has been shown to associate with genes transcribed by RNA Polymerase (Pol) I and II. Here the authors provide mechanistic insights into this process and reveal that Chd1 is involved in protecting genome integrity at promoter regions by preventing DNA break accumulation.

Introduction Carbapenem-resistant gram-negative bacilli are a worldwide concern because of high morbidity and mortality rates. Additionally, the increasing prevalence of these bacteria is dangerous. To investigate the extent of antimicrobial resistance and prioritize the utility of novel drugs, we evaluated the molecular characteristics and antimicrobial susceptibility profiles of carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii in Ecuador in 2022. Methods Ninety-five clinical isolates of carbapenem non-susceptible gram-negative bacilli were collected from six hospitals in Ecuador. Carbapenem resistance was confirmed with meropenem disk diffusion assays following Clinical Laboratory Standard Institute guidelines. Carbapenemase production was tested using a modified carbapenemase inactivation method. Antimicrobial susceptibility was tested with a disk diffusion assay, the Vitek 2 System, and gradient diffusion strips. Broth microdilution assays were used to assess colistin susceptibility. All the isolates were screened for the bla KPC, bla NDM, bla OXA-48, bla VIM and bla IMP genes . In addition, A. baumannii isolates were screened for the bla OXA-23 , bla OXA -58 and bla OXA-24/40 genes. Results Carbapenemase production was observed in 96.84% of the isolates. The bla KPC , bla NDM and bla OXA-48 genes were detected in Enterobacterales, with bla KPC being predominant. The bla VIM gene was detected in P. aeruginosa, and bla OXA-24/40 predominated in A. baumannii . Most of the isolates showed co-resistance to aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. Both ceftazidime/avibactam and meropenem/vaborbactam were active against carbapenem-resistant gram-negative bacilli that produce serin-carbapenemases. Conclusion The epidemiology of carbapenem resistance in Ecuador is dominated by carbapenemase-producing K. pneumoniae harbouring bla KPC . Extensively drug resistant (XDR) P. aeruginosa and A. baumannii were identified, and their identification revealed the urgent need to implement strategies to reduce the dissemination of these strains.

Before implantation in the uterus, mammalian embryos set aside trophoblast stem cells that are maintained in the extraembryonic ectoderm (ExE) during gastrulation to generate the fetal portion of the placenta. Their proliferation depends on diffusible signals from neighboring cells in the epiblast, including fibroblast growth factor 4 (Fgf4). Here, we show that Fgf4 expression is induced by the transforming growth factor β-related protein Nodal. Together with Fgf4, Nodal also acts directly on neighboring ExE to sustain a microenvironment that inhibits precocious differentiation of trophoblast stem cells. Because the ExE itself produces the proteases Furin and PACE4 to activate Nodal, it represents the first example, to our knowledge, of a stem cell compartment that actively maintains its own microenvironment.