Current Research in Biotechnology最新文献

Leishmaniasis is recognised as the second largest parasitic disease worldwide and yet a neglected disease. The current pharmacological treatments are associated with significant challenges, including high toxicity, high cost and parasitic resistance. Considering the potential of isopentyl caffeate (ICaf) as an anti-leishmanial agent, the present work evaluated the in vivo toxicity of ICaf and the absorption, distribution, metabolism, and excretion (ADME) properties in silico, aiming at the treatment of Leishmania amazonensis. For the in vivo toxicity testing, Swiss mice (Mus musculus) were treated with a single dose of ICaf. During the 14-day evaluation period, the animals underwent assessments including hippocratic screening, weight measurement, as well as histological and hematological evaluations. Analysis of ADME properties of ICaf was conducted to evaluate its pharmacokinetic characteristics and bioavailability. Characteristics, such as molar refractivity through Lipinski's Rule of Five, were identified. The in silico results showed that ICaf is considered to have good oral bioavailability and has potential to be considered as a new drug. From the in vivo toxicity testing, none of the evaluated parameters revealed toxicity of ICaf to the animals when treated intraperitoneally. The in vivo treatment reduced the lesion and the parasite load at the tested doses, corroborating the assumption that ICaf may be a potential pharmacological alternative against L. amazonensis.

In the last years, many research efforts have been applied for the development of filamentous fungi as hosts for heterologous protein production. Aspergillus vadensis CBS 113365, a close relative of the industrial workhorse Aspergillus niger, has been suggested as a more suitable cell factory as it does not acidify the culture medium and produces very low levels of secreted proteases. Therefore, efficient methods and tools that allow the genetic manipulation and exploitation of this biotechnologically relevant fungus are needed. To date, only protoplast-mediated transformation and classical cloning strategies have been implemented for A. vadensis genetic modification, which decreases the exploitation capacity of this fungus at the industrial level. In this study, we have adapted and implemented an Agrobacterium tumefaciens-mediated transformation protocol for A. vadensis for the first time, and applied the FungalBraid system to genetically modify this species by means of synthetic biology. As proof of concept, we have successfully complemented and fluorescently labelled a uridine auxotrophic A. vadensis pyrA^- strain and generated A. vadensis mutants carrying the Penicillium expansum-based expression cassette for the heterologous production of the antifungal protein PeAfpA from P. expansum. Even though we have yet to find the conditions that trigger PeAfpA production in this species, the implementation of the ATMT method reported here, along with the application of the FungalBraid system, will greatly aid in this task and will facilitate the exploitation of A. vadensis as a fungal workhorse for protein production for multiple biotechnological applications.

Anaerobic ammonium oxidation (anammox)-based process has become a method for achieving carbon-neutral wastewater treatment. However, in mainstream wastewater with a low-strength ammonium, obtaining partial nitritation (PN) for the anammox process can be challenging and often result in NO₃^– accumulation. The recently proposed partial denitrification (PD), which reduces NO₃^– back to NO₂^–, can provide NO₂^– for anammox. For a successful PD-anammox (PD/A), it is crucial to efficiently cultivate PD bacteria (PDNB) and maintain a balance between the activities of PDNB and anammox bacteria (AnAOB). In this study, an efficient PDNB enrichment was cultivated for a long period of 400 days in a sequential batch reactor (SBR) by feeding it with acetate (300 mg COD/L) and nitrate (100 mg NO₃^–-N/L) at an exchange ratio of 50 %. The nitrite accumulation efficiency (NAE) gradually increased to >90 %, with Thauera phenylacetica identified as the key species for achieving high NAE. When PDNB was applied with AnAOB to remove 50 mg NH₄⁺-N/L and 50 mg NO₃^–-N/L, different total nitrogen (TN) removal efficiencies were observed depending on the mixing ratio of PDNB and AnAOB (1:5–1:20). The most rapid and complete TN removal was achieved at the mixing ratio of 1:15. At lower mixing ratios of 1:5 and 1:10, PDNB activity exceeded that of AnAOB, resulting in incomplete TN removal. Conversely, at a higher inoculation ratio of 1:20, AnAOB activity surpassed of PDNB, leading to delayed T-N removal. These results indicate that maintaining a balance between NO₂^– accumulation by PDNB and NO₂^– consumption by AnAOB is essential for successful PD/A process operation. The findings of this study can be utilized as fundamental data for the operational strategy of an anammox-based process to achieve a carbon-neutral wastewater treatment.

Biomolecular interactions among proteins are fundamental for all cellular functions. The chromosome segregation proteins are the key regulators of inherent functions in the living cells. Aurora kinases have drawn much interest as possible drug targets in higher eukaryotes. The human pathogen, E. histolytica is the causative agent of amoebiasis, and a major health concern in developing countries. However, there is no vaccine against it and the popular drugs- metronidazole, tinidazole etc. show significant side effects in humans. To identify new controlling agents, we must have a thorough knowledge about the cell cycle regulatory proteins of E. histolytica, as many unusual cell cycle events can be found in this parasite, that do not happen in human cells. This study describes the first comprehensive analysis of the interaction between an aurora kinase protein and a BAR homology domain containing protein. Fes/CIP4 and EFC/F-BAR homology domain (FCH) containing protein, EhABP has been identified as a novel interactor of EhAK7, an aurora kinase homolog from E. histolytica by yeast two-hybrid screening against the cDNA library of E. histolytica and their interaction has been proved by in vitro binding assay. Both the N and C-terminus of EhAK7 are responsible for this interaction. We found the reduced expression of EhAK7 and EhABP genes, defects in actin filament organization and irregular-shaped nucleus in the trophozoites treated with an aurora kinase inhibitor VX-680. This indicates that EhAK7 play an important role in the cytokinesis of E. histolytica through the interaction with a BAR homology domain containing protein, EhABP.

The recombinant 40 kDa BoExXyl43A glycoside hydrolase family 43 (GH43) from bacterium Bacteroides ovatus exhibited highest specific activity (U/mg) against corn cob xylan (136.8), followed by Beechwood xylan (81.1), Carbosynth xylan (69.3), 4-O-D-methylglucuronoxylan (61.4) and Birchwood xylan (59.9). BoExXyl43A demonstrated optimal performance at 37 °C and pH 7.6 with V_max and K_m of 141.8 U/mg and 4.0 mg/mL as well as 64.1 U/mg and 6.0 mg/mL against corn cob and Birchwood xylan, respectively. The activity of BoExXyl43A increased by 48 % by addition of 10 mM Ca²⁺ ions, while 1 mM EDTA or 1 mM EGTA decreased its activity by 100 % or 42.5 %, respectively, highlighting its calcium-ion dependence. Thin-layer chromatography (TLC) analysis of BoExXyl43A hydrolysates of Birchwood and Beechwood xylan as well as that of various xylooligosaccharides (DP2-DP9) from corn cob xylan showed the release of D-xylose, identifying it as an exo-β-1,4-xylosidase/exo-β-1,4-xylanase (EC 3.2.1.-/3.2.1.37). Moreover, the time-dependent TLC analysis of xylobiose hydrolysis showed release of D-xylose units, confirming its β-xylosidase activity. BoExXyl43A also exhibited exo-1,4-β-xylosidase activity on Larchwood and Carbosynth xylans. Notably, it released D-xylose from α-L-Araf²-xylotriose demonstrating its activity against decorated xylooligosaccharides. BoExXyl43A's exo-1,4-β-xylosidase and residual β-xylosidase activity on xylan and xylobiose, respectively, could potentially enhance xylan saccharification efficiency in bioethanol-based refineries. The molecular modeling showed that BoExXyl43A has 5-bladed β-propeller structure with a very shallow active-site having −1, +1 and + 2 subsites, which could accommodate three D-xylose units of longer xylan like xylododecaose thus supporting its exoxylosidase activity.

Biodiesels constitute a growing class of fuel in a world that is increasingly inclined towards more ecological and sustainable energy. Despite their many advantages, biodiesels have limited cold flow properties and larger NO_X emissions. These limitations are mostly attributed to the chemical compositions of biodiesels which are dictated by the chemical compositions of their feedstock oils. Accordingly, this study presents a novel approach to produce Genetically Engineered Biodiesel (GEB) whose chemical composition can be controlled by the genetic manipulation of oleaginous yeast oils for the production of designer biodiesels with improved properties and performances. Using full-factorial central composite design, the best chemical composition of an optimal biodiesel was predicted. Then, simple and combined MFE1, PEX10 and POX2 mutants of the oleaginous yeast Yarrowia lipolytica were constructed. These mutants showed interesting lipid profiles where their biodiesels are predicted to have better cold flow properties. These mutants showed also higher lipid titers by 2–3 folds compared to the parent strain. This study provides an approach for tailor designing of biodiesel properties and performances via genetic engineering. Moreover, it provides solutions potentially enabling biodiesel to be used as a standalone fuel in cold climates without any mixing with petrodiesel.

The demand for broad-spectrum antibacterial agents continues with increasing rates of resistance of microbial pathogens to traditional antibiotics. Peptides and lipopeptides are gaining traction as promising novel, class-reference antibiotics for tackling difficult-to-treat infections caused by multi-drug resistant bacteria. To identify novel candidates and expand treatment options in clinical settings, we explored the in vitro antibacterial potential and mode of action of a short octapeptide combining a cationic block of four lysines and a highly hydrophobic segment of four phenylalanines (K4F4), and two K4F4-inspired lipopeptides (Palmitoyl-K4F4 and K4-NH-Palmitoyl). Preliminary AI-based screening had revealed the antimicrobial potential of the K4F4 peptide coupled with limited haemolytic activity. Broth dilution and haemolytic assays have confirmed these in silico predictions. Overall, our lipidated peptides were more active at lower MIC values compared to non-lipidated species, indicating the beneficial impact of tailing lipidation on design of peptide-based antimicrobials. An integrated view of the membrane-active mechanism of these novel therapeutic templates was obtained using a combination of flow cytometry, fluorescence microscopy and dye-based permeabilization assays. K4F4 and its lipidated derivatives act via a fast-disrupting mechanism without inducing bacterial resistance mechanisms in a long-term exposure assay. A K4F4-inspired lipopeptide together with its shorter version (K4-NH-Palmitoyl), were more stable in environments closer emulating physiological conditions, showing a higher antibacterial response in physiological salts and serum than their parent peptide. Our findings reveal the antibacterial and antibiofilm potential of a novel polylysine-polyphenyalanine peptide and highlight the significant contribution of lipidation and shortening as molecular engineering strategies to improve and guide the future design of next-generation membrane-targeting antibiotics.

Fucoxanthin (FX) is a carotenoid of marine origin primarily distributed in brown seaweeds and has garnered interest for its antioxidative, anti-inflammatory, and anticancer properties. Despite its potential, a comprehensive understanding of its anticancer effects and mechanisms of action remains elusive.

The aim of this review is to present novel insights into the anticancer effects of FX, shedding light on previously unexplored molecular mechanisms and its synergistic potential with established chemotherapeutic agents.

A comprehensive search was conducted employing databases like PubMed/MedLine, Scopus, and Web of Science to aggregate relevant pharmacological experimental studies. The results of the studies showed that FX exhibits anticancer activity against various cancer types, including breast, colorectal, and lung cancer, through multiple pathways: cell cycle arrest, apoptosis induction, and inhibition of angiogenesis. Additionally, FX potentiates the effects of existing chemotherapeutic agents, making it a potential candidate for combination therapies. The evidence suggests that FX possesses considerable anticancer properties, acting through diverse molecular mechanisms; the heterogeneity of study designs and the limited number of clinical trials make it hard to conclude. Further in-depth studies, particularly randomized controlled trials, are essential for validating FX's efficacy and for paving the way for its integration into standard cancer treatment regimens; additional research is needed to explore its pharmacokinetics, safety profile, and potential synergistic effects with existing chemotherapeutics.

miRNA is a type of classic non-coding RNA which is enriched in cancer. Various studies reported that miRNA be involved in the progression and metastasis of TSCC. Exploring pivotal miRNAs and elaborating the mechanisms inducing metastasis are important for improving the prognosis of TSCC patients. Here, Let-7b miRNA was identified as a significant factor contributing to metastasis of TSCC via directly targeting the 3′UTR of MOR mRNA, which leads to abnormally high expression of MOR protein. MOR might bind with SRC and potentially activate p-EGFR through SRC. As a treatment modality, Let-7b miRNA mimics were encapsulated into the endosomal pH-responsive nanoparticles. NPs (Let-7b miRNA mimics) significantly inhibited Cal27 cells in vitro and in vivo metastatic model. Altogether, our data revealed that Let-7b miRNA-MOR-SRC-EGFR axis might be an effective therapeutic target in TSCC metastasis.