Protein Recovery Research Articles

Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2–3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.

Therapeutic benefit of oregano oil in the acute idiosyncratic hepatotoxicity induced by carbon tetrachloride in rats: Adverse effects of high dose of oreganum.

Toxicity caused by carbon tetrachloride (CCl4) can lead to serious liver injury. The aim of the study is to investigate the protective effects of oregano oil (Origanum minutiflorum extract oil) against CCl4-induced liver injury. Two doses of oregano oil were used in the experiment: a low dose (LD; 20 mg/kg) and a high dose (HD; 60 mg/kg) during 2 weeks. CCl4 caused severe liver damage, nucleolus destruction in hepatocytes and cytogenetic changes in the nucleus. Indirectly, CCl4 causes decreased protein synthesis and significantly high creatinine and urea values. Hematological disorders have been recorded, such as decreased RBC and hemoglobin concentration, increased WBC and deformability of the erythrocyte membrane. Both doses of oregano oil had protective effects. Improved protein synthesis and high globulins level, creatinine and urea were found in both groups. Cytogenetic changes in the nucleus of hepatocytes were reduced. A high dose of oregano oil had maximal protective effects for RBC, but a very weak effect on hemoglobin synthesis. Also, WBC and lymphocyte values were low. Origanum stimulates protein synthesis and recovery of hepatocytes after liver injury, reduces the deformability of the erythrocyte membrane. High doses of oregano oil decreased WBC and lymphocytes which may lead to a weakening of the immune response. However, high doses are more effective against severe platelet aggregation than low doses, suggesting an effective treatment against thrombocytosis.

Production of techno-functional proteins and plant biostimulants from Nannochloropsis gaditana

Nannochloropsis gaditana is produced mainly for its lipid content and fatty acid profile. However, its protein content is comparable to or even higher than that of most conventional protein sources. In this work, the microalga N. gaditana BEA 1202B purchased from the Spanish Bank of Algae (BEA) was produced using 3.4 m3 tubular photobioreactors achieving a biomass productivity of 0.1 g L−1·day−1 and a protein content of 29.2 ± 4.5 % (d.w.). The biomass produced was used as a source of proteins; the protein recovery was optimised following a response surface methodology. Under optimal conditions, which were solubilisation at pH = 10.2 and precipitation at pH = 3.1, the protein recovery yield was 17.5 g·100 g−1. The extract exhibited a water-holding and an oil-holding capacity of 2.1 and 3.0 g g−1, respectively. The leftovers from protein extraction were assessed as plant biostimulants and demonstrated a positive effect on root development, weight gain, and chlorophyll retention. The process described in this work presents a sustainable and environmentally friendly approach to harness the potential of the microalga N. gaditana in producing high-value food proteins and plant biostimulants. The integration of this microalgal-based biorefinery concept could contribute to more sustainable agriculture and address the growing global demand for protein-rich products and plant promoters.

Effect of thermal and defatting treatments on the composition, protein profile and structure of house cricket (Acheta domesticus) protein extracts

This study investigated the impact of blanching (100 °C, 40 s), defatting method (maceration, Soxhlet) and solvent polarity (hexane, ethanol) on the profile, structure and solubility of house cricket protein extracts. Blanching and Soxhlet using ethanol impacted the protein profile, with a lower content of myosin heavy chain and a higher abundance of low molecular weight proteins (<25 kDa). Moreover, ethanol induced aggregation of non-blanched cricket proteins, with a 13–72% reduction in protein recovery yield. The protein secondary structure of non-blanched extracts was also affected by ethanol with 18% more β-sheets. Furthermore, blanching resulted in a lower protein surface hydrophobicity by a factor of 3 to 7, with no impact of solvent polarity. Finally, the solubility of protein extracts remained >75%, regardless of the blanching and defatting methods. These findings, combined with the evaluation of techno-functional properties, could be used for the development of cricket-based protein ingredients for food formulations.

EhVps35, a retromer component, is involved in the recycling of the EhADH and Gal/GalNac virulent proteins of Entamoeba histolytica

The retromer is a highly conserved eukaryotic complex formed by the cargo selective complex (CSC) and the sorting nexin (SNX) dimer subcomplexes. Its function is protein recycling and recovery from the endosomes to conduct the target molecules to the trans-Golgi network or the plasma membrane. The protozoan responsible for human amoebiasis, Entamoeba histolytica, exhibits an active membrane movement and voracious phagocytosis, events in which the retromer may be fully involved. In this work, we studied the structure of EhVps35 the central member of the CSC retromeric subcomplex as it binds EhVps26 and EhVps29, the other two CSC members, allowing the position of the retromer in the membranes. We also studied the EhVps35 role in the recycling of virulence proteins, particularly those involved in phagocytosis. Confocal microscopy assays revealed that EhVps35 is located in the plasmatic and endosomal membranes and in the phagocytic cups and channels. In addition, it follows the target cell from the moment it is in contact with the trophozoites. Molecular docking analyses, immunoprecipitation assays, and microscopy studies revealed that EhVps35 interacts with the EhADH, Gal/GalNac lectin, and actin proteins. In addition, experimental evidence indicated that it recycles surface proteins, particularly EhADH and Gal/GalNac proteins, two molecules highly involved in virulence. Knockdown of the Ehvps35 gene induced a decrease in protein recycling, as well as impairments in the efficiency of adhesion and the rate of phagocytosis. The actin cytoskeleton was deeply affected by the Ehvps35 gene knockdown. In summary, our results revealed the participation of EhVps35 in protein recycling and phagocytosis. Furthermore, altogether, our results demonstrated the concert of finely regulated molecules, including EhVps35, EhADH, Gal/GalNac lectin, and actin, in the phagocytosis of E. histolytica.

Manipulation Strategy to Increase Expression Level of Soluble Recombinant Protein Penicillin G Acylase (PGA) in Bacterial Host Escherichia coli: A Review Article

The strategy of producing PGA on a massive scale with high levels of soluble protein can be through recombinant genetic techniques and expressed in a certain host. E. coli is still a popular bacterial host to produce a recombinant protein which has advantages such as fast growth, low production cost, and high expression rate. Apart from its advantages, E. coli as a production host also has disadvantages including the expression of recombinant proteins often failing to form the proper folding conformation which makes the protein biologically inactive. Many strategies can be developed to overcome these problems, such as the selection of the host strain (E. coli HB101 &amp; JM109), fusion protein to enhance the recovery of soluble protein (MBP &amp; NusA), optimization of fermentation (low-temperature incubation), and optimization of the protein isolation process for the recovery of active PGA (Freeze-thawing method).

Abstract 1631: A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture

Abstract We describe a novel homogeneous cell-based assay developed for the quantitative detection of Ki-67 protein, a recognized marker of cell proliferation, utilizing Lumit™ immunoassay technology. This luminescent assay has been effectively multiplexed with a fluorescent measure of cell death, employing a cell-impermeant fluorogenic DNA dye. This multiplex allows for the clear distinction between antiproliferative activity and overt cytotoxicity in cell culture models. Through varying lytic conditions, we demonstrated effective cell lysis and detection of nuclear Ki-67 protein. Specifically, Lumit™ Lysis Buffer II provided efficient recovery of Ki-67 protein without compromising assay performance, including the requisite binding of labeled antibodies to the target protein. This assay exhibited excellent signal-to-background (up to 40-fold) and signal linearity (R^2 = 0.99) in wildtype HeLa cells (~1000 to 80,000/well), while the signal in Ki-67 knockout HeLa cells was comparable to the no-cell background, indicating excellent specificity for Ki-67 protein. Due to the unavailability of recombinant full-length Ki-67 protein, two different recombinant Ki-67 protein fragments were used to corroborate assay sensitivity of &amp;lt;1 pM Ki-67 protein. The assay's suitability for human Ki-67 protein detection, not mouse, was observed through further cell-based testing. The proliferation/cytotoxicity assay multiplex was validated in both suspension and adherent cell lines, as well as primary cell culture, detecting antiproliferative and proliferative responses. CDK4/6 inhibitors are a class of antiproliferative agents of significant interest for their potential in treating cancer. In Jurkat cells, treatment with the CDK4/6 inhibitor palbociclib led to a dose-dependent reduction in Ki-67 protein expression with an EC50 of 88 nM, and total Ki-67 elimination at 2 μM compound concentration. Notably, there was no cytotoxicity detected at the tested doses and treatment duration (up to 48 h), but a significant reduction in viable cell number at maximal palbociclib concentrations, aligning with an antiproliferative mechanism. This work establishes a rapid, simple, luminescent assay for determining Ki-67 levels that can be paired with a fluorogenic readout of cell death, facilitating efficient and quantitative discernment of altered proliferation in mammalian cell culture using standard microplate readers. Citation Format: Dan F. Lazar, Kevin R. Kupcho, Andrew L. Niles, James J. Cali. A homogeneous assay system for discernment of proliferative, antiproliferative, and cytotoxic effects in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1631.

Abstract 3746: Identification of cancer specific extracellular vesical biomarkers to aid in diagnosis of cancer

Abstract The limitations of serial tumor biopsies are well known, thus liquid biopsies which allow for repeated plasma-based biomarker analysis are urgently needed. Extracellular vesicles (EVs) represent an extremely stable and emerging proteomic substrate which allows for deep enrichment of biologically relevant signals from plasma, and broad analysis of tumor associated and host response proteins. To identify novel biomarkers, we performed proteomics analysis on EVs purified using size exclusion chromatography and a proprietary buffer system which increases EV and corona protein recovery. Initial DIA mass spectrometry (MS) analysis was performed on EVs from 125 patients (25 each from CRC, breast, NSCLC, ovarian, and normal patients). A subset of 25 normal and 25 ovarian was subjected to a second deeper analysis on a much more powerful DIA platform. Initial analysis identified 204 unique proteins differentially expressed in ovarian cancer patients (&amp;gt;2-fold change; p&amp;lt;0.05). Repeat analysis of the ovarian vs normal cohorts on the Biognosys TrueDiscovery™ platform identified 645 differentially expressed, a significant increase in depth of analysis. These cancer exosome associated proteins represent a broad range of host response signals. Taking ~20 of the top proteins, we extended our analysis using ELISAs to determine if the results by MS were reproducible in a simpler format. The ELISA analysis recapitulated the MS results. This confirms that the TrueDiscovery™ platform can easily be translated into a simple, quick and distributable ELISA platform. Extending these results, a blinded cohort of ovarian and normal EVs was analyzed using a predefined cutoff for a host immune response biomarker. This blinded analysis demonstrated sensitivity of 1.0 and specificity of 0.9, exceptionally high values for a single analyte assay, underscoring the potential value of analyzing the host/immune response proteins in the context of the EV microenvironmentKey biomarkers from cancer patient EVs suggests it is possible to track host immunosuppressive environment via antigen presenting cell and check point antagonist biomarkers. In both cases, highly significant differences are seen comparing EVs from normal patient plasma versus cancer patient plasma. Interestingly, when these biomarkers are evaluated in pre-EV enriched plasma, no significant differences are seen. This underscores the unique nature of the EV microenvironment, and suggests that host immune response and tumor microenvironment may be recapitulated in the EV/host response proteome using our unique methods. These data confirm that the EV microenvironment represents a unique oncological ecosystem with highly specific and sensitive disease biomarkers which are not dysregulated in complete plasma. This novel approach to assessing cancer proteomics has great promise for real time, longitudinal analysis of cancer patient tumor/host immune biology. Citation Format: Todd A. Hembrough, Zachary Opheim, Cheryl Bandoski, Issa Isaac, Martin Mehnert, Alan Ezrin. Identification of cancer specific extracellular vesical biomarkers to aid in diagnosis of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3746.

Extraction and purification of seaweed protein from Ulva sp. - Challenges to overcome

Ulva sp. has gained attention as a promising and sustainable protein source, due to its high essential amino acid content. This study investigated traditional extraction methods using a double screw press followed by acid or heat precipitation. Furthermore, the screw pressing was combined by a novel purification method that combines mild heat treatment with microfiltration for removal of chlorophyll, to produce white Ulva protein. The double screw press followed by acid precipitation or heat denaturation showed low total protein yields, with 14% of the biomass protein extracted, of which half was soluble. The highest total protein yield (5.5%) was achieved with acid precipitation at pH 2, while heat denaturation at 90 °C resulted in a total protein yield of 4.1%. Acid precipitation proved to be effective with ∼ 81% precipitation of solubilized protein compared to ∼ 43% for heat denaturation. While microfiltration effectively retained chlorophyll in the retentate, the recovery of protein in the permeate remained challenging. SDS-PAGE analysis of proteins in different microfiltration-fractions showed that larger protein complexes (>250 kDa) were unable to transmit through the membranes, resulting in a maximum recovery of 24.9% of the feed-protein in the permeate. Further refinement of Ulva fractionation protocols is imperative for improvement.

Increased reactive carboxyl and free alfa-amino groups from fish type I collagen peptides by Alcalase® hydrolysis exhibit higher antibacterial and antioxidant activities

Abstract This study aimed to generate low molecular weight peptides (LMWP) from fish collagen type I hydrolysed by increasing activity of Alcalase® from 0.0 to 12.0 (AU-A per 100 g) at 55 °C and 7.4 of pH for 3 h 40 min. The results showed that all enzyme activity levels caused 34–55 % reductions in protein recovery, 1.0–3.0 folds’ increase in free α-amino groups and 1.7–3.2 folds’ increase in carboxyl groups. Degree of hydrolysis ranged from 20 to 30 % with increasing enzyme activity. The number average molecular weight significantly reduced from 3200 g/mol in 0.0 AU-A per 100 g enzyme activity to 1151, 1398, 1175, 1040 and 1246 g/mol in 2.4, 4.8, 7.2, 9.6 and 12.0 AU-A per 100 g enzyme activities, respectively. Depending upon enzyme activity level, the produced LMWP with reactive carboxyl and amino end-groups exhibited 2.5- to 4.0-fold increases in antioxidant capacity and 1.0–3.5 log cfu/ml inhibition of four pathogen bacteria. Highest inhibition of 2.5 log cfu/ml in Escherichia coli was obtained from 2.4 AU-A per 100 g enzyme activity and 3.5 log cfu/ml in Listeria monocytogenes from 9.6 and 12.0 AU-A per 100 g enzyme activity levels. Infrared spectroscopy clearly identified reactive end-groups and showed remarkably differences in molar absorptivity of various molecular regions between non-enzyme and enzyme treated collagen type I molecule. A 9.6 and 12.0 AU-A per 100 g enzyme activity levels were found optimally effective to generate LMWP. In conclusion, LMWP exhibited high antioxidant and antibacterial activity due to increased functional reactive end-groups, and these bio-active peptides may have greater potentialities in various food and pharmaceutical applications.

Protein recovery from yellow peas (Pisum sativum L.) for enhanced processing sustainability and functional properties

This research focuses on sustainable protein recovery methods from a new yellow pea variety by examining alternativepH-shifting processes. The study focuses on reducing water consumption during alkaline extraction by adjustingsolid-liquid ratios, and evaluating the impact of various isoelectric precipitants, including lactic acid and lactic acidbacteria (Lactobacillus plantarum and Lactobacillus lactis), on the functional and antioxidant properties of productsacross a wide range of pH values. It was here found that the process alternative with three 1:10 (w/v) extractioncycles and lactic acid bacteria as precipitant agent achieved high process productivity (0.36 kg protein product/kgpea flour) and low specific water consumption (94.9 kg water/kg protein product). No significant differences wereobserved in protein content and yield when compared to other flour-water ratios with higher water consumptionor less eco-friendly precipitants. Products precipitated with lactic acid bacteria formed stable emulsions even at theisoelectric point, exhibited superior free radical scavenging activity, although solubility and water holding capacitywere lower, and no differences were noted in oil holding capacity, foaming capacity, and foam stability.

Environmental and economic life cycle assessment of emerging sludge treatment routes

Before implementing large-scale emerging sludge treatment methods, it is crucial to conduct a comprehensive analysis of their environmental impacts and economic costs at the system level. This study utilizes standardized Life Cycle Assessment (LCA) and Life Cycle Cost (LCC) accounting methods to evaluate four emerging sludge treatment routes: anaerobic fermentation (AF), pyrolysis (PY), hydrothermal carbonization (HTC), and alkaline thermal hydrolysis (ATH). These routes are compared with two traditional sludge treatment routes, anaerobic digestion (AD) and aerobic composting (AC). The results indicate that the conventional route AD is the optimal choice in terms of environmental pollution, carbon emission control, and life cycle cost. The emerging routes AF and ATH have significant environmental benefits from carbon source recovery and protein recovery. However, they require approximately twice the cumulative energy demand of AD and additional consumption of chemicals, which imposes a greater environmental burden. While PY and HTC outperform the conventional sludge treatment routes in terms of the total environmental impact (1.79E-10, 1.72E-10) due to energy recovery and the use of clean energy sources. AF and PY demonstrate better economic performance (842.11 and 712.00 CNY) compared to the traditional route AC (905.06 CNY). However, both HTC and ATH incur costs approximately 1.3 times higher than AC due to their high energy and material demands. Furthermore, sensitivity analysis reveals that emerging treatment routes are highly influenced by energy and resource recovery and consumption. Therefore, reducing the energy and resource inputs for emerging routes could significantly decrease their environmental impact and cost, maximizing their resource recovery advantages.

Enhancing the potential of rapeseed cake as protein-source food by γ-irradiation.

Rapeseed cake serves as a by-product in the oil extraction industry, characterized by its elevated protein content. However, the presence of antinutritional factors limits the utilization of rapeseed cake as a viable protein source. In this study, different doses of γ-irradiation were used to irradiate rapeseed cake and rapeseed protein isolate was extracted through a modified alkaline solution and acid precipitation method from rapeseed cake. The chemical composition and in vivo acute toxicity of rapeseed protein isolate were determined. The protein recovery rate of rapeseed protein isolate was 39.08 ± 3.01% after irradiation, while the content of antinutritional factors was significantly reduced. Moreover, γ-irradiation did not have any experimentally related effects on clinical observations or clinicopathology in mice. Overall, the reduced antinutrients and increased functional properties suggest that the irradiation of rapeseed cake (<9 kGy) could be utilized as a pre-treatment in the development of rapeseed cake-based value-added protein products.

Comparing green extraction methods for the recovery of protein-rich fraction from peach seeds (Prunus persica)

Peach processing generates significant volume of coproducts, such as the underutilized peach seeds, which are rich in fatty acids and protein. The recovery of protein-rich fractions from peach seeds is highly relevant because of the increasing demand for alternative non-animal protein sources. This study aimed to investigate different extraction techniques for protein recovery from the underestimated peach seeds, and evaluate the functionalities of the protein fraction. The peach seeds were defatted using the supercritical fluid extraction (SFE-CO2), with oily recovery up to 29.53% ± 0.45. Then, the protein fraction was recovered using the following methods: heat-stirred extraction (HSE) as a conventional method, and, as alternative procedures, microwave-assisted extraction (MAE), pressure liquid extraction (PLE), and ultrasound-assisted extraction (UAE) using NaOH solution as solvent. The PLE provided the highest protein content (95.10 % ± 0.98), followed by the HSE technique (55.09% ± 0.35). Furthermore, the PLE showed the best performance in techno-functional properties of the protein fraction: emulsion activity, foaming, turbidity, free sulfhydryl content, hydrophobicity, and color, compared to the classical HSE technique. Therefore, it is suggested that PLE (10 min) method recovered an isolated protein with better functional properties, providing promising result for protein purification. Therefore, PLE can be considered an alternative method to add value to underestimated peach seeds, conferring the upcycling concept.

Evaluation of cell disruption methods for protein and coenzyme Q10 quantification in purple non-sulfur bacteria.

A recent focus has been on the recovery of single-cell protein and other nutritionally valuable bioproducts, such as Coenzyme Q10 (CoQ10) from purple non-sulfur bacteria (PNSB) biomass following wastewater treatment. However, due to PNSB's peculiar cell envelope (e.g., increased membrane cross-section for energy transduction) and relatively smaller cell size compared to well-studied microbial protein sources like yeast and microalgae, the effectiveness of common cell disruption methods for protein quantification from PNSB may differ. Thus, this study examines the efficiency of selected chemical (NaOH and EDTA), mechanical (homogenization and bead milling), physical (thermal and bath/probe sonication), and combined chemical-mechanical/physical treatment techniques on the PNSB cell lysis. PNSB biomass was recovered from the treatment of gas-to-liquid process water. Biomass protein and CoQ10 contents were quantified based on extraction efficiency. Considering single-treatment techniques, bead milling resulted in the best protein yields (p < 0.001), with the other techniques resulting in poor yields. However, the NaOH-assisted sonication (combined chemical/physical treatment technique) resulted in similar protein recovery (p = 1.00) with bead milling, with the former having a better amino acid profile. For example, close to 50% of the amino acids, such as sensitive ones like tryptophan, threonine, cystine, and methionine, were detected in higher concentrations in NaOH-assisted sonication (>10% relative difference) compared to bead-milling due to its less disruptive nature and improved solubility of amino acids in alkaline conditions. Overall, PNSB required more intensive protein extraction techniques than were reported to be effective on other single-cell organisms. NaOH was the preferred chemical for chemical-aided mechanical/physical extraction as EDTA was observed to interfere with the Lowry protein kit, resulting in significantly lower concentrations. However, EDTA was the preferred chemical agent for CoQ10 extraction and quantification. CoQ10 extraction efficiency was also suspected to be adversely influenced by pH and temperature.

Protein hydrolysates prepared by Alcalase using ultrasound and microwave pretreated almond meal and their characterization.

The study aimed to optimize ultrasonic (US: 40kHz/200W for 10, 20, 30, 40, and 50min), and microwave (MW: 160W for 45, 90, 125, 180, and 225s) pretreatment conditions on protein extraction yield and degree of protein hydrolysis (DH) from almond de-oiled meal, an industrial by-product. First order model was used to describe the kinetics of almond protein hydrolysates obtained with Alcalase. The highest DH, 10.95% was recorded for the US-50min and 8.87% for MW-45s; while it was 5.76% for the untreated/control sample. At these optimized pretreatment conditions, a 1.16- and 1.18-fold increment in protein recovery was observed for theUS and MW pretreatments, respectively in comparison to the conventional alkaline extraction. The molecular weight distribution recorded for pretreated samples disclosed a significant reduction in the band thickness in comparison with control. Both the pretreatments resulted in a significant increase (P < 0.05) in the antioxidant activity, and TCA solubility index when compared with the control. Results evinced that US and/or MW pretreatments before enzymatic hydrolysis can be a promising approach for the valorization of almond meal for its subsequent useas an ingredient for functional foods/nutraceuticals which otherwise fetches low value as an animal feed.

Systematic and quantitative analysis of stop codon readthrough in Rett syndrome nonsense mutations

Rett syndrome (RTT) is a neurodevelopmental disorder resulting from genetic mutations in the methyl CpG binding protein 2 (MeCP2) gene. Specifically, around 35% of RTT patients harbor premature termination codons (PTCs) within the MeCP2 gene due to nonsense mutations. A promising therapeutic avenue for these individuals involves the use of aminoglycosides, which stimulate translational readthrough (TR) by causing stop codons to be interpreted as sense codons. However, the effectiveness of this treatment depends on several factors, including the type of stop codon and the surrounding nucleotides, collectively referred to as the stop codon context (SCC). Here, we develop a high-content reporter system to precisely measure TR efficiency at different SCCs, assess the recovery of the full-length MeCP2 protein, and evaluate its subcellular localization. We have conducted a comprehensive investigation into the intricate relationship between SCC characteristics and TR induction, examining a total of 14 pathogenic MeCP2 nonsense mutations with the aim to advance the prospects of personalized therapy for individuals with RTT. Our results demonstrate that TR induction can successfully restore full-length MeCP2 protein, albeit to varying degrees, contingent upon the SCC and the specific position of the PTC within the MeCP2 mRNA. TR induction can lead to the re-establishment of nuclear localization of MeCP2, indicating the potential restoration of protein functionality. In summary, our findings underscore the significance of SCC-specific approaches in the development of tailored therapies for RTT. By unraveling the relationship between SCC and TR therapy, we pave the way for personalized, individualized treatment strategies that hold promise for improving the lives of individuals affected by this debilitating neurodevelopmental disorder. Key messagesThe efficiency of readthrough induction at MeCP2 premature termination codons strongly depends on the stop codon context.The position of the premature termination codon on the transcript influences the readthrough inducibility.A new high-content dual reporter assay facilitates the measurement and prediction of readthrough efficiency of specific nucleotide stop contexts.Readthrough induction results in the recovery of full-length MeCP2 and its re-localization to the nucleus.MeCP2 requires only one of its annotated nuclear localization signals.

Piperine, a phytochemical prevents the biofilm city of methicillin-resistant Staphylococcus aureus: A biochemical approach to understand the underlying mechanism

Methicillin-resistant Staphylococcus aureus (MRSA), a drug-resistant human pathogen causes several nosocomial as well as community-acquired infections involving biofilm machinery. Hence, it has gained a wide interest within the scientific community to impede biofilm-induced MRSA-associated health complications. The current study focuses on the utilization of a natural bioactive compound called piperine to control the biofilm development of MRSA. Quantitative assessments like crystal violet, total protein recovery, and fluorescein-di-acetate (FDA) hydrolysis assays, demonstrated that piperine (8 and 16 μg/mL) could effectively compromise the biofilm formation of MRSA. Light and scanning electron microscopic image analysis confirmed the same. Further investigation revealed that piperine could reduce extracellular polysaccharide production by down-regulating the expression of icaA gene. Besides, piperine could reduce the cell-surface hydrophobicity of MRSA, a crucial factor of biofilm formation. Moreover, the introduction of piperine could interfere with microbial motility indicating the interaction of piperine with the quorum-sensing components. A molecular dynamics study showed a stable binding between piperine and AgrA protein (regulator of quorum sensing) suggesting the possible meddling of piperine in quorum-sensing of MRSA. Additionally, the exposure to piperine led to the accumulation of intracellular reactive oxygen species (ROS) and potentially heightened cell membrane permeability in inhibiting microbial biofilm formation. Besides, piperine could reduce the secretion of diverse virulence factors from MRSA. Further exploration revealed that piperine interacted with extracellular DNA (e-DNA), causing disintegration by weakening the biofilm architecture. Conclusively, this study suggests that piperine could be a potential antibiofilm molecule against MRSA-associated biofilm infections.

Effects of alkaline and ultrasonication on duckweed (Wolffia arrhiza) protein extracts’ physicochemical and techno-functional properties

Wolffia is a protein-rich aquatic plant with the potential to help address food sustainability issues; however, a more efficient extraction process must be sought due to limited yield with conventional methods. This study aimed to investigate the effects of duckweed forms (fresh and dried), duckweed-to-aqueous solution ratio (DSR), as well as alkaline and ultrasonication extractions on yields, physicochemical and techno-functional properties of duckweed protein extracts (DPE). Three extraction methods were used: alkaline extraction (AE), ultrasound-assisted alkaline extraction (UAAE), and ultrasound-assisted water extraction (UAWE). The study showed fresh duckweed resulted in a 2.5-fold higher yield and protein recovery than oven-dried duckweed. UAE significantly enhanced extraction yield and protein recovery. The optimal extraction process was a DSR of 1:6 using UAAE at pH 8.5, resulting in a 16% yield and 34% protein recovery. Furthermore, the study found that UAE facilitated the extraction of non-polar/hydrophobic amino acids while AE proved to be efficient in extracting sulfur-containing amino acids. This study, for the first time, revealed the role of UAE and AE in promoting the extraction of different profiles of amino acids. The DPE produced using UAAE contained 65% protein, meeting the WHO/FAO/UNU recommended protein intake for preschool-aged children. Furthermore, the DPE exhibited excellent emulsifying properties and oil-holding capacity comparable to commercial soy protein isolates. Overall, UAAE was identified as a promising approach for producing techno-functional and nutritious protein ingredients from Wolffia. The resulting DPE proved to show great potential for functional plant-based food and feed applications.

Protein and water recovery from tuna defrosting wastewater

The objective of this research was to recover protein and water from tuna defrosting wastewater. Tuna defrosting wastewater (TDW) was concentrated, and salt protein residue (PR) was separated from concentrate TDW (cTDW). Protein in the cTDW was precipitated (PP). Salt was removed from PR and PP by using hot water (60 °C). PR and PP were dried at 50 °C before analysis for total protein, amino acid profile and salt content. Salty protein solution (PS) following salt removal from the precipitate was collected and concentrated. Then salt cPS was desalted by Sephadex G-25, and the elution was collected and concentrated. The resulting cPS was analyzed for pH, total protein, salt content, amino acid profile and antioxidant properties. Water from the protein recovery procedure was collected and analyzed for biological qualities (heterotrophic plate count, coliform bacteria, E. coli, Staphylococcus aureus, Salmonella spp. and Clostridium perfringens), physical qualities (apparent color, turbidity, pH) and chemical qualities (total dissolved solids, total hardness and sulfate).The results showed that cTDW contained 11.57 ± 0.03 % protein and 3.36 ± 0.03% NaCl. After salt was removed, the dried PR and PP contained 33.10 ± 0.16% and 6.92 ± 0.13% protein, respectively, and 0.23 ± 0.00% and 0.05 ± 0.00% NaCl, respectively. Dried PR contained 9 essential amino acids at higher concentrations than in PP. Concentrated PS contained 3.15 ± 0.12% protein and no NaCl. Histidine (254.15 mg/100 g) was the dominant amino acid in cPS. Antioxidant properties are shown by values for DPPH, ABTS and FRAP. The physical, chemical and bacterial parameters of recovered water met the guidelines for drinking water quality. These results indicate that recovery of protein and water is possible in fish processing, which could reduce costs for processors and benefit the environment.