A state-of-the-art review of the recent advances in exosome isolation and detection methods in viral infection.

Proteins, RNA, DNA, lipids, and carbohydrates are only some of the molecular components found in exosomes released by tumor cells. They play an essential role in healthy and diseased cells as messengers of short- and long-distance intercellular communication. However, since exosomes are released by every kind of cell and may be found in blood and other bodily fluids, they may one day serve as biomarkers for a wide range of disorders. In many pathological conditions, including cancer, inflammation, and infection, they play a role. It has been shown that the biogenesis of exosomes is analogous to that of viruses and that the exosomal cargo plays an essential role in the propagation, dissemination, and infection of several viruses. Bidirectional modulation of the immune response is achieved by the ability of exosomes associated with viruses to facilitate immunological escape and stimulate the body's antiviral immune response. Recently, exosomes have received a lot of interest due to their potential therapeutic use as biomarkers for viral infections such as human immunodeficiency virus (HIV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Epstein-Barr virus (EBV), and SARS-CoV-2. This article discusses the purification procedures and detection techniques for exosomes and examines the research on exosomes as a biomarker of viral infection.

Purification and biochemical characterization of pullulanase produced from Bacillus sp. modified by ethyl-methyl sulfonate for improved applications.

Strain improvement via chemical mutagen could impart traits with better enzyme production or improved characteristics. The present study sought to investigate the physicochemical properties of pullulanase produced from the wild Bacillus sp and the mutant. The pullulanases produced from the wild and the mutant Bacillus sp. (obtained via induction with ethyl methyl sulfonate) were purified in a-three step purification procedure and were also characterized. The wild and mutant pullulanases, which have molecular masses of 40 and 43.23 kDa, showed yields of 2.3% with 6.0-fold purification and 2.0% with 5.0-fold purification, respectively, and were most active at 50 and 40 °C and pH 7 and 8, respectively. The highest stability of the wild and mutant was between 40 and 50 °C after 1 h, although the mutant retained greater enzymatic activity between pH 6 and 9 than the wild. The mutant had a decreased Km of 0.03 mM as opposed to the wild type of 1.6 mM. In comparison to the wild, the mutant demonstrated a better capacity for tolerating metal ions and chelating agents. These exceptional characteristics of the mutant pullulanase may have been caused by a single mutation, which could improve its utility in industrial and commercial applications.

Iron Oxide Nanoparticle-Based Ferro-Nanofluids for Advanced Technological Applications.

Iron oxide nanoparticle (ION)-based ferro-nanofluids (FNs) have been used for different technological applications owing to their excellent magneto-rheological properties. A comprehensive overview of the current advancement of FNs based on IONs for various engineering applications is unquestionably necessary. Hence, in this review article, various important advanced technological applications of ION-based FNs concerning different engineering fields are critically summarized. The chemical engineering applications are mainly focused on mass transfer processes. Similarly, the electrical and electronics engineering applications are mainly focused on magnetic field sensors, FN-based temperature sensors and tilt sensors, microelectromechanical systems (MEMS) and on-chip components, actuators, and cooling for electronic devices and photovoltaic thermal systems. On the other hand, environmental engineering applications encompass water and air purification. Moreover, mechanical engineering or magneto-rheological applications include dampers and sealings. This review article provides up-to-date information related to the technological advancements and emerging trends in ION-based FN research concerning various engineering fields, as well as discusses the challenges and future perspectives.

Purification of damson plum polyphenol oxidase by affinity chromatography and investigation of metal effects on enzyme activity.

Polyphenol oxidase (PPO) was firstly purified from damson plum as a high antioxidant source. PPO was treated by 0-80% ammonium sulfate precipitation and dialysis. Characterization results were determined for catechol, 4-methyl catechol, pyrogallol and caffeic acid as 0.05 M/pH: 7.2/25 °C; 0.2 M/pH: 4.5/10 °C; 0.01 M/pH: 6.8/5 °C, and 0.2 M/pH: 8.5/10 °C, respectively. Vmax and KM values were calculated for same substrates as 17,219.97 U/(mL*min) and 11.67 mM; 7309.72 U/(mL*min) and 5 mM; 12,580.12 U/(mL*min) and 3.74 mM; 12,100.41 U/(mL*min) and 6.25 mM, respectively. Catechol gave the highest Vmax value among substrates. Affinity purification was performed by using Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose 6B-L-Tyrosine-p-aminobenzoic acid. Single bands were approximately observed at 50 kDa for each affinity sample in SDS-PAGE and Native-PAGE. 93.88 and 10.46 purification-folds were obtained for PPO by reference Sepharose-4B and original Sepharose-6B gels. Metal effects upon PPO activity were also investigated due to the importance of enzymatic browning in foods. Cu+2 activation and Fe+2 inhibition were observed with a final metal concentration of 1 mM at 219.66 and 43.18%, respectively. PPO purification from damson plum by affinity chromatography, its characterization, stability evaluation by statistically, and effects of metal ions on damson plum PPO have not been investigated in the literature.

RNA-Centric Methods: Toward the Interactome of Specific RNA Transcripts.

RNA-protein interactions play an important role in numerous cellular processes in health and disease. In recent years, the global RNA-bound proteome has been extensively studied, uncovering many previously unknown RNA-binding proteins. However, little is known about which particular proteins bind to which specific RNA transcript. In this review, we provide an overview of methods to identify RNA-protein interactions, with a particular focus on strategies that provide insights into the interactome of specific RNA transcripts. Finally, we discuss challenges and future directions, including the potential of CRISPR-RNA targeting systems to investigate endogenous RNA-protein interactions.

[Expert consensus on special blood purification technics in patients with corona virus disease 2019].

The outbreak of corona virus disease 2019 (COVID-19) has spread to more than 70 countries worldwide and there was a higher mortality in those who developed serious illness.Cytokine storm syndrome is an important pathophysiological basis for COVID-19 patients developing into severe or critical conditions. It was indicated in the diagnosis and treatment scheme, by the National Health Commission of the People's Republic of China, that blood purifications such as plasma exchange, plasma adsorption, hemoperfusion, hemofiltration and plasmafiltration could be considered for use in the critical patients with cytokine storm syndrome. This expert consensus, proposed by the Chinese Society of Nephrology and the Nephrology Committee of Chinese Research Hospital Association, is to guide and standardize the clinical application of blood purifications in the treatment of severe or critical patients with COVID-19.

Truncated domains of human serum albumin improves the binding efficiency of uremic toxins: A surface plasmon resonance and computational approach.

Albumin binding is the major cause for the toxicity of protein bound uremic toxins (PBUTs) in uremic patients. Albumin binding property is exploited to address this issue, as some of the extracorporeal dialysis systems use albumin as dialysate. In this line, a detailed study about binding of PBUTs to human serum albumin (HSA) and its domains gives valuable information. The focus of this work emphasizes the mechanism of binding of HSA and its domains with a few selected PBUTs such as hippuric acid (HA), indole acetic acid (IAA) and melatonin. The HSA domains (D2, D3 and D2-3) were expressed in Pichia pastoris and purified by using Albupure matrix. The binding of the expressed domains and HSA, with PBUTs, was measured using surface plasmon resonance and analyzed. All the three domains have significant affinity towards PBUTs, while D3 had greater affinity for all the three selected PBUTs. Docking studies showed that the basic amino acid, lysine, was forming hydrogen bond with PUBTs inorder to stabile these complex. This study would be having therapeutic importance for preparing the extracorporeal dialysis systems, in combination of different domains of HSA to remove the PBUTs.

Doing more with less: Evaluation of the use of high linear velocities in preparative supercritical fluid chromatography.

The evaluation of higher than typical linear velocities is discussed for supercritical fluid chromatographic purifications on the preparative scale. SFC separation efficiency suffers far less at high linear velocities than HPLC by the rapid mass transfer of analytes carried by compressed CO2 through the stationary phase. The technique is discussed using chiral test compounds and columns. In many cases, running at high linear velocities can yield significant time savings and decreased consumption of mobile phase solvent, while also lowering energy consumption. Within the practical limitations of commercial instrumentation, using 20 µm particles can aid in achieving higher linear velocities not attainable with smaller 5 µm particles, particularly when running with high percentages of organic co-solvent. Use of larger particles for the stationary phase also lowers the associated column cost. These benefits can yield an overall purification process that is more productive and environmentally friendly.

Analyzing Phage-Host Protein-Protein Interactions Using Strep-tag® II Purifications.

After injecting their genome into the bacterial host cell, bacteriophages need to convert the host metabolism toward efficient phage production. For this, specific proteins have evolved which interact with key host proteins to inhibit, activate or redirect the function of these proteins. Since 70% of the currently annotated phage genes are hypothetical proteins of unknown function, the identification and characterization of these phage proteins involved in host-phage protein-protein interactions remains challenging. Here, we describe a method to identify phage proteins involved in host-phage protein-protein interactions using a combination of affinity purifications and mass spectrometry analyses. A bacterial strain is engineered in which a bacterial target protein is fused to a Strep-tag® II at the C-terminal end. This strain is infected with a specific bacteriophage, followed by an affinity purification of the tagged protein which allows the copurification of all bacterial and phage specific interacting proteins. After SDS-PAGE analysis and an in-gel trypsin digestion, the purified interacting proteins are identified by mass spectrometry analysis. The identification of phage proteins involved in interactions provides first hints toward the elucidation of the biological function of these proteins.

Enhancement of a protocol purifying T1 lipase through molecular approach.

T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.

Mass Spectrometry-Based Proteomics to Unveil the Non-coding RNA World.

The interaction between non-coding RNAs (ncRNAs) and proteins is crucial for the stability, localization and function of the different classes of ncRNAs. Although ncRNAs, when embedded in various ribonucleoprotein (RNP) complexes, control the fundamental processes of gene expression, their biological functions and mechanisms of action are still largely unexplored. Mass Spectrometry (MS)-based proteomics has emerged as powerful tool to study the ncRNA world: on the one hand, by identifying the proteins interacting with distinct ncRNAs; on the other hand, by measuring the impact of ncRNAs on global protein levels. Here, we will first provide a concise overview on the basic principles of MS-based proteomics for systematic protein identification and quantification; then, we will recapitulate the main approaches that have been implemented for the screening of ncRNA interactors and the dissection of ncRNA-protein complex composition. Finally, we will describe examples of various proteomics strategies developed to characterize the effect of ncRNAs on gene expression, with a focus on the systematic identification of microRNA (miRNA) targets.

A carbon nanoparticles-based solid-phase purification method facilitating sensitive MALDI-MS analysis of permethylated N-glycans.

In recent decades, MALDI-MS has been extensively used for the analysis of glycans. However, native glycans usually have low ionization efficiency in MS, which hinders the direct analysis. Permethylation of glycans is a solution for this issue, but a significant amount of salt is introduced during this process, which can further suppress the MS signals. Thus, it is necessary to purify the glycans prior to MALDI-MS analysis. In this study, we developed a carbon nanoparticles-based solid-phase purification method to enable direct MALDI-MS analysis of permethylated glycans. Two carbon nanomaterials, carbon nanoparticles (CNPs) and graphene nanosheets (GNs), and two conventional carbon materials, activated charcoal and porous graphitic carbon (PGC), were investigated as sorbents to purify permethylated N-glycans derived from ribonuclease B and fetuin. The results confirmed the superior performance of CNPs over the other carbon materials. Additionally, our method was also employed to purify glycans released from human sera in different esophageal disease stages. The obtained data confirmed 16 and 18 structures in adenocarcinoma and Barret's sera with significantly different relative intensities versus disease-free sera. Comparing the performance of CNPs-based solid-phase purification method employed in this study to online purification suggested more than 97% recovery rate. The results of this study demonstrate that CNPs have the potential to be a better alternative to existing solid-phase purification sorbents.

The Toxic Truth About Carbon Nanotubes in Water Purification: a Perspective View.

Without nanosafety guidelines, the long-term sustainability of carbon nanotubes (CNTs) for water purifications is questionable. Current risk measurements of CNTs are overshadowed by uncertainties. New risks associated with CNTs are evolving through different waste water purification routes, and there are knowledge gaps in the risk assessment of CNTs based on their physical properties. Although scientific efforts to design risk estimates are evolving, there remains a paucity of knowledge on the unknown health risks of CNTs. The absence of universal CNT safety guidelines is a specific hindrance. In this paper, we close these gaps and suggested several new risk analysis roots and framework extrapolations from CNT-based water purification technologies. We propose a CNT safety clock that will help assess risk appraisal and management. We suggest that this could form the basis of an acceptable CNT safety guideline. We pay particular emphasis on measuring risks based on CNT physico-chemical properties such as diameter, length, aspect ratio, type, charge, hydrophobicity, functionalities and so on which determine CNT behaviour in waste water treatment plants and subsequent release into the environment.

Possible Case of Halogen Bond-Driven Self-Disproportionation of Enantiomers (SDE) via Achiral Chromatography.

The major breakthrough reported in this work is the discovery of likely halogen bond-driven self-disproportionation of enantiomers (SDE). Taking into account that the halogen-bonding interactions can be rationally designed and can match, or even exceed, the strength of the more familiar hydrogen bond, this discovery clearly opens an unexpected new direction of research in the areas of molecular chirality and the SDE phenomenon.

Purification of PEDOT:PSS by Ultrafiltration for Highly Conductive Transparent Electrode of All-Printed Organic Devices.

A highly conductive, air stable and scalable poly(3,4-ethylenedioxythiophene) (PEDOT): poly(4-styrenesulfonate) (PEDOT:PSS) are prepared by using mass production ultrafiltration. By effectively removing excess PSS and various reaction impurities using repeated 100 nm pore membrane filtration, purified PEDOT:PSS exhibit conductivity as high as 2000 S cm-1 .

Systematic identification of hypothetical bacteriophage proteins targeting key protein complexes of Pseudomonas aeruginosa.

Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism, and cell division during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.).

Preparative two-dimensional liquid chromatography/mass spectrometry for the purification of complex pharmaceutical samples.

A new preparative two-dimensional liquid chromatography/mass spectrometry system (2D LC-LC/MS) has been designed and implemented to enhance capability and resolving power for the separation and purification of pharmaceutical samples. The system was constructed by modifications of a conventional preparative LC/MS instrument with the addition of a set of switching valves and a sample loop, as well as interfacing a custom software program with MassLynx. The system integrates two chromatographic separations from the first and second dimensions into a single automated run to perform the purification of a target compound from a complex mixture without intermediate steps of sample preparation. The chromatography in the first dimension, operated in the heart-cutting mode, separates the target compound from the impurities by mass-triggered fractionation based on its molecular weight. This purified fraction from the first dimension is stored in the sample loop, and then gets transferred to the second column by using at-column dilution. A control software program, coined Prep 2D LCMS, was designed to integrate with MassLynx to retrieve data acquisition status. All of the chromatographic hardware components used in this preparative 2D LC-LC/MS system are from the original open access preparative LC/MS system, which has high level of robustness and affords easy and user-friendly operation. The new system is very versatile and capable of collecting multiple fractions with different masses under various purification modes as configured in the methods, such as conventional one-dimensional (1D) purification and/or 2D purification. This new preparative 2D LC-LC/MS system is therefore the ideal tool for medicinal chemistry lab in drug discovery environment.

Purification and characterization of the human high-molecular-weight salivary mucin / 实用口腔医学杂志

砄bjective:To purify and characterize human high molecular eight salivary mucin(MG1).Methods: MG1 was purified by 10%(w/v) cetyltrimethylammonium bromid precipitation, CM Sephadex ion exchange chromatography and sephadax G 200 gel filtration chromatography. The protein content was studied with Folinin Lowrys analysis and characterized by PAGE and SDS PAGE electrophoresis.Results:The data of PAGE showed that the purified glycoprotein was free of contaminating proteins;those SDS PAGE showed that the melocular weight of the glycoprotein was between Mr 500 000 and 1 000 000.Protein quantitative analysis showed that it contained 14.17% of protein.Amino acid analysis revealed that it contained 17 kinds of amino acid;Thr,Ser,Pro and ALa were the dominant amino acid(45.6% of the total).Conclusion: The data indicate the applied technique is reliable for purification MG1 from saliva.