Gel Filtration Research Articles

Protein purification via consecutive histidine-polyphosphate interaction.

While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.

Human Plasma‐Derived Extracellular Vesicles Protect Against Cerebral Ischemia‐Reperfusion Injury via HSP27 Phosphorylation

AbstractIschemic stroke (IS) has become a serious public health problem, with patients undergoing endovascular treatment experiencing ischemia‐reperfusion (I/R) injury, which exacerbates cerebrovascular diseases. Circulating extracellular vesicles (EVs) have shown potential for treating cerebral I/R injury. In this study, the therapeutic effect of human plasma‐derived EVs and protective mechanisms in cerebral I/R injury is explored. An oxygen‐glucose deprivation/reperfusion (OGD/R) model is used to treat SH‐SY5Y cells in vitro, and an I/R injury model is constructed by transient middle cerebral artery occlusion (tMCAO) in mice. Human plasma‐derived EVs are extracted by size exclusion chromatography. Western blot, Immunofluorescence Staining, and 2,3,5‐Triphenyltetrazolium Chloride (TTC) Staining are employed to observe the effects of EVs on the neuroinflammatory response and infarct volumes in tMCAO mice, while TUNEL Staining, Flow Cytometry, and Western Blot are employed to assess cell apoptosis. Human plasma‐derived EVs alleviated apoptosis in SH‐SY5Y cells under OGD/R stress and exerted a protective effect against brain I/R injury in tMCAO mice. Mechanistically, EVs protected against cerebral I/R injury via HSP27 phosphorylation, and the HSP27 phosphorylation inhibitor KRIBB3 attenuated the anti‐apoptotic effects of EVs. Human plasma‐derived EVs activated the phosphorylation of HSP27, thereby inhibiting cell apoptosis and protecting against cerebral I/R injury.

Preparation and Characterization of H-Shaped Polylactide.

An H-polymer has an architecture that consists of four branches symmetrically attached to the ends of a polymer backbone, similar in shape to the letter "H". Here, a renewable H-polymer efficiently synthesized using only ring-opening transesterification is demonstrated. The strategy relies on a tetrafunctional poly(±-lactide) macroinitiator, from which four poly(±-lactide) branches are grown simultaneously. 1H NMR spectroscopy, size exclusion chromatography (SEC), and matrix-assisted laser desorption/ionization (MALDI) spectrometry were used to verify the macroinitiator purity. Branch growth was probed using 1H NMR spectroscopy and SEC to reveal unique transesterification phenomena that can be controlled to yield architecturally pure or more complex materials. H-shaped PLA was prepared at the multigram scale with a weight-average molar mass Mw > 100 kg/mol and low dispersity Đ < 1.15. Purification involved routine precipitations steps, which yielded products that were architecturally relatively pure (∼93%). Small-amplitude oscillatory shear and extensional rheology measurements demonstrate the unique viscoelastic behavior associated with the H-shaped architecture.

Proteomic characterization and cytotoxic potential of proteins from Cuscuta (Cuscuta epithymum (L.) crude herbal product against MCF-7 human breast cancer cell line

BackgroundThe burden of breast cancer, the second leading cause of death worldwide, is increasing at an alarming rate. Cuscuta, used in traditional medicine for different ailments, including cancer, is known for containing phytochemicals that exhibit anticancer activity; however, the bioactivities of proteins from this plant remain unexplored. This study aimed to screen the cytotoxic potential of proteins from the crude herbal product of Cuscuta epithymum(L.) (CE) harvested from the host plants Alhagi maurorum and Medicago sativa.MethodsThe proteins from CE were extracted using a salting-out method, followed by fractionation with a gel filtration chromatography column. Gel-free shotgun proteomics was subsequently performed for protein characterization. The viability assay using MTT was applied to deduce the cytotoxic potential of proteins against MCF-7 breast cancer cells, with further exploration of the effect of treatment on the expression of the apoptotic mediator BCL2-associated X protein (BAX) and B-cell lymphoma protein 2 (BCL-2) proteins, using western blotting to strengthen the findings from the in vitro viability assay.ResultsThe crude proteins (CP) of CE were separated into four protein peaks (P1, P2, P3, and P4) by gel filtration chromatography. The evaluation of potency showed a dose-dependent decline in the MCF-7 cell line after CP, P1, P2, and P3 treatment with the respective IC50 values of 33.8, 43.1, 34.5, and 28.6 µg/ml. The percent viability of the cells decreased significantly upon treatment with 50 µg/ml CP, P1, P2, and P3 (P < 0.001). Western-blot analysis revealed upregulation of proapoptotic protein BAX in the cells treated with CP, P3 (P < 0.01), and P2 (P < 0.05); however, the antiapoptotic protein, BCL-2 was downregulated in the cells treated with CP and P3 (P < 0.01), but no significant change was detected in P2 treated cells. The observed cytotoxic effects of proteins in the CP, P1, P2, and P3 from the in vitro viability assay and western blot depicted the bioactivity potential of CE proteins. The database search revealed the identities of functionally important proteins, including nonspecific lipid transfer protein, superoxide dismutase, carboxypeptidase, RNase H domain containing protein, and polyribonucleotide nucleotidyltransferase, which have been previously reported from other plants to exhibit anticancer activity.ConclusionThis study indicated the cytotoxic activity of Cuscuta proteins against breast cancer MCF-7 cells and will be utilized for future investigations on the mechanistic effect of active proteins. The survey of CE proteins provided substantial data to encourage further exploration of biological activities exhibited by proteins in Cuscuta.

Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS.

Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 μg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab')2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab')2 subunits is much slower than the other four F(ab')2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.

Bioassay-guided isolation and identification of antibacterial compounds from Piper betle leaf with inhibitory activity against the Vibrio species in shrimp

Bioassay-guided isolation and identification of antibacterial compounds from <i>Piper betle</i> leaf with inhibitory activity against the <i>Vibrio</i> species in shrimp

Macrophage PET imaging in mouse models of cardiovascular disease and cancer with an apolipoprotein-inspired radiotracer

Macrophages are key inflammatory mediators in many pathological conditions, including cardiovascular disease (CVD) and cancer, the leading causes of morbidity and mortality worldwide. This makes macrophage burden a valuable diagnostic marker and several strategies to monitor these cells have been reported. However, such strategies are often high-priced, non-specific, invasive, and/or not quantitative. Here, we developed a positron emission tomography (PET) radiotracer based on apolipoprotein A1 (ApoA1), the main protein component of high-density lipoprotein (HDL), which has an inherent affinity for macrophages. We radiolabeled an ApoA1-mimetic peptide (mA1) with zirconium-89 (89Zr) to generate a lipoprotein-avid PET probe (89Zr-mA1). We first characterized 89Zr-mA1’s affinity for lipoproteins in vitro by size exclusion chromatography. To study 89Zr-mA1’s in vivo behavior and interaction with endogenous lipoproteins, we performed extensive studies in wildtype C57BL/6 and Apoe-/- hypercholesterolemic mice. Subsequently, we used in vivo PET imaging to study macrophages in melanoma and myocardial infarction using mouse models. The tracer’s cell specificity was assessed by histology and mass cytometry (CyTOF). Our data show that 89Zr-mA1 associates with lipoproteins in vitro. This is in line with our in vivo experiments, in which we observed longer 89Zr-mA1 circulation times in hypercholesterolemic mice compared to C57BL/6 controls. 89Zr-mA1 displayed a tissue distribution profile similar to ApoA1 and HDL, with high kidney and liver uptake as well as substantial signal in the bone marrow and spleen. The tracer also accumulated in tumors of melanoma-bearing mice and in the ischemic myocardium of infarcted animals. In these sites, CyTOF analyses revealed that natZr-mA1 was predominantly taken up by macrophages. Our results demonstrate that 89Zr-mA1 associates with lipoproteins and hence accumulates in macrophages in vivo. 89Zr-mA1’s high uptake in these cells makes it a promising radiotracer for non-invasively and quantitatively studying conditions characterized by marked changes in macrophage burden.

Preliminary Purification and Partial Characterization of a Functional Bacteriocin of Lacticaseibacillus paracasei Zhang and Mining for its Gene Cluster.

Bacteriocins produced by lactic acid bacteria (LAB) have good potential for use as food biopreservatives. Lacticaseibacillus paracasei Zhang (L. paracasei Zhang) is both a food use and a probiotic bacterium. This study aimed to purify and preliminary characterize the active antibacterial metabolite of L. paracasei Zhang. The cell-free supernatant of L. paracasei Zhang was collected and purified by ultrafiltration and gel filtration chromatography. The 1-3kDa active fraction could inhibit the growth of Staphylococcus aureus but not Escherichia coli. Further antibacterial activity assays revealed its capacity to suppress various foodborne and human opportunistic pathogens (including Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Listeria monocytogenes, and Bacillus cereus), but not fungi. The antibacterial activity showed good tolerance to heat (40 to 100°C), acid-base (pH 2-3 and pH 6-10), and digestions by a number of industrial and animal/human enzymes (such as trypsin, pepsin, α-amylase, and protease K, except papain); these desired properties make it a suitable biopreservative to be used in harsh and complex industrial production processes. The high papain sensitivity suggested a proteinaceous/peptide nature of the bioactivity. Moreover, our genomic data mining for bacteriocin through BAGEL4 revealed an area of interest encoding a complete set of putative genes required for bacteriocin production. In conclusion, our study showed that L. paracasei Zhang can produce extracellular functional antibacterial metabolite, likely a class II bacteriocin. Our preliminary extraction and characterization of the active metabolite demonstrated that it has good potential to be used as a biopreservative or an agent for suppressing gastrointestinal infections.

Characterization of the 263K-derived microsomal fraction: a source of prions for nanofiltration validation studies.

The manufacturing processes of plasma products include steps that can remove prions. The efficacy of these steps is measured in validation studies using animal brain-derived prion materials called spikes. Because the nature of the prion agent in blood is not known, the relevance of these spikes, particularly with steps that are based on retention mechanisms such as nanofiltration, is important to investigate. The aggregation and sizes of PrPres assemblies of microsomal fractions (MFs) extracted from 263K-infected hamster brains were analyzed using velocity gradients. The separated gradient fractions were either inoculated to Tg7 mice expressing hamster-PrPc to measure infectivity or used in Protein Misfolding Cyclic Amplification for measuring seeding activity. The collected data allowed for reanalyzing results from previous nanofiltration validation studies. A significant portion of MFs was found to be composed of small PrPres assemblies, estimated to have a size ≤24 mers (~22-528 kDa), and to contain a minimum of 20% of total prion infectivity. With this data we could calculate reductions of 4.10 log (15 N), 2.53 log (35 N), and 1.77 log (35 N) from validation studies specifically for these small PrPres objects. Our gradient data provided evidence that nanofilters can remove the majority of the smallest PrPres entities within microsomes spikes, estimated to be in a size below 24 mers, giving insight about the fact that, in our conditions, size exclusion may not be the only mechanism for retention nanofiltration.

Insights into copper sensing and tolerance in Pneumocystis species

IntroductionPneumocystis species are pathogenic fungi known to cause pneumonia in immunocompromised mammals. They are obligate to their host, replicate extracellularly in lung alveoli and thrive in the copper-enriched environment of mammalian lungs. In this study, we investigated the proteome of Pneumocystis murina, a model organism that infects mice, in the context of its copper sensing and tolerance.Methods and resultsThe query for copper-associated annotations in FungiDB followed by a manual curation identified only 21 genes in P. murina, significantly fewer compared to other clinically relevant fungal pathogens or phylogenetically similar free-living fungi. We then employed instrumental analyses, including Size-Exclusion Chromatography Inductively Coupled Plasma Mass Spectrometry (SEC-ICP-MS), Immobilized Metal Affinity Chromatography (IMAC), and Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS), to isolate and identify copper-binding proteins from freshly extracted organisms, revealing 29 distinct cuproproteins. The RNA sequencing (RNA-seq) analysis of P. murina exposed to various CuSO4 concentrations at three temporal intervals (0.5, 2, and 5 h) indicated that significant gene expression changes occurred only under the highest CuSO4 concentration probed (100 μM) and the longest exposure duration (5 h). This stimulus led to the upregulation of 43 genes and downregulation of 27 genes compared to untreated controls. Quantitative PCR (qPCR) confirmed the expression of four out of eight selected upregulated genes, including three assumed transcription factors (PNEG_01236, PNEG_01675, and PNEG_01730) and a putative copper transporter (PNEG_02609). Notably, the three applied methodologies — homology-based annotation, SEC-ICP-MS/IMAC/LC–MS/MS, and RNA-seq — yielded largely distinct findings, with only four genes (PNEG_02587, PNEG_03319, PNEG_02584, and PNEG_02989) identified by both instrumental methods.DiscussionThe insights contribute to the broader knowledge of Pneumocystis copper homeostasis and provide novel facets of host-pathogen interactions for extracellular pathogens. We suggest that future studies of Pneumocystis pathogenicity and copper stress survival should consider the entire spectrum of identified genes.

A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies

Background: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. Methods: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. Results: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. Conclusions: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.

Impact on Quality during In-Use Preparation of an Antibody Drug Conjugate with Eight Different Closed System Transfer Device Brands.

The aim of this study was to investigate the in-use compatibility of eight commercially available closed system transfer device brands (CSTDs) with a formulated model antibody drug conjugate (ADC). Overall, in-use simulated dosing preparation applying the CSTD systems investigated raised concerns for several product quality attributes. The incompatibilities observed were mainly associated with increased visible and subvisible particles formation as well as significant changes in holdup volumes. Visible and subvisible particles contained heterogeneous mixtures of particle classes, with the majority of subvisible particles associated with silicone oil leaching from CSTD systems during simulated dose preparation upon contact with the ADC formulation. These observations demonstrate that CSTD use may adversely impact product quality and delivered dose which could potentially lead to safety and efficacy concerns during administration. Other product quality attributes measured including turbidity, color, ADC recovery, and purity by size exclusion HPLC, did not show relevant changes. It is therefore strongly recommended to test and screen the compatibility of CSTDs with the respective ADC, in a representative in-use simulated administration setting, during early CMC development, i.e., well before the start of clinical studies, to include information about compatibility and to ensure that the CSTD listed in the manuals of preparation for clinical handling has been thoroughly assessed before human use.

Interaction of heparin with human cardiac troponin complex and its influence on the immunodetection of troponins in human blood samples.

Heparin is a highly charged polysaccharide used as an anticoagulant to prevent blood coagulation in patients with presumed myocardial infarction and to prepare heparin plasma samples for laboratory tests. There are conflicting data regarding the effects of heparin on the measurement of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT), which are used for the immunodiagnosis of acute myocardial infarction. In this study, we investigated the influence of heparin on the immunodetection of human cardiac troponins. Gel filtration (GF) techniques and sandwich fluoroimmunoassay were performed. The regions of сTnI and cTnT that are affected by heparin were investigated with a panel of anti-cTnI and anti-cTnT monoclonal antibodies, specific to different epitopes. Heparin was shown to bind to the human cardiac full-size ternary troponin complex (ITC-complex) and free cTnT, which increased their apparent molecular weights in GF studies. Heparin did not bind to the low molecular weight ITC-complex and to binary cTnI-troponin С complex. We did not detect any sites on cTnI in the ITC-complex that were specifically affected by heparin. In contrast, cTnT regions limited to approximately 69-99, 119-138 and 145-164 amino acid residues (aar) in the ITC-complex and a region that lies approximately between 236 and 255 aar of free cTnT were prone to heparin influence. Heparin binds to the ITC-complex via cTnT, interacting with several sites on the N-terminal and/or central parts of the cTnT molecule, which might influence the immunodetection of analytes in human blood.

(Thermo)mechanical and chemical characteristics of photochemically crosslinked acrylates from bio‐ and fossil‐based origin

AbstractThe acrylates with oligomers and monomers from (partially) bio‐based feedstock become available at (semi‐)industrial scale, which can be processed through photochemical crosslinking for applications in coatings, additive manufacturing, electronics, or inks. Although fossil‐ and bio‐based acrylates may have a similar chemical composition, it requires good understanding of processing and structure–property relationships as minor changes in microstructure may strongly alter the performance. A comparative study on mechanical properties and chemical structure of bio‐ and fossil‐based acrylates with different functionalities and backbone structures reveals higher ductility of bio‐based acrylates, in relation with a more complex organization of the intrinsic molecular structure. The latter is confirmed by mechanical testing and visco‐elastic characteristics (dynamic mechanical analysis) yielding lower stiffness and higher dampening of bio‐based acrylates, in parallel with a lower glass transition temperature (differential scanning calorimetry). The complex molecular arrangements include a nanoscale morphology with ordered structure (X‐ray diffraction), conformational changes (infrared spectroscopy), and a residual high‐molecular weight fraction (size exclusion chromatography). The visco‐elastic calculations indicate only 4% to 5% lower crosslinking density and around 10% higher mean molar mass of the polymer chains segments between chemical crosslinks and trapped chain entanglements, which explain the unique structure and performance of bio‐based acrylates.

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites

BackgroundGlycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated.MethodsWe enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC–MS/MS.ResultsHuman albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated.ConclusionsAlbumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Interfacial Super‐Assembly of Vacancy Engineered Ultrathin‐Nanosheets toward Nanochannels for Smart Ion Transport and Salinity Gradient Power Conversion

Ion‐selective nanochannel membranes assembled from two‐dimensional (2D) nanosheets hold immense promise for power conversion using salinity gradient. However, they face challenges stemming from insufficient surface charge density, which impairs both permselectivity and durability. Herein, we present a novel vacancy‐engineered, oxygen‐deficient NiCo layered double hydroxide (NiCoLDH)/cellulose nanofibers‐wrapped carbon nanotubes (VOLDH/CNF‐CNT) composite membrane. This membrane, featuring abundant angstrom‐scale, cation‐selective nanochannels, is designed and fabricated through a synergistic combination of vacancy engineering and interfacial super‐assembly. The membrane shows interlayer free‐spacing of ~3.62 Å, which validates the membrane size exclusion selectivity.This strategy, validated by DFT calculations and experimental data, improves hydrophilicity and surface charge density, leading to the strong interaction with K+ ions to benefit the low ion transport resistance and exceptional charge selectivity. When employed in an artificial river water|seawater salinity gradient power generator, it delivers a high‐power density of 5.35 W/m2 with long‐term durability (20,000s), which is almost 400% higher than that of the pristine NiCoLDH membrane. Furthermore, it displays both pH‐ and temperature‐sensitive ion transport behavior, offering additional opportunities for optimization. This work establishes a basis for high‐performance salinity gradient power conversion and underscores the potential of vacancy engineering and super‐assembly in customizing 2D nanomaterials for diverse advanced nanofluidic energy devices.

Interfacial Super-Assembly of Vacancy Engineered Ultrathin-Nanosheets toward Nanochannels for Smart Ion Transport and Salinity Gradient Power Conversion.

Ion-selective nanochannel membranes assembled from two-dimensional (2D) nanosheets hold immense promise for power conversion using salinity gradient. However, they face challenges stemming from insufficient surface charge density, which impairs both permselectivity and durability. Herein, we present a novel vacancy-engineered, oxygen-deficient NiCo layered double hydroxide (NiCoLDH)/cellulose nanofibers-wrapped carbon nanotubes (VOLDH/CNF-CNT) composite membrane. This membrane, featuring abundant angstrom-scale, cation-selective nanochannels, is designed and fabricated through a synergistic combination of vacancy engineering and interfacial super-assembly. The membrane shows interlayer free-spacing of ~3.62 Å, which validates the membrane size exclusion selectivity.This strategy, validated by DFT calculations and experimental data, improves hydrophilicity and surface charge density, leading to the strong interaction with K+ ions to benefit the low ion transport resistance and exceptional charge selectivity. When employed in an artificial river water|seawater salinity gradient power generator, it delivers a high-power density of 5.35 W/m2 with long-term durability (20,000s), which is almost 400% higher than that of the pristine NiCoLDH membrane. Furthermore, it displays both pH- and temperature-sensitive ion transport behavior, offering additional opportunities for optimization. This work establishes a basis for high-performance salinity gradient power conversion and underscores the potential of vacancy engineering and super-assembly in customizing 2D nanomaterials for diverse advanced nanofluidic energy devices.

Purification, Characterization and Antifungal Activity of the Aspergillus niveus Chitinase Produced Using Shrimp Shells

Chitinases are biotechnologically relevant enzymes that can be applied in such different sectors as pharmaceutical, food, environmental management, the biocontrol of pests and in the paper and cellulose industry. Microorganisms as filamentous fungi are the most important source of these biomolecules. The fungus Aspergillus niveus produces extracellular chitinase when cultured under submerged fermentation using shrimp shells, a residue generated by the fish industry, as a carbon source, for 96 h at 30 °C and 100 rpm. The particle size and concentration of the shrimp shells affected enzyme production. The chitinase was purified until electrophoretic homogeneity through the use of a Sephadex G-100 chromatographic column. It is a monomeric glycoprotein with a molecular mass of 47 kDa estimated using SDS-PAGE and 49.3 kDa determined using gel filtration. The carbohydrate content was 22.8%. The best temperature and pH for enzyme activity were 65 °C and 6.0, respectively. Approximately 80% of the enzymatic activity was preserved at pH 4.0 and 5.0 for 48 h, and the half-life (t50) was maintained for 48 h at 40 °C. Salts, EDTA and β-mercaptoethanol did not affect chitinase activity significantly, but organic solvents reduced it. The kinetic parameters determined using p-NPGlycNac were Km of 2.67 mmol L−1, Vmax of 12.58 U mg of protein−1, Kcat of 2.47 s−1 and K cat/Km of 0.93 s−1 mmol L−1. The A. niveus chitinase inhibited the growth of all fungal strains used, especially Trichoderma harzianum (MIC = 22.4 μg mL−1) and Penicillium purpurogenum (MIC = 11.2 μg mL−1). The chitinase produced by A. niveus presented interesting characteristics that indicate its potential of application in different areas.

Phytochemical evaluation of Ziziphus mucronata and Xysmalobium undulutum towards the discovery and development of anti-malarial drugs

BackgroundThe development of resistance by Plasmodium falciparum is a burdening hazard that continues to undermine the strides made to alleviate malaria. As such, there is an increasing need to find new alternative strategies. This study evaluated and validated 2 medicinal plants used in traditional medicine to treat malaria.MethodsInspired by their ethnobotanical reputation of being effective against malaria, Ziziphus mucronata and Xysmalobium undulutum were collected and sequentially extracted using hexane (HEX), ethyl acetate (ETA), Dichloromethane (DCM) and methanol (MTL). The resulting crude extracts were screened for their anti-malarial and cytotoxic potential using the parasite lactate dehydrogenase (pLDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. This was followed by isolating the active compounds from the DCM extract of Z. mucronata using silica gel chromatography and structural elucidation using spectroscopic techniques (NMR: 1H, 12C, and DEPT). The active compounds were then targeted against P. falciparum heat shock protein 70–1 (PfHsp70-1) using Autodock Vina, followed by in vitro validation assays using ultraviolet–visible (UV–VIS) spectroscopy and the malate dehydrogenase (MDH) chaperone activity assay.ResultsThe extracts except those of methanol displayed anti-malarial potential with varying IC50 values, Z. mucronata HEX (11.69 ± 3.84 µg/mL), ETA (7.25 ± 1.41 µg/mL), DCM (5.49 ± 0.03 µg/mL), and X. undulutum HEX (4.9 ± 0.037 µg/mL), ETA (17.46 ± 0.024 µg/mL) and DCM (19.27 ± 0.492 µg/mL). The extracts exhibited minimal cytotoxicity except for the ETA and DCM of Z. mucronata with CC50 values of 10.96 and 10.01 µg/mL, respectively. Isolation and structural characterization of the active compounds from the DCM extracts revealed that betulinic acid (19.95 ± 1.53 µg/mL) and lupeol (7.56 ± 2.03 µg/mL) were responsible for the anti-malarial activity and had no considerable cytotoxicity (CC50 > µg/mL). Molecular docking suggested strong binding between PfHsp70-1, betulinic acid (− 6.8 kcal/mol), and lupeol (− 6.9 kcal/mol). Meanwhile, the in vitro validation assays revealed the disruption of the protein structural elements and chaperone function.ConclusionThis study proves that X undulutum and Z. mucronata have anti-malarial potential and that betulinic acid and lupeol are responsible for the activity seen on Z. mucronata. They also make a case for guided purification of new phytochemicals in the other extracts and support the notion of considering medicinal plants to discover new anti-malarials.

From laboratory to industrial use: Understanding process variation during enzymatic protein hydrolysis with dry film fourier-transform infrared spectroscopy

Industries are continuously looking for dedicated sensor solutions for evaluating the chemical composition of products that can provide valuable insights into process control, process optimization, and product quality. A rapid and robust analytical method, Fourier-Transform Infrared (FTIR) spectroscopy, holds significant potential in this regard as it provides detailed compositional values of food nutrients. The main aim of the present study was to use dry film FTIR spectroscopy as an analytical tool to characterize products from an industrial enzymatic protein hydrolysis process and link this to understand industrial process variations. For this purpose, 463 protein hydrolysate samples were obtained from an industrial enzymatic protein hydrolysis plant. In the same period, systematic variations in process parameters such as raw material composition, enzyme type, and water addition, were performed. All samples were analyzed using dry film FTIR spectroscopy. Two subsets containing 200 and 68 hydrolysate samples were chosen for measuring average molecular weight (AMW) and collagen content respectively, providing reference data for constructing calibration models based on partial least squares regression (PLSR). The percentage of low molecular weight constituents was derived from the molecular weight distribution of size exclusion chromatograms of protein hydrolysates and also used in the modeling. Calibration models for the prediction of AMW, low molecular weight constituents, and collagen content were obtained with a good fit and lower estimation errors. Principal component analysis (PCA) of protein hydrolysates' FTIR spectra effectively differentiated process variations (enzyme types and raw materials) without extensive reference analysis. One-factor analysis of variance (ANOVA) tests was used to observe the impact of process variation on product quality. FTIR proved to be a sensitive method for liquid protein analysis and process control with a significant potential in process optimization approaches. To the authors’ knowledge, this is the first time dry film FTIR spectroscopy has been used to evaluate an industrial bioprocess with designed process variations.