Green and fast prediction of crude protein contents in bee pollen based on digital images combined with Random Forest algorithm.

Bee pollen is considered an excellent dietary supplement with functional characteristics, and it has been employed in food and cosmetics formulations and in biomedical applications. Therefore, understanding its chemical composition, particularly crude protein contents, is essential to ensure its quality and industrial application. For the quantification of crude protein in bee pollen, this study explored the potential of combining digital image analysis and Random Forest algorithm for the development of a rapid, cost-effective, and environmentally friendly analytical methodology. Digital images of bee pollen samples (n = 244) were captured using a smartphone camera with controlled lighting. RGB channels intensities and color histograms were extracted using open source softwares. Crude protein contents were determined using the Kjeldahl method (reference) and in combination with RGB channels and color histograms data from digital images, they were used to generate a predictive model through the application of the Random Forest algorithm. The developed model exhibited good performance and predictive capability for crude protein analysis in bee pollen (R2 = 80.93 %; RMSE = 1.49 %; MAE = 1.26 %). Thus, the developed analytical methodology can be considered environmentally friendly according to the AGREE metric, making it an excellent alternative to conventional analysis methods. It avoids the use of toxic reagents and solvents, demonstrates energy efficiency, utilizes low-cost instrumentation, and it is robust and precise. These characteristics indicate its potential for easy implementation in routine analysis of crude protein in bee pollen samples in quality control laboratories.

Capillary electrophoresis in the analysis of therapeutic peptides-A review.

Therapeutic peptides are a growing class of innovative drugs with high efficiency and a low risk of adverse effects. These biomolecules fall within the molecular mass range between that of small molecules and proteins. However, their inherent instability and potential for degradation underscore the importance of reliable and effective analytical methods for pharmaceutical quality control, therapeutic drug monitoring, and compliance testing. Liquid chromatography-mass spectrometry (LC-MS) has long time been the "gold standard" conventional method for peptide analysis, but capillary electrophoresis (CE) is increasingly being recognized as a complementary and, in some cases, superior, highly efficient, green, and cost-effective alternative technique. CE can separate peptides composed of different amino acids owing to differences in their net charge and size, determining their migration behavior in an electric field. This review provides a comprehensive overview of therapeutic peptides that have been used in the clinical environment for the last 25 years. It describes the properties, classification, current trends in development, and clinical use of therapeutic peptides. From the analytical point of view, it discusses the challenges associated with the analysis of therapeutic peptides in pharmaceutical and biological matrices, as well as the evaluation of CE as a whole and the comparison with LC methods. The article also highlights the use of microchip electrophoresis, nonaqueous CE, and nonconventional hydrodynamically closed CE systems and their applications. Overall, the article emphasizes the importance of developing new CE-based analytical methods to ensure the high quality, safety, and efficacy of therapeutic peptides in clinical practice.

Pharmacoinformatic Investigation of Silymarin as a Potential Inhibitor against Nemopilema nomurai Jellyfish Metalloproteinase Toxin-like Protein.

Jellyfish stings pose a major threat to swimmers and fishermen worldwide. These creatures have explosive cells containing one large secretory organelle called a nematocyst in their tentacles, which contains venom used to immobilize prey. Nemopilema nomurai, a venomous jellyfish belonging to the phylum Cnidaria, produces venom (NnV) comprising various toxins known for their lethal effects on many organisms. Of these toxins, metalloproteinases (which belong to the toxic protease family) play a significant role in local symptoms such as dermatitis and anaphylaxis, as well as systemic reactions such as blood coagulation, disseminated intravascular coagulation, tissue injury, and hemorrhage. Hence, a potential metalloproteinase inhibitor (MPI) could be a promising candidate for reducing the effects of venom toxicity. For this study, we retrieved the Nemopilema nomurai venom metalloproteinase sequence (NnV-MPs) from transcriptome data and modeled its three-dimensional structure using AlphaFold2 in a Google Colab notebook. We employed a pharmacoinformatics approach to screen 39 flavonoids and identify the most potent inhibitor against NnV-MP. Previous studies have demonstrated the efficacy of flavonoids against other animal venoms. Based on our analysis, Silymarin emerged as the top inhibitor through ADMET, docking, and molecular dynamics analyses. In silico simulations provide detailed information on the toxin and ligand binding affinity. Our results demonstrate that Silymarin's strong inhibitory effect on NnV-MP is driven by hydrophobic affinity and optimal hydrogen bonding. These findings suggest that Silymarin could serve as an effective inhibitor of NnV-MP, potentially reducing the toxicity associated with jellyfish envenomation.

Inflammation-related proteomics demonstrate landscape of fracture blister fluid in patients with acute compartment syndrome.

Background: Blisters are tense vesicles or bullae that arise on swollen skin and are found in a wide range of injuries. As a complication of fracture, fracture blisters are considered soft tissue injuries, which often lead to adverse effects such as prolonged preoperative waiting time and increased risk of surgical site infection. However, our previous study found that in patients with acute compartment syndrome, fracture blisters may be a form of compartment pressure release, but the specific mechanism has not been revealed. Here, we mapped out the proteomic landscape of fracture blister fluid for the first time and compared its expression profile to cupping and burn blisters. Methods: First, fluid samples were collected from 15 patients with fracture blisters, 7 patients with cupping blisters, and 9 patients with burn blisters. Then, the expression levels of 92 inflammatory proteins were measured using the Olink Target 96 Inflammation panel. Protein profiles were compared across the three groups using Differential Protein Expression Analysis and Principal Component Analysis (PCA). Results: Fracture blisters had significantly higher levels of 50 proteins in comparison to cupping and 26 proteins in comparison to burn blisters. Notably, PCA showed fracture blisters closely resembled the protein expression profile of burn blisters but were distinct from the protein expression profile of cupping blisters. Conclusion: Our study provides the first characterization of fracture blister fluid using proteomics, which provides a valuable reference for further analysis of the difference between blisters caused by fractures and those caused by other pathogenic factors. This compendium of proteomic data provides valuable insights and a rich resource to better understand fracture blisters.

Speeding up SDS-PAGE: Theory and experiment.

In order to accelerate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), here we propose an optimized version of the technique enabled by experimental tuning reinforced by theoretical description. In the resulting system, the gel buffer was diluted twofold and supplemented with glycine at a low concentration, whereas a higher voltage was applied. This approach reduced runtime from 90 to 18 min. It is important to emphasize that, despite the high voltage applied to the gel, the resolution of the bands did not decrease compared to the original Laemmli method. The proposed acceleration approach can be used in other variants of SDS-PAGE.

Bee pollen and bee bread nutritional potential: Chemical composition and macronutrient digestibility under in vitro gastrointestinal system.

Bee pollen (BP) and bee bread (BB) have been often investigated as potential functional foods. Both bee products are generally characterized by their high nutritional content, with BB being referred as more digestible than BP, however, there is a lack of scientific studies proving this claim. Here, we present a comparative evaluation of the macronutrient digestibility of BP and BB after applying a simulated in vitro gastrointestinal digestive system, together with the evaluation of its nutritional value and chemical composition. The digestibility scores for protein content were calculated on average as 69% and 76% for BP and BB, respectively, whereas digestibility scores for soluble sugars varied depending on bee product and sugar type. The results demonstrated that the nutritional values of both bee products changed depending on their botanical origin but BB is more accessible in the intestinal lumen, especially regarding protein.

Effects of ultrasound assisted cell wall disruption on physicochemical properties of camellia bee pollen protein isolates.

Camellia bee pollen protein isolates were extracted by cell wall disruption using ultrasonication, freeze-thawing, enzymatic hydrolysis, and their combinations. The effects of these methods on microstructure of cell wall, protein release, protein yield, physiochemical properties and structure of proteins were investigated. As compared with physical treatments (ultrasonication, freeze-thawing and their combination), the enzymatic hydrolysis significantly improved the yield of proteins, because it not only promoted the release of proteins from the inside of pollen, but also released proteins in pollen wall. The proteins extracted by enzymatic hydrolysis method also exhibited better solubility, emulsifying and gelation properties due to the partial hydrolysis of proteins by protease. In addition, when ultrasound was combined with freeze-thawing or enzymatic hydrolysis, it could further improve the yield of proteins and the functional properties of proteins, which was mainly related to the changes of protein structure induced by cavitation effect of ultrasound.

Physicochemical, functional, and digestive characteristics of tea seed cake protein obtained by ultrafiltration.

To make full use of tea seed cake protein (TSCP), this study investigated the physicochemical and functional properties of TSCP, including the TSCP extract and three ultrafiltration fractions TSCP-1 (Mw > 10 kDa), TSCP-2 (3.5 kDa < Mw < 10 kDa), and TSCP-3 (Mw < 3.5 kDa). After ultrafiltration, the content, thermal stability, and surface hydrophobicity of TSCP were increased, and the molecular weight distribution and structure of TSCP showed significant differences. In terms of functionality, each fraction showed its advantages. Specifically, compared with the others, TSCP had better solubility and foaming properties, and TSCP-1 had significantly higher oil absorption capacity, and TSCP-2 had better water absorption capacity and emulsifying properties, and TSCP-3 can flow more easily (p < 0.05). In terms of nutritional value, the content of essential amino acids in all samples was sufficient. The degree of hydrolysis of TSCP was highest (80.98 ± 1.50%), and ultrafiltration decreased digestibility. These results indicated that ultrafiltration effectively improved the structure and functional properties of TSCP, and the obtained fractions can be applied to different scenarios. Practical Application: Tea seed cakes are rich in protein and usually regarded as byproducts during oil processing. Because of its good functional properties, tea seed cake proteins obtained by ultrafiltration have the potential to be used as ingredients for food.

Endogenous Fluorescent Proteins in the Mucus of an Intertidal Polychaeta: Clues for Biotechnology.

The vast ocean holds many unexplored organisms with unique adaptive features that enable them to thrive in their environment. The secretion of fluorescent proteins is one of them, with reports on the presence of such compounds in marine annelids being scarce. The intertidal Eulalia sp. is an example. The worm secretes copious amounts of mucus, that when purified and concentrated extracts, yield strong fluorescence under UV light. Emission has two main maxima, at 400 nm and at 500 nm, with the latter responsible for the blue-greenish fluorescence. Combining proteomics and transcriptomics techniques, we identified ubiquitin, peroxiredoxin, and 14-3-3 protein as key elements in the mucus. Fluorescence was found to be mainly modulated by redox status and pH, being consistently upheld in extracts prepared in Tris-HCl buffer with reducing agent at pH 7 and excited at 330 nm. One of the proteins associated with the fluorescent signal was localized in secretory cells in the pharynx. The results indicate that the secretion of fluorescent proteinaceous complexes can be an important defense against UV for this dweller. Additionally, the internalization of fluorescent complexes by ovarian cancer cells and modulation of fluorescence of redox status bears important considerations for biotechnological application of mucus components as markers.

Holistic analytical characterization and risk assessment of residual host cell protein impurities in an active pharmaceutical ingredient synthesized by biocatalysts.

Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately controlled to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taking into consideration the residual HCP profile in the product, the dose, dosage form, administration route, and so forth. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of an investigational stimulator of interferon genes protein agonist, MK-1454, which is a cyclic dinucleotide synthesized using Escherichia coli cell lysate overexpressing cyclic GMP-AMP synthase as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools including nanoscale liquid chromatography coupled to tandem mass spectrometry, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 active pharmaceutical ingredient.

Microbial Diversity and Characteristics of Kombucha as Revealed by Metagenomic and Physicochemical Analysis.

Kombucha is a fermented tea made from a Symbiotic Culture of Bacteria and Yeast (SCOBY) with a long history of use as a health tonic. It is likely that most health benefits come from the tea and fermentation metabolites from specific microbial communities. Despite its growing importance as a functional health drink, the microbial ecosystem present in kombucha has not been fully documented. To characterize the microbial composition and biochemical properties of 'The Good Brew' original base kombucha, we used metagenomics amplicon (16S rRNA and ITS) sequencing to identify the microbial communities at the taxonomic level. We identified 34 genera with 200 microbial species yet described in kombucha. The dominance of organic acid producing microorganisms Acetobacter, Komagataeibacter and Starmerella are healthy for the human gut and their glucose metabolising activities have a putative role in preventing conditions such as diabetes and obesity. Kombucha contains high protein (3.31 µg/mL), high phenolic content (290.4 mg/100 mL) and low sugars (glucose: 1.87 g/L; sucrose 1.11 g/L; fructose: 0.05 g/L) as compared to green tea. The broad microbial diversity with proven health benefits for the human gut suggests kombucha is a powerful probiotic. These findings are important to improve the commercial value of kombucha and uncover the immense prospects for health benefits.

What about Dinner? Chemical and Microresidue Analysis Reveals the Function of Late Neolithic Ceramic Pans.

The Late Neolithic palafitte site, Ustie na Drim, in the northern part of Lake Ohrid (North Macedonia), excavated in 1962, offered ceramic fragments of large, flat, elongated pans. These artifacts could be dated by relative chronology to roughly around 5200-5000 BC. According to their shape and technological traits, the ceramic pans were probably used for baking. The attached materials on the surface of studied pan fragments were sampled for consequent chemical and microscopical analyses (i.e., analyses of starch, phytoliths, and microscopic animal remains). An immunological method revealed the presence of pork proteins in samples. The presence of organic residues of animal origin was, moreover, confirmed by the detection of cholesterol using gas chromatography coupled to mass spectrometry. Analysis of detected microscopic botanical objects revealed starch grains of several plants (i.e., oak, cattail, and grasses). An interesting find was the hair of a beetle larva, which could be interpreted contextually as the khapra beetle, a pest of grain and flour. Based on our data, we suppose that the ceramic pans from Ustie na Drim were used for the preparation of meals containing meat from common livestock in combination with cereals and wild plants.

Ultrafiltration strategy combined with nanoLC-MS/MS based proteomics for monitoring potential residual proteins in TCMIs.

Traditional Chinese medicine injections (TCMIs) containing complex constituents frequently cause unpredictable adverse reactions. The residual heterologous proteins in TCMIs may be one kind of the sensitized constituents. However, few methods were developed to identify and monitor the residual proteins of TCMIs in industry. Here, we described a method combining the advantages of ultrafiltration and mass spectrometry-based proteomics for monitoring the potential residual proteins in Re Du Ning injection (RDNI) intermediates and preparations. We identified and quantified both de novo peptides and the proteins matched against databases of three raw plants by using PEAKS software. Interesting, we found there was a significant decrease of peptides and proteins in No. 3-5 of RDNI intermediates and some even disappeared. Besides, we found this method could greatly reduce the interference of contaminants in proteomics experiments. The rapid and accurate method proposed in this paper could be used for monitoring potential residual proteins in TCMIs to guarantee their quality and safety.

Antioxidant capacity of honey from the urban apiary: a comparison with honey from the rural apiary.

Honey is a source of natural antioxidant compounds exerting several health-beneficial effects. Since urban beekeeping is quite common, the fear among potential consumers about the quality and the safety of honey produced exclusively in the cities is observed. However, the antioxidant properties of urban honey have not yet been tested. We described the antioxidant properties of linden honey from urban and rural areas. We analyzed the total phenolic content, DPPH• radical scavenging activity, Trolox equivalent antioxidant activity assay, the protein content, and catalase activity. The analysis showed that all tested parameters were significantly higher in honey from rural areas than in urban samples. The differences in the obtained results are certainly not the effect of the floral composition of honey, but rather due to the location of the honeybee colonies. It seems that the consumption of honey from urban areas for health purposes should be considered.

Improved Protein and PTM Characterization with a Practical Electron-Based Fragmentation on Q-TOF Instruments.

Electron-based dissociation (ExD) produces uncluttered mass spectra of intact proteins while preserving labile post-translational modifications. However, technical challenges have limited this option to only a few high-end mass spectrometers. We have developed an efficient ExD cell that can be retrofitted in less than an hour into current LC/Q-TOF instruments. Supporting software has been developed to acquire, process, and annotate peptide and protein ExD fragmentation spectra. In addition to producing complementary fragmentation, ExD spectra enable many isobaric leucine/isoleucine and isoaspartate/aspartate pairs to be distinguished by side-chain fragmentation. The ExD cell preserves phosphorylation and glycosylation modifications. It also fragments longer peptides more efficiently to reveal signaling cross-talk between multiple post-translational modifications on the same protein chain and cleaves disulfide bonds in cystine knotted proteins and intact antibodies. The ability of the ExD cell to combine collisional activation with electron fragmentation enables more complete sequence coverage by disrupting intramolecular electrostatic interactions that can hold fragments of large peptides and proteins together. These enhanced capabilities made possible by the ExD cell expand the size of peptides and proteins that can be analyzed as well as the analytical certainty of characterizing their post-translational modifications.

High photosynthetic plasticity may reinforce invasiveness of upside-down zooxanthellate jellyfish in Mediterranean coastal waters.

Ecological profiling of non-native species is essential to predict their dispersal and invasiveness potential across different areas of the world. Cassiopea is a monophyletic taxonomic group of scyphozoan mixotrophic jellyfish including C. andromeda, a recent colonizer of sheltered, shallow-water habitats of the Mediterranean Sea, such as harbors and other light-limited, eutrophic coastal habitats. To assess the ecophysiological plasticity of Cassiopea jellyfish and their potential to spread across the Mare Nostrum by secondary introductions, we investigated rapid photosynthetic responses of jellyfish to irradiance transitions-from reduced to increased irradiance conditions (as paradigm of transition from harbors to coastal, meso/oligotrophic habitats). Laboratory incubation experiments were carried out to compare oxygen fluxes and photobiological variables in Cassiopea sp. immature specimens pre-acclimated to low irradiance (PAR = 200 µmol photons m-2 s-1) and specimens rapidly exposed to higher irradiance levels (PAR = 500 µmol photons m-2 s-1). Comparable photosynthetic potential and high photosynthetic rates were measured at both irradiance values, as also shown by the rapid light curves. No significant differences were observed in terms of symbiont abundance between control and treated specimens. However, jellyfish kept at the low irradiance showed a higher content in chlorophyll a and c (0.76±0.51SD vs 0.46±0.13SD mg g-1 AFDW) and a higher Ci (amount of chlorophyll per cell) compared to jellyfish exposed to higher irradiance levels. The ratio between gross photosynthesis and respiration (P:R) was >1, indicating a significant input from the autotrophic metabolism. Cassiopea sp. specimens showed high photosynthetic performances, at both low and high irradiance, demonstrating high potential to adapt to sudden changes in light exposure. Such photosynthetic plasticity, combined with Cassiopea eurythermal tolerance and mixotrophic behavior, jointly suggest the upside-down jellyfish as a potentially successful invader in the scenario of a warming Mediterranean Sea.

Proximate composition, polyphenols, and antioxidant activity of solid state fermented peanut press cake.

The current research was led to assess the influence of solid-state fermentation (SSF) with Aspergillus oryzae (MTCC 3107) on polyphenols, antioxidant activities, and proximate composition from peanut press cake of variety HNG-10. Total phenolic, flavonoid, and tannin contents were calculated for polyphenols quantification whereas DPPH, ABTS, FRAP, and metal chelating assay were performed for antioxidant activity. Quantification of polyphenols was confirmed by High Performance Liquid Chromatography technique. Maximum value of total phenolic, flavonoid, and tannin content was found to be 25.55 µM/g GAE, 101.17 µM/g QE, and 245.33 µg/g TAE, respectively. The highest inhibition of free radicals scavenging was noticed on the 5th day of fermentation after that decreased gradually with the increase of fermentation time. Significant increase in fat, i.e. 7.05-12.80% and protein content i.e. 44.05-49.60% was observed. Significant difference in proximate composition of fermented and non-fermented press cake concluded that the progressive role of fermentation improved or transformed physico-chemical properties of substrates.

Development of a new atmospheric pressure plasmaspray ionization for ambient mass spectrometry.

A new atmospheric pressure ionization method, plasmaspray ionization, termed as PSI, was developed to be an alternative ambient ion source for mass spectrometry. It comprises a plasma jet device and a sample spray part. While the nonthermal plasma jet strikes the surface of stainless steel tube out of the spray capillary, the sprayed sample will be ionized with the assistant of auxiliary gas. Although PSI is a little bit more complex than electrospray ionization (ESI) in instrument, it shows both better linearity and higher sensitivity for organic compounds. For protein samples, it presents wider distributions of multiply charged ions and higher mass resolution without sacrificing any sensitivity. For the mechanism of PSI, the charge build-up process on the tip of capillary should play a key role for the ion formation, and the stimulated pulsed voltage on the flow tube will promote the ion aggregation speed until the charge density is high enough. PSI source contains the features of plasma ionization and ESI and can be considered as a novel combo bridging these techniques. These results reflect that this method of PSI can be applied and further developed as a versatile new ion source for a wild range of organic and biological samples.

Intact Protein Mass Spectrometry for Therapeutic Protein Quantitation, Pharmacokinetics, and Biotransformation in Preclinical and Clinical Studies: An Industry Perspective.

Recent advancements in immunocapture methods and mass spectrometer technology have enabled intact protein mass spectrometry to be applied for the characterization of antibodies and other large biotherapeutics from in-life studies. Protein molecules have not been traditionally studied by intact mass or screened for catabolites in the same manner as small molecules, but the landscape has changed. Researchers have presented methods that can be applied to the drug discovery and development stages, and others are exploring the possibilities of the new approaches. However, a wide variety of options for assay development exists without clear recommendation on best practice, and data processing workflows may have limitations depending on the vendor. In this perspective, we share experiences and recommendations for current and future application of mass spectrometry for biotherapeutic molecule monitoring from preclinical and clinical studies.

[Content of prostate small extracorporeal protein in the urine as an index in evaluating the therapeutic effect on chronic prostatitis].

OBJECTIVE: To investigate the clinical value of the prostate small extracorporeal protein (PSEP) level in the urine in evaluating the therapeutic effect on chronic prostatitis (CP). METHODS: Totally 188 CP patients were treated with minocycline and Ningmitai Capsules in our hospital and regularly returned for follow-up examination from November 2017 to November 2018. Based on the results of treatment after 4 and 8 weeks of medication, we divided the patients into a cured, an effective and an ineffective group and compared the contents of PSEP in the urine samples of the three groups of patients before and after treatment. RESULTS: Compared with the baseline, the PSEP content in the urine after 4 weeks of medication was decreased in the cured group (n = 20) (ï¼»3.63 ± 3.81ï¼½ vs ï¼»1.16 ± 0.41ï¼½ ng/ml, P < 0.05), effective group (n = 85) (ï¼»4.13 ± 4.05ï¼½ vs ï¼»2.97 ± 2.89ï¼½ ng/ml, P > 0.05) and ineffective group (n = 83) (ï¼»4.72 ± 2.98ï¼½ vs ï¼»3.74 ± 1.31ï¼½ ng/ml, P > 0.05), and so was that after 8 weeks of treatment in the cured group (n = 48) (ï¼»3.72 ± 3.51ï¼½ vs ï¼»0.89 ± 0.37ï¼½ ng/ml, P < 0.05), effective group (n = 106) (ï¼»4.37 ± 3.93ï¼½ vs ï¼»1.83 ± 0.71ï¼½ ng/ml, P < 0.05) and ineffective group (n = 34) (ï¼»4.61 ± 3.59ï¼½ vs ï¼»3.58 ± 1.15ï¼½ ng/ml, P > 0.05). CONCLUSIONS: The PSEP level in the urine can be used as an index for clinical evaluation of the therapeutic effect on chronic prostatitis.