Total Protein Extracts Research Articles

Efficient enhancement of the antimicrobial activity of Chlamydomonas reinhardtii extract by transgene expression and molecular modification using ionizing radiation

BackgroundIonizing radiation has been used for mutagenesis or material modification. The potential to use microalgae as a platform for antimicrobial production has been reported, but little work has been done to advance it beyond characterization to biotechnology. This study explored two different applications of ionizing radiation as a metabolic remodeler and a molecular modifier to enhance the antimicrobial activity of total protein and solvent extracts of Chlamydomonas reinhardtii cells.ResultsFirst, highly efficient transgenic C. reinhardtii strains expressing the plant-derived antimicrobial peptides, AtPR1 or AtTHI2.1, were developed using the radiation-inducible promoter, CrRPA70Ap. Low transgene expression was significantly improved through X-irradiation (12–50 Gy), with peak activity observed within 2 h. Protein extracts from these strains after X-irradiation showed enhanced antimicrobial activity against the prokaryotic bacterium, Pseudomonas syringae, and the eukaryotic fungus, Cryptococcus neoformans. In addition, X-irradiation (12 Gy) increased the growth and biomass of the transgenic strains. Second, C. reinhardtii cell extracts in ethanol were γ-irradiated (5–20 kGy), leading to molecular modifications and increased antimicrobial activity against the phytopathogenic bacteria, P. syringae and Burkholderia glumae, in a dose-dependent manner. These changes were associated with alterations in fatty acid composition. When both transgenic expression of antimicrobial peptides and molecular modification of bioactive substances were applied, the antimicrobial activity of C. reinhardtii cell extracts was further enhanced to some extent.ConclusionOverall, these findings suggest that ionizing radiation can significantly enhance the antimicrobial potential of C. reinhardtii through efficient transgene expression and molecular modification of bioactive substances, making it a valuable source of natural antimicrobial agents. Ionizing radiation can act not only as a metabolic remodeler of transgene expression in microalgae but also as a molecular modifier of the bioactive substances.

Extracellular Proteomic Profiling from the Erythrocytes Infected with Plasmodium Falciparum 3D7 Holds Promise for the Detection of Biomarkers.

Plasmodium falciparum (P. falciparum), which causes the most severe form of malaria, if left untreated, has 24h window in which it can cause severe illness and even death. The aim of this study was to create the most comprehensive and informative secretory-proteome possible by combining high-accuracy and high-sensitivity protein identification technology. In this study, we used Plasmodium falciparum 3D7 (Pf3D7) as the model parasite to develop a label-free quantification proteomic strategy with the main goal of identifying Pf3D7 proteins that are supposed to be secreted outside the infected erythrocytes in the spent media culture during the in-vitro study. The spent culture media supernatant was subjected to differential and ultra-centrifugation steps followed by total protein extraction, estimation, and in-solution digestion using trypsin, digested peptides were analyzed using Nano-LC coupled with ESI for MS/MS. MS/MS spectra were processed using Maxquant software (v2.1.4.0.). Non-infected erythrocytes incubated spent cultured media supernatant were considered as control. Out of discovered 38 proteins, proteins belonging to P. falciparum spp. were EGF-like protein (C0H544), Endoplasmic reticulum chaperone GRP170 (C0H5H0), Small GTP-binding protein sar1 (Q8I1S0), Erythrocyte membrane protein 1, PfEMP1 (Q8I639), aldehyde reductase (Q8ID61), Conserved Plasmodium proteins (Q8IEH3, Q8ILD1), Antigen 332, DBL-like protein (Q8IHN4), Fe-S cluster assembly protein (Q8II78), identified and chosen for further in-depth investigation. This study highlights the value of secretory Plasmodium proteins play crucial roles in various aspects of the disease progression and host-pathogen interactions which can serve as diagnostic markers for malaria infection.

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins of organelles, cellular signaling, bioenergetic metabolism and molecular chaperoning.

During the natural aging process, frailty is often associated with abnormal muscular performance. Although inter-individual differences exit, in most elderly the tissue mass and physiological functionality of voluntary muscles drastically decreases. In order to study age-related contractile decline, animal model research is of central importance in the field of biogerontology. Here we have analyzed wild type mouse muscle to establish a proteomic map of crude tissue extracts. Proteomics is an advanced and large-scale biochemical method that attempts to identify all accessible proteins in a given biological sample. It is a technology-driven approach that uses mass spectrometry for the characterization of individual protein species. Total protein extracts were used in this study in order to minimize the potential introduction of artefacts due to excess subcellular fractionation procedures. In this report, the proteomic survey of aged muscles has focused on organellar marker proteins, as well as proteins that are involved in cellular signaling, the regulation of ion homeostasis, bioenergetic metabolism and molecular chaperoning. Hence, this study has establish a proteomic reference map of a highly suitable model system for future aging research.

Identification of horn-derived animal medicines based on detection of keratin-derived characteristic peptides

Due to the highly stable structure of keratin, the extraction and dissolution steps of animal medicines rich in keratin are complex, which seriously restricts the detection efficiency and flux. Therefore, this study simplified the pre-treatment steps of horn samples and optimized the detection methods of characteristic peptides to improve the efficiency of identifying the specificity of horn-derived animal medicines. For detection of the characteristic peptides in horn-derived animal medicines treated with/without iodoace-tamide(IAA), the ion pair conditions of the characteristic peptides were optimized, and the retention time, intensity and other data of the specific peptides were compared between the samples treated with/without IAA. Two pre-treatment methods, direct enzymatic hydrolysis and total protein extraction followed by enzymatic hydrolysis, were used to prepare horn-derived animal medicine samples. The effects of different methods on the detection of specific peptides in the samples of Saiga antelope horn, water buffalo horn, goat horn, and yak horn were compared regarding the retention time of specific peptides and ion intensity. The results indicated that after direct enzymatic hydrolysis, the specific peptides in the samples without IAA treatment can be detected. Compared with the characteristic peptides in the samples treated with IAA, their retention time shifted back and the mass spectrometry response slightly decreased. The specific peptides of the samples without IAA treatment had good specificity and did not affect the specificity identification of horn-derived animal medicines. Overall, the process of direct enzymatic hydrolysis can be used to treat horn samples, omitting the steps of protein extraction and dithiothreitol and IAA treatment, significantly improving the pre-treatment efficiency without affecting the specificity identification of horn-derived animal medicines. This study provides ideas for quality research and standard improvement of horn-derived animal medicines.

A smartphone-based solution for fluorescence-powered protein aggregate detection

The level of protein aggregates can be used as an indicator of some diseases, including neurodegenerative disorders such as Alzheimer’s disease, and may be closely related to the efficacy of anti-cancer treatments. Moreover, strict control over protein aggregate levels is essential in pharmaceutical manufacturing to prevent potential side effects in the human body. In this study, we present a low-cost solution to evaluate protein aggregate levels in liquid samples using a smartphone-based fluorescence detection device. Initially, protein aggregate levels were assessed in bovine serum albumin samples. Then, this analysis was conducted using total protein extracts from breast cancer cells. The results obtained with the device were consistent with commercial spectrometer measurements, resulting in mean absolute errors of 0.098for bovine serum albumin samples and 0.152 for the proteins extracted from cancer cells, corresponding to differences of 9.8 % and 15.2 % between the commercial spectrometer and the developed device in the considered scales.

The benefits of experimental breeding research genotypes for the genetic resources of spring durum wheat

AbstractOur study aimed to incorporate 60 newly acquired spring durum wheat genotypes into the Prague Gene Bank collection, focusing on 54 experimental breeding research genotypes and their technological quality contributions for conservation and future use. We analyzed a total of 11 field and 6 technological parameters of grain quality. Additionally, we identified the composition of high molecular weight glutenin subunits (HMW-GSs) in 13 accessions with contrasting technological characteristics. Two different extraction procedures were employed to detect HMW-GSs: one based on total protein extraction and the other on gliadin pre-extraction. The tested parameters exhibited a significant range of variability, with the relative standard deviation ranging from 2.1% for starch content to 96.7% for the gluten index. Additionally, six breeding research genotypes exhibited high technological grain quality comparable to the two modern durum wheat cultivars. Two accessions: M90-99–2 and IG 142076 showed above-average Zeleny sedimentation values, probably positively influenced by the presence of HMW-GSs A1: (2*). Genotypes PI 675012 and IG 142039 displayed the highest grain stability in technological parameters. Both extraction methods also detected the specific subunit B1: (6 + 8*) in the genotype IG 142039. Preserving these genotypes thus represents a substantial expansion of genetic resources in the genebank.

Effects of arbuscular mycorrhizal fungi on the reduction of arsenic accumulation in plants: a meta-analysis.

Arsenic (As) accumulation in plants is a global concern. Although the application of arbuscular mycorrhizal fungi (AMF) has been suggested as a potential solution to decrease As concentration in plants, there is currently a gap in a comprehensive, quantitative assessment of the abiotic and biotic factors influencing As accumulation. A meta-analysis was performed to quantitatively investigate the findings of 76 publications on the impacts of AMF, plant properties, and soil on As accumulation in plants. Results showed a significant dose-dependent As reduction with higher mycorrhizal infection rates, leading to a 19.3% decrease in As concentration. AMF reduced As(V) by 19.4% but increased dimethylarsenic acid (DMA) by 50.8%. AMF significantly decreased grain As concentration by 34.1%. AMF also improved plant P concentration and dry biomass by 33.0% and 62.0%, respectively. The most significant reducing effects of As on AMF properties were seen in single inoculation and experiments with intermediate durations. Additionally, the benefits of AMF were significantly enhanced when soil texture, soil organic carbon (SOC), pH level, Olsen-P, and DTPA-As were sandy soil, 0.8%-1.5%, ≥7.5, ≥9.1 mg/kg, and 30-60 mg/kg, respectively. AMF increased easily extractable glomalin-related soil protein (EE-GRSP) and total glomalin-related soil protein (T-GRSP) by 23.0% and 28.0%, respectively. Overall, the investigated factors had significant implications in developing AMF-based methods for alleviating the negative effects of As stress on plants.

Alterations in HCN1 expression and distribution during epileptogenesis in rats

BackgroundThe hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) is predominantly located in key regions associated with epilepsy, such as the neocortex and hippocampus. Under normal physiological conditions, HCN1 plays a crucial role in the excitatory and inhibitory regulation of neuronal networks. In temporal lobe epilepsy, the expression of HCN1 is decreased in the hippocampi of both animal models and patients. However, whether HCN1 expression changes during epileptogenesis preceding spontaneous seizures remains unclear. ObjectiveThe aim of this study was to determine whether the expression of HCN1 is altered during the epileptic prodromal phase, thereby providing evidence for its role in epileptogenesis. MethodsWe utilized a cobalt wire-induced rat epilepsy model to observe changes in HCN1 during epileptogenesis and epilepsy. Additionally, we also compared HCN1 alterations in epileptogenic tissues between cobalt wire- and pilocarpine-induced epilepsy rat models. Long-term video EEG recordings were used to confirm seizures development. Transcriptional changes, translation, and distribution of HCN1 were assessed using high-throughput transcriptome sequencing, total protein extraction, membrane and cytoplasmic protein fractionation, western blotting, immunohistochemistry, and immunofluorescence techniques. ResultsIn the cobalt wire-induced rat epilepsy model during the epileptogenesis phase, total HCN1 mRNA and protein levels were downregulated. Specifically, the membrane expression of HCN1 was decreased, whereas cytoplasmic HCN1 expression showed no significant change. The distribution of HCN1 in the distal dendrites of neurons decreased. During the epilepsy period, similar HCN1 alterations were observed in the neocortex of rats with cobalt wire-induced epilepsy and hippocampus of rats with lithium pilocarpine-induced epilepsy, including downregulation of mRNA levels, decreased total protein expression, decreased membrane expression, and decreased distal dendrite expression. ConclusionsAlterations in HCN1 expression and distribution are involved in epileptogenesis beyond their association with seizure occurrence. Similarities in HCN1 alterations observed in epileptogenesis-related tissues from different models suggest a shared pathophysiological pathway in epileptogenesis involving HCN1 dysregulation. Therefore, the upregulation of HCN1 expression in neurons, maintenance of the HCN1 membrane, and distal dendrite distribution in neurons may represent promising disease-modifying strategies in epilepsy.

An efficient protocol for extracting thylakoid membranes and total leaf proteins from Posidonia oceanica and other polyphenol-rich plants

BackgroundThe extraction of thylakoids is an essential step in studying the structure of photosynthetic complexes and several other aspects of the photosynthetic process in plants. Conventional protocols have been developed for selected land plants grown in controlled conditions. Plants accumulate defensive chemical compounds such as polyphenols to cope with environmental stresses. When the polyphenol levels are high, their oxidation and cross-linking properties prevent thylakoid extraction.ResultsIn this study, we developed a method to counteract the hindering effects of polyphenols by modifying the grinding buffer with the addition of both vitamin C (VitC) and polyethylene glycol (PEG4000). This protocol was first applied to the marine plant Posidonia oceanica and then extended to other plants synthesizing substantial amounts of polyphenols, such as Quercus pubescens (oak) and Vitis vinifera (grapevine). Native gel analysis showed that photosynthetic complexes (PSII, PSI, and LHCII) can be extracted from purified membranes and fractionated comparably to those extracted from the model plant Arabidopsis thaliana. Moreover, total protein extraction from frozen P. oceanica leaves was also efficiently carried out using a denaturing buffer containing PEG and VitC.ConclusionsOur work shows that the use of PEG and VitC significantly improves the isolation of native thylakoids, native photosynthetic complexes, and total proteins from plants containing high amounts of polyphenols and thus enables studies on photosynthesis in various plant species grown in natural conditions.

Culture of Hoffa fat pad mesenchymal stem/stromal cells on microcarrier suspension in vertical wheel bioreactor for extracellular vesicle production

BackgroundMesenchymal stromal cells (MSCs) are increasingly employed in regenerative medicine approaches for their immunomodulatory and anti-inflammatory properties, which are encoded in their secretome including extracellular vesicles (EVs). The Hoffa fat pad (HFP) located infrapatellarly harbours MSCs that could assist in tissue homeostasis in osteoarthritic joints. Intraarticular injection therapies based on blood products could modulate the populations of released HFP-MSC-EVs in a quantitative manner.MethodsTo obtain amounts of HFP-MSC-derived EVs that allow pre-clinical evaluation, suitable EV production systems need to be developed. This work investigates the release of EVs from primary HFP-MSCs cultivated in a 3D environment using microcarrier suspension culture in a vertical wheel bioreactor in comparison to conventional 2D culture. To simulate an intraarticular blood product therapy, cultures were treated with citrate-anticoagulated platelet-rich plasma (CPRP) or hyperacute serum (hypACT) before EV collection. HFP-MSC-EVs are enriched via ultrafiltration and characterised via Western Blot, nanoparticle tracking analysis in scatter as well as fluorescence mode. EV potency was determined via RT-qPCR analysing the expression of type II and X collagen (COL2 and COL10), as well as inducible nitric oxide synthase (iNOS) in primary OA chondrocytes.ResultsBlood product supplementation elevated HFP-MSC metabolic activity as determined via XTT assay over the course of 14 days. 3D culture resulted in a roughly 100-fold EV yield compared to 2D culture and elevated number of EVs released per cell. Total protein content correlated with the EV concentration. While typical EV marker proteins such as CD9, CD63 or Alix were detected in total protein extracts, CD9 and CD73 colocalised on individual EVs highlighting their cell origin. The type of blood product treatment did not affect the size or concentration of EVs obtained from HFP-MSCs. Assessing potency of 3D culture EVs in comparison to 2D EVs revealed superior biological activity with regard to inhibition of inflammation, inhibition of chondrocyte hypertrophy and induction of cartilage-specific ECM production.ConclusionsHFP-MSCs proliferate in presence of human blood products indicating that animal serum in culture media can be avoided in the future. The culture of HFP-MSCs in the employed bioreactor was successfully used to generate quantities of EVs that could allow evaluation of HFP-MSC-EV-mediated effects in pre-clinical settings. In addition, EV potency of 3D EVs is superior to EVs obtained in conventional 2D culture flasks.

Extraction of proteins and location of the L-ype Voltage-Dependent Ca2+ channel (CaV1.2 α1c) in pig sperm extracted from fresh, frozen and permeated semen

Ion channels are protein structures located in the cell plasma membrane. Sperm cells require calcium ions (Ca2+) to carry out fundamental physiological processes, such as hypermotility, capacitation and acrosomal reaction. Protein extraction is the first, and most critical, stage of molecular tests (Western Blot, proteomic tests, etc). The implementation of these techniques in the study of sperm cells will allow us to understand their physiology (biomarkers) and ways of manipulation (refrigeration, cryopreservation, permeation using streptolysin O (SLO), etc.). The goal of this project was to determine whether if changes in the plasma membrane, brought on by cryopreservation or permeation with SLO, modify the Ca2+ CaV1.2 α1C channel in pig sperm cells. 2 ½ pig ejaculates were taken as working simples, and they were treated as follows: -Control (C): refrigerated semen (SR) 16°/24h; Treatment 1 (T1): SR with SLO T2: cryopreserved semen (SC); T3: SC, previously treated with SLO. It was determined that a better quantitation of proteins from the samples (p<0.001) is obtained with a (total) protein extraction buffer composed of SDS 2% + β-Mercaptoethanol 3%. The detection of the CaV 1.2 α1C protein was done through Western Bloting, using the Rabbit anti-CaV 1.2 α1C (USA, Sigma-AldrichTM) antigen. Better visibility of the protein band of the Ca2+ channel was achieved in the C and T1 experimental groups; whereas its’ presence was not observed in T2 and T3. It was determined that the Ca2+ CaV 1.2 α1C was present in the sperm cells, and that it deteriorates once the sperm cell is frozen and permeated with SLO, which is linked to the decrease of cell viability percentages. alibri font, size 10, and single line spacing.

Recognition of Immunoreactive Proteins in Leishmania infantum Amastigote-Like and Promastigote Using Sera of Visceral Leishmaniasis Patients: a Preliminary Study.

Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets.

Improved methods for total and chloroplast protein extraction from Cajanus species for two-dimensional gel electrophoresis and mass spectrometry.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6-7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research.

Mustard seed major allergen Sin a1 activates intestinal epithelial cells and also dendritic cells that drive type 2 immune responses.

Mustard seeds belong to the food category of mandatory labelling due to the severe reactions they can trigger in allergic patients. However, the mechanisms underlying allergic sensitization to mustard seeds are poorly understood. The aim of this work is to study type 2 immune activation induced by the mustard seed major allergen Sin a1 via the intestinal mucosa, employing an in vitro model mimicking allergen exposure via the intestinal epithelial cells (IECs). Sin a1 was isolated from the total protein extract and exposed to IEC, monocyte derived dendritic cells (DCs) or IEC/DC co-cultures. A system of consecutive co-cultures was employed to study the generic capacity of Sin a1 to induce type 2 activation leading to sensitization: IEC/DC, DC/T-cell, T/B-cell and stem cell derived mast cells (MCs) derived from healthy donors. Immune profiles were determined by ELISA and flow cytometry. Sin a1 activated IEC and induced type-2 cytokine secretion in IEC/DC co-culture or DC alone (IL-15, IL-25 and TSLP), and primed DC induced type 2 T-cell skewing. IgG secretion in the T-cell/B-cell phase was enhanced in the presence of Sin a1 in the first stages of the co-culture. Anti-IgE did not induce degranulation but promoted IL-13 and IL-4 release by MC primed with the supernatant from B-cells co-cultured with Sin a1-IEC/DC or -DC primed T-cells. Sin a1 enhanced the release of type-2 inflammatory mediators by epithelial and dendritic cells; the latter instructed generic type-2 responses in T-cells that resulted in B-cell activation, and finally MC activation upon anti-IgE exposure. This indicates that via activation of IEC and/or DC, mustard seed allergen Sin a1 is capable of driving type 2 immunity which may lead to allergic sensitization.

Biochemical and techno-functional investigation of argan press-cake proteins foreseeing food application

Argan press-cake (APC) is reported to contain a significant content of proteins, comparable to conventional sources of protein isolates, such as soybean. In this regard, the present study evaluated the biochemical and techno-functional properties of major APC proteins fractions, foreseeing food application. Albumins and globulins represented approximately 41% and 50% of total extractable proteins, respectively. Globulins were prone to aggregation, potentially by hydrophobic interactions, resulting in limited solubility in water in the range of pH 3 to pH 11. Nevertheless, the limited solubility of globulins in water at pH ≤ 9 could be improved, up to 8-fold, by pH12-shifting treatment, that is, unfolding the proteins structure at pH 12, followed by refolding at pH 9 or lower. pH12-shifted albumins and globulins presented similar surface activity in the range of pH 3 to pH 11 and were able to form and stabilize foams and emulsions. Comprehensively, both fractions reduced the interfacial tension and surface tension up to 8 mN/m and 48 mN/m, respectively, and formed stable emulsions, with a mean droplet volume diameter (d4,3) of approximately 16 μm, using rotor-stator homogenization. These results indicate that albumins and globulins extracted from argan by-products could have potential applications as techno-functional food ingredients.

An Efficient Protein Extraction Method from Astragalus armatus Willd. Roots for Proteomic Analysis

Abstract The first step of the proteomic study is the extraction and the success of this technique was based mainly on the choice of the best extraction. The purpose of this study was to determine the simplest and lowest-cost method of total protein extraction. Initially, various extraction methods were performed for protein extraction from roots of Astragalus armatus Willd. The methods employed were extraction by RIPA buffer, hypotonic buffer and distilled water. After determination of protein concentration by Bradford method and SDS PAGE electrophoretic analysis, the quantity and quality of extracted proteins using different protocols from A. armatus were determined and compared. The protein yield of RIPA buffer method was higher than the yields of hypotonic buffer method and distilled water method. The best protein patterns were produced by RIPA buffer method. The extract obtained by RIPA buffer was the optimal protocol for protein extraction.

Proteomic Analyses of the Mouse Brain.

Proteomics is a scientific field that aims to identify and characterize all proteins within a biological system, including their posttranslational modifications (PTMs), quantitative changes, and protein-protein interactions. Over the last two decades, proteomic approaches have been widely used in neuroscience research, providing multidimensional insights into the biology and pathology of the brain.Here, we present a basic protocol for profiling protein expression in the mouse brain, which involves total protein extraction, fractionation, digestion, and identification through liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This method is compatible with many prevalent techniques used for protein quantitation, PTM analysis, and protein-protein interaction mapping.

Unconventional Extraction Methods of Oleaginous Yeast Cell Pretreatment and Disruption

Extraction is one of the most commonly used methods for obtaining and purifying chemical compounds for commercial usage. The aim of this study was to evaluate the effect of unconventional permeabilization and cell disruption methods on the yield of lipid extraction from cells of the oleaginous yeast Yarrowia lipolytica. Batch cultures in a medium with molasses and waste post-frying oil were carried out. The biomass was subjected to pulsed electric field (PEF), high-pressure processing (HPP), ultrasounds (US), and several conventional processing techniques with chemical and mechanical agents (glass beads, acetone, Triton and Tween surfactants). The effectiveness of the applied methods, either on cell permeabilization or cell disruption, was investigated by analyzing the oil and total protein extraction yield and oil leaching efficiency, as well as by using microscope images. The PEF and US treatments proved to be effective permeabilization methods as a step of sample pretreatment for extraction. These unconventional physical methods could efficiently increase intracellular lipid extraction yield in solvent applications.

Tree growth and density enhanced, while diversity and spatial clustering reduced soil mycorrhizal C and N sequestration: Strong interaction with soil properties in northeastern China

In this paper, the effects of species diversity, tree growth, and spatial clustering on mycorrhizal carbon and nitrogen sequestration and the interaction of soil physicochemical properties in Northeast China were investigated. Based on 720 10 m ∗ 10 m plots in Harbin Experimental Forest Farm of Northeast Forestry University, we determined mycorrhizal biomarkers of easily extractable Glomalin-related soil protein (EEG) and total Glomalin-related soil protein (TG). Four plant diversity indices, seven structural metrics, and five soil properties were also measured. We found that: 1) The low tree diversity plots had 1.08–1.23 times higher TG, EEG, TG-N/TN (proportion of N in TG to TN), and TG-C/SOC (proportion of C in TG to SOC) than the high plots. 2) Tree diameter was negatively correlated with EEG and TG, but positively correlated with the EEG and TG contribution to soil TN and SOC. Soil EEG and TG were positively correlated with under-branch height and tree density. W (Uniform Angle Index, higher W indicates more clustering of tree distribution in the plot) was negatively correlated with the above four ratios and positively correlated with EEG/TG. 3) pH was the most powerful explainer for the GRSP variations (6.8 %, strongest negative association with GRSP/TN, R2 > 0.13), followed by soil electrical conductance (6.5 %, positive relation with TG, p < 0.05), AP (3.2 %). 4) Plant diversity mainly affected GRSP traits through the interaction with soils (0.07), tree growth and density directly increased TG, TG-N/TN, and TG-C/SOC, while tree spatial distribution directly reduced TG-N/TN. Our finding highlighted the important effects of tree diversity and forest structural traits on GRSP amount, carbon sequestration, and nutrient retentions, and could support glomalin-related forest soil management of temperate forests in the high-latitude northern hemisphere.

Influence of whey protein concentrates as a novel binder on physiochemical, textural and ultrastructural properties of restructured buffalo meat slices

SummaryThe present study was carried out to determine the effect of whey protein concentrates (WPC; 0%, 1%, 2% and 3%) as a novel binder for restructured buffalo meat slices (RBMS). 3% WPC had significantly (P &lt; 0.01) higher cooking yield, batter stability, water‐holding capacity and pH and lower (%) diameter shrinkage as compared to other formulations. The addition of WPC did not influence the total protein extractability, collagen content and collagen solubility of RBMS. However, the addition of 3% WPC significantly (P &lt; 0.01) improved the textural characteristics, instrumental colour and sensory scores and enhanced the meat protein–milk protein gel network. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed the matrix and gel networking of myofibrillar proteins and whey proteins, which caused more cohesiveness and compactness of the product. It was concluded that the addition of 3% WPC acted as an excellent binder for RBMS which can be used for commercial applications in the processed meat industry.