Mg Of Protein Research Articles

Antibiotics Resistance and PGPR Traits of Endophytic Bacteria Isolated in Arid Region of Morocco

This study aimed to characterize endophytic bacteria isolated from legume nodules and roots in the rhizosphere soils of Acacia trees in Morocco’s arid regions. The focus was on identifying bacterial strains with plant growth-promoting rhizobacteria (PGPR) traits and antibiotic resistance, which could enhance legume productivity under various abiotic stresses. Autochthonous legumes were used to harbor the endophytic bacteria, including chickpea (Cicer arietinum), faba bean (Vicia faba), lentil (Lens culinaris), and common bean (Phaseolus vulgaris). In a previous study, seventy-two isolates were obtained, and molecular characterization grouped them into twenty-two bacterial isolates. These twenty-two bacterial isolates were then further analyzed for their antibiotic resistance and key PGPR traits, such as phosphate solubilization, indole-3-acetic acid (IAA) production, and siderophore production. The results revealed that 86.36% of the isolates were resistant to erythromycin, 45.45% to ciprofloxacin, 22.73% to ampicillin-sulbactam, and 9.09% to tetracycline, with ciprofloxacin and tetracycline being the most effective. All isolates produced IAA, with HN51 and PN105 exhibiting the highest production at 6 µg of IAA per mg of protein. The other isolates showed varying levels of IAA production, ranging from moderate to low. Siderophore production, assessed using CAS medium, indicated that the strains PN121, LR142, LNR146, and HR26 exhibited high production, while the rest demonstrated moderate to low capacities. Additionally, 18.2% of the isolates demonstrated phosphate solubilization on YED-P medium, with PR135 and LNR135 being the most efficient, achieving solubilization indices of 2.14 and 2.13 cm, respectively. LR142 and LNR146 showed a moderate solubilization efficiency. Overall, these findings indicate that these isolated endophytic bacteria possess significant potential as biofertilizers, owing to their antibiotic resistance, IAA production, siderophore production, and phosphate solubilization abilities. These characteristics position them as promising candidates for enhancing legume growth under abiotic stress and contributing to sustainable agriculture in arid regions.

Continuous Flow Synthesis of Hexyl Laurate Using Immobilized Thermomyces Lanuginosus Lipase from Residual Babassu Mesocarp.

Brazil has one of the greatest biodiversities on the planet, where various crops play a strategic role in the country's economy. Among the highly appreciated biomasses is babassu, whose oil extraction generates residual babassu mesocarp (BM), which still needs new strategies for valorization. This work aimed to use BM as a support for the immobilization of Thermomyces lanuginosus lipase (TLL) in an 8.83 mL packed-bed reactor, followed by its application as a biocatalyst for the synthesis of hexyl laurate in an integrated process. Initially, the percolation of a solution containing 5 mg of TLL at 25 °C and flows ranging from 1.767 to 0.074 mL min-1 was investigated, where at the lowest flow rate tested (residence time of 2 h), it was possible to obtain an immobilized derivative with hydrolytic activity of 504.7 U g-1 and 31.7 % of recovered activity. Subsequent studies of treatment with n-hexane, as well as the effect of temperature on the immobilization process, were able to improve the activities of the final biocatalyst BM-TLLF, achieving a final hydrolysis activity of 7023 U g-1 and esterification activity of 430 U ⋅ g-1 against 142 U g-1 and 113.5 U g-1 respectively presented by the commercial TLIM biocatalyst. Desorption studies showed that the TL IM has 18 mg of protein per gram of support, compared to 4.92 mg presented by BM-TLL. Both biocatalysts were applied to synthesize hexyl laurate, achieving 98 % conversion at 40 °C within 2 h. Notably, BM-TLLF displayed exceptional recyclability, maintaining catalytic efficiency over 12 cycles. This reflects a productivity of 180 mg of product ⋅ h-1 U-1 of the enzyme, surpassing 46 mg h-1 U-1 obtained for TLIM. These results demonstrate the efficacy of continuous flow technology in creating a competitive and integrated process offering an exciting alternative for the valorization of residual lignocellulosic biomass.

Characterisation of seryl tRNA synthetase (srs-2) in Haemonchus contortus and Teladorsagia circumcincta

The aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76–93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The Km and Vmax for ATP were 3.9 ± 1.0 μM and 2.7 ± 0.1 μmol min−1 mg−1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins.

Neuronal tissue collection from intra-cranial instruments used in deep brain stimulation surgery for Parkinson’s disease with implications for study of alpha-synuclein

Alpha-synuclein (αSyn) forms pathologic aggregates in Parkinson’s disease (PD) and is implicated in mechanisms underlying neurodegeneration. While pathologic αSyn has been extensively studied, there is currently no method to evaluate αSyn within the brains of living patients. Patients with PD are often treated with deep brain stimulation (DBS) surgery in which surgical instruments are in direct contact with neuronal tissue; herein, we describe a method by which tissue is collected from DBS surgical instruments in PD and essential tremor (ET) patients and demonstrate that αSyn is detected. 24 patients undergoing DBS surgery for PD (17 patients) or ET (7 patients) were enrolled; from patient samples, 81.2 ± 44.8 µg of protein (n = 15), on average, was collected from surgical instruments. Light microscopy revealed axons, capillaries, and blood cells as the primary components of purified tissue (n = 3). ELISA assay further confirmed the presence of neuronal and glial tissue in DBS samples (n = 4). Further analysis was conducted using western blot, demonstrating that multiple αSyn antibodies are reactive in PD (n = 5) and ET (n = 3) samples; truncated αSyn (1–125 αSyn) was significantly increased in PD (n = 5) compared to ET (n = 3), in which αSyn misfolding is not expected (0.64 ± 0.25 vs. 0.25 ± 0.12, P = 0.046), thus showing that multiple forms of αSyn can be detected from living PD patients with this method.

Cellular and enzymatic features of thrombi in humans are vascular bed dependent

AbstractMechanisms behind vascular remodeling following thrombosis are unclear. Although acute arterial thrombosis in the cerebrovascular circulation has devastating consequences and requires immediate attention, the management of venous thromboembolism (VTE) varies significantly. Our goal was to determine the molecular signatures and cellular content of thrombus extracted using a catheter to gain insight into vascular remodeling. Twenty-five patients underwent catheter-directed thrombectomy (CDT); 13 for cerebrovascular accident (CVA), 8 for pulmonary embolism, and 4 for deep vein thrombosis. Protein and RNA were extracted from thrombi to enable immunoblotting, RNA sequencing, and quantification of gene expression. The time from symptom onset to thrombus extraction was 7.7 ± 1.9 hours for CVA and 109 ± 55 hours for VTE. Protein concentration, white blood cell content (monocytes), and red blood cell content were greater in venous thrombus than in arterial thrombus, whereas the platelet content was similar. Both venous and arterial thrombi contained several zinc endopeptidases belonging to the matrix metalloproteinase (MMP) family. MMP9 activity in venous thrombus was greater than arterial thrombus (61 ± 9 ng/mL per μg protein vs 25 ± 6 ng/mL per μg protein; P = .005). Arterial and venous thrombi displayed surprisingly different phenotypes, with biologically active enzymes promoting blood vessel remodeling and enzymatic activity proportional to thrombus age extracted from the veins. These mechanistic data may support the role of early CDT in venous circulation to avoid irreversible vascular remodeling.

A highly sensitive approach for the analysis of tyrosine phosphoproteome in primary T cells

Tyrosine phosphorylation, a common post-translational modification process for proteins, is involved in a variety of biological processes. However, the abundance of tyrosine-phosphorylated proteins is very low, making their identification by mass spectrometry (MS) is difficult; thus, milligrams of the starting material are often required for their enrichment. For example, tyrosine phosphorylation plays an important role in T cell signal transduction. However, the number of primary T cells derived from biological tissue samples is very small, and these cells are difficult to culture and expand; thus, the study of T cell signal transduction is usually carried out on immortalized cell lines, which can be greatly expanded. However, the data from immortalized cell lines cannot fully mimic the signal transduction processes observed in the real physiological state, and they usually lead to conclusions that are quite different from those of primary T cells. Therefore, a highly sensitive proteomic method was developed for studying tyrosine phosphorylation modification signals in primary T cells. To address the issue of the limited T cells numbers, a comprehensive protocol was first optimized for the isolation, activation, and expansion of primary T cells from mouse spleen. CD3+ primary T cells were successfully sorted; more than 91% of the T cells collected were well activated on day 2, and the number of T cells expanded to over 7-fold on day 4. Next, to address the low abundance of tyrosine-phosphorylated proteins, we used SH2-superbinder affinity enrichment and immobilized Ti4+affinity chromatography (Ti4+-IMAC) to enrich the tyrosine-phosphorylated polypeptides of primary T cells that were co-stimulated with anti-CD3 and anti-CD28. These polypeptides were resolved using nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Finally, 282 tyrosine phosphorylation sites were successfully identified in 1 mg of protein, including many tyrosine phosphorylation sites on the immunoreceptor tyrosine-based activation motif (ITAM) in the intracellular region of the T cell receptor membrane protein CD3, as well as the phosphotyrosine sites of ZAP70, LAT, VAV1, and other proteins related to signal transduction under costimulatory conditions. In summary, to solve the technical problems of the limited number of primary cells, low abundance of tyrosine-phosphorylated proteins, and difficulty of detection by MS, we developed a comprehensive proteomic method for the in-depth analysis of tyrosine phosphorylation modification signals in primary T cells. This protocol may be applied to map signal transduction networks that are closely related to physiological states.

Response of selected scion and rootstock grape (<i>Vitis </i>spp.) genotypes to induced drought stress

Climate change is expected to elevate drought frequency, straining agricultural freshwater resources. Developing drought-tolerant grapevine varieties is crucial. This study examined grape scion and rootstock genotypes under well-watered (WW) and induced-drought (ID) conditions. ID treatment reduced vine length by 11.34-35.15%, with Vitis parviflora, 110R, and Male Hybrid rootstocks showing superior growth. Root length increased under ID, indicating an adaptive moisture-seeking response. The ID treatment led to substantial reduction in leaf count and average leaf area, especially in Flame Seedless (27.71 and 19.07 cm2, respectively). Drought stress elevated chlorophyll a:b ratio, affecting chlorophyll degradation in different genotypes. Significant variations were observed in leaf and root iron (Fe) and zinc (Zn) contents. Enzyme activities particularly peroxidase and polyphenol oxidase especially rose under drought, particularly in V. parviflora (3.39 μM guaiacol min-1 mg-1 protein and 1.33EU/ml/min respectively) likely to be contributing in drought tolerance mechanism. Principal component analysis (PCA) highlighted impact of traits on genotypes, emphasizing V. parviflora, Male Hybrid and Pusa Navrang as superior drought stress tolerant genotypes. Genotype clustering confirmed distinct groupings, while, correlation analysis unveiled intricate trait interactions.

Plant Cadmium Toxicity and Biomarkers Are Differentially Modulated by Degradable and Nondegradable Microplastics in Soil.

The impact of microplastics (MPs) as emerging pollutants on plant heavy metal toxicity has been extensively reported in vegetable-soil systems over recent years. However, little attention has been given to cultivar variations between degradable and non-degradable MPs. This study investigated the effects of degradable polylactic acid (PLA) and nondegradable polypropylene (PP) MPs on plant growth and biomarker (malonaldehyde (MDA) and antioxidant enzymes) performance in Cd-contaminated arable soil. The results show that both types of MPs significantly impacted plant biomass and biomarker contents across all three Cd levels. The degree of impact was significantly sensitive to both the type and dose of MPs, as they reduced the soil pH and cation exchange capacity (CEC) while increasing soil dissolved organic carbon (DOC), microbial biomass carbon, and nitrogen. PP exhibited greater root growth inhibition and phytotoxicity at higher doses of 1% and 5% compared to PLA. Specifically, the highest MDA contents were 1.44 and 2.20 mmol mg-1 protein for shoots and roots, respectively, in the 5% PLA treatment under a 10.1 mg kg-1 Cd level, which were 1.22 and 1.18 times higher than those in corresponding treatments of 5% PP. Overall, PLA had less significant effects on plant phytotoxicity, Cd availability, and soil properties compared to PP. Regression pathway analysis indicated that MPs increased shoot Cd uptake by altering both soil physical-chemical and microbial characteristics. Among the soil variables, pH, CEC, and Cd bioavailability were found to play vital roles. Yet, no single variable acts alone in the mechanism for plant Cd uptake. PLAs are suggested to replace conventional non-biodegradable plastics to control environmental MP pollution, particularly in agricultural systems with higher Cd contamination. However, the long-term effects of the by-products generated during the biodegradation process require further investigation.

Rapid Protein-Ligand Affinity Determination by Photoinduced Hyperpolarized NMR.

The binding affinity determination of protein-ligand complexes is a cornerstone of drug design. State-of-the-art techniques are limited by lengthy and expensive processes. Building upon our recently introduced novel screening method utilizing photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR, we provide the methodological framework to determine binding affinities within 5-15 min using 0.1 mg of protein. The accuracy of our method is demonstrated for the affinity constants of peptides binding to a PDZ domain and fragment ligands binding to the protein PIN1. The method can also be extended to measure the affinity of nonphoto-CIDNP-polarizable ligands in competition binding experiments. Finally, we demonstrate a strong correlation between the ligand-reduced signals in photo-CIDNP-based NMR fragment screening and the well-established saturation transfer difference (STD) NMR. Thus, our methodology measures protein-ligand affinities in the micro- to millimolar range in only a few minutes and informs on the binding epitope in a single-scan experiment, opening new avenues for early stage drug discovery approaches.

Isolation and characterization of a β-galactosidase from Lactobacillus helveticus for industrial processing

In this study, a thermostable β-galactosidase from Lactobacillus helveticus (Lb. helveticus) OSU-PECh-4A has been isolated through diafiltration and size-exclusion chromatography. The enzyme consists of a heterodimer with a molecular mass of 110 kDa, with a small and large subunit of 36 kDa and 74 kDa, respectively. The Km and Vmax values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) hydrolysis were, respectively, 29.87 ± 1.05 mM, 1.88 ± 0.02 μmol D-glucose released per min per mg of protein, and 0.067 ± 0.003 mM, 1.70 ± 0.05 μmol o-nitrophenol (oNP) released per min per mg of protein. This β-galactosidase is significantly activated by Mg+2 (2–10 mM) and slightly inhibited by D-Glucose. The enzyme can also hydrolyze 57 ± 3% of lactose after 12 h of reaction at 45°C and under high lactose concentration. We propose that this enzyme provides a significant advantage from a practical and consumer point of view due to its origins as a probiotic source and improved features for important industrial applications, such as lactose hydrolysis and the potential to produce galacto-oligosaccharides (GOS).

Analysis of alpha-synuclein harvested from intracranial instruments used in deep brain stimulation surgery for Parkinson's disease.

Alpha-synuclein (αSyn) forms pathologic aggregates in Parkinson's disease (PD) and is implicated in mechanisms underlying neurodegeneration. While pathologic αSyn has been extensively studied, there is currently no method to evaluate αSyn within the brains of living patients. Patients with PD are often treated with deep brain stimulation (DBS) surgery in which surgical instruments are in direct contact with neuronal tissue; herein, we describe a method by which tissue is purified from DBS surgical instruments in PD and essential tremor (ET) patients and demonstrate that αSyn is robustly detected. 24 patients undergoing DBS surgery for PD (17 patients) or ET (7 patients) were enrolled; from patient samples, 81.2 ± 44.8 μg protein (n=15) is able to be purified, with immunoblot assays specific for αSyn reactive in all tested samples. Light microscopy revealed axons and capillaries as the primary components of purified tissue (n=3). Further analysis was conducted using western blot, demonstrating that truncated αSyn (1-125 αSyn) was significantly increased in PD (n=5) compared to ET (n=3), in which αSyn misfolding is not expected (0.64 ± 0.25 vs. 0.25 ± 0.12, P = 0.046), thus showing that pathologic αSyn can be reliably purified from living PD patients with this method.

Purification of Recombinant E. Coli Topoisomerase III for Structure-Based Drug Design Using Protein Crystallization

Type IA Topoisomerases are ubiquitous enzymes found throughout all life forms and species. These topoisomerases relieve the topographical constrains formed by DNA during processes like replication and transcription via a cleavage-religation mechanism performed through a catalytically active tyrosine residue in the primary structure of the enzyme. E. coli Topoisomerase III (EtopIII) is a type of Type IA topoisomerase, and its main function in the cell is as a decatenase, which means that it unlinks circular or intertwined pieces of genetic material and creates two unlinked segments of DNA from a singular linked chain. Structure-based determination of the enzyme’s three-dimensional structure via crystallization could be beneficial in finding a novel antibiotic drug that is able to inhibit this enzyme in vivo, a solution that is sorely needed in a world where antibiotic-resistant bacteria are an ever-growing problem. The crystallization of the enzyme can be undertaken by truncating a part of the C-terminal of the enzyme that makes it harder for it to crystallize and introduce a DNA/RNA ligand to observe its binding interactions with said substrate. For this to happen, the truncated recombinant mutant must be purified and concentrated to desirable levels for crystallization. The results of the experiment show that out of 4L of bacterial starting culture, a total of 34.57 mg of protein was able to be purified. This protein was then sent for crystallization experiments, of which results are still pending, but preliminary results indicate that the protein quickly aggregates in one of the RNA ligand conditions, which elicits a change in protocol for future purifications. Furthermore, results from a relaxation assay indicate that the protein exhibits some relaxation activity even after the truncation of the C-terminal chain. These results could expand the knowledge of this enzyme for future structure-based drug development.

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.

Dehydrodieugenol isolated from Ocotea cymbarum induces cell death in human breast cancer cell lines by dysregulation of intracellular copper concentration

In this work, two neolignans – dehydrodieugenol (1) and dehydrodieugenol B (2) – were isolated from leaves of Ocotea cymbarum (H. B. K.) Ness. (Lauraceae). When tested against two human breast cancer cell lines (MCF7 and MDA-MB-231), compound 1 was inactive (IC50 > 500 μM) whereas compound 2 displayed IC50 values of 169 and 174 μM, respectively. To evaluate, for the first time in the literature, the synergic cytotoxic effects of compounds 1 and 2 with ion Cu2+, both cell lines were incubated with equimolar solutions of these neolignans and Cu(ClO4)2·6H2O. Obtained results revealed no differences in cytotoxicity upon the co-administration of compound 2 and Cu2+. However, the combination of compound 1 and Cu2+ increases the cytotoxicity against MCF7 and MDA-MB-231 cells, with IC50 values of 165 and 204 μM, respectively. The activity of compound 1 and Cu2+ in MCF7 spheroids regarding the causes/effects considering the tumoral microenvironment were accessed using fluorescence staining and imaging by fluorescence microscopy. This analysis enabled the observation of a higher red filter fluorescence intensity in the quiescence zone and the necrotic core, indicating a greater presence of dead cells, suggesting that the combination permeates the spheroid. Finally, using ICP-MS analysis, the intracellular copper disbalance caused by mixing compound 1 and Cu2+ was determined quantitatively. The findings showcased a 50-fold surge in the concentration of Cu2+ compared with untreated cells (p > 0.0001) – 18.7 ng of Cu2+/mg of proteins and 0.37 ng of Cu2+/mg of protein, respectively. Conversely, the concentration of Cu2+ in cells treated with compound 1 was similar to values of the negative control group (0.29 ng of Cu2+/mg of protein). This alteration allowed us to infer that compound 1 combined with Cu2+ induces cell death through copper homeostasis dysregulation.

L-cysteine-coated magnetite nanoparticles as a platform for enzymes immobilization: Amplifying biocatalytic activity of Candida antarctica Lipase A

This study presents the synthesis of magnetite nanoparticles coated with L-cysteine (Fe3O4@LC) and their subsequent utilization as a support matrix for the immobilization of Candida antarctica Lipase A (CALA). The immobilization process involved physical interactions and covalent bonding mediated by glutaraldehyde. Comprehensive characterization was conducted using techniques including X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and vibrating sample magnetometry (VSM), providing compelling evidence of a successful synthesis. Under optimized conditions, employing 1 mg of protein per gram of support, pH 7 in a 25 mM phosphate buffer, and a 5 h reaction at 25 °C, immobilization on glutaraldehyde-activated support achieved an impressive yield of 85.0 % ± 2.6, accompanied by a specific activity of 212.5 ± 1.3 U/g, outperforming the physical adsorption approach. Remarkably, the immobilized enzyme exhibited higher activity than the free enzyme at alkaline and acidic pH levels. Furthermore, thermal and pH inactivation studies revealed that the biocatalyst's half-life exceeded that of free CALA by more than 8 times at pH 10. These results underscore the potential of the Fe3O4@LC-GLU-CALA system as a robust biocatalytic matrix with promising applications in biodiesel production, ester synthesis, and pharmaceutical manufacturing.

Leptin Induces Evidence of Placental Mitochondrial Dysfunction in a Mouse Model of Preeclampsia

Preeclampsia (PE) affects 5-10% of pregnancies worldwide that increase maternal and fetal morbidity and mortality. PE is often characterized by hypertension, endothelial dysfunction, and restriction of intrauterine fetal growth. Our lab established that mid-gestation leptin infusion in pregnant mice induces a model of PE, notably reducing fetal growth in late gestation. Clinical studies indicate that PE patients demonstrate elevated levels of leptin compared to healthy pregnancies as well as present with evidence of placental mitochondrial dysfunction. Therefore, we hypothesize that mid-late gestation leptin infusion induces placental mitochondrial dysfunction in pregnant mouse placenta. We infused timed-pregnant C57/Bl6 mice with saline (sham) or leptin (0.9 mg/kg/day) via s.c. osmotic minipump from gestation day (GD)11-18 (n=4-8). We collected placentas on GD18 for analysis. We isolate mitochondria from mouse placenta via differential centrifugation, after douncing pulverized tissue. From the isolated mitochondria, we assessed morphology via electron microscopy (EM) and performed amplex red assay for reactive oxygen species (ROS) measurements and tetramethyl rhodamine methyl ester (TMRM) dye was used to measure membrane potential. Pairwise comparisons were by student’s t-test. Our data indicates that leptin decreased membrane potential in placenta mitochondria compared to the sham placentas (1466±16.42 sham vs 1353±32.48 leptin A.U./mg of protein, **p<0.01). In comparison, there were no differences in H2O2 production between the sham and leptin-infused placental mitochondrial isolations (7381±125.2 leptin vs 7311±180.3 sham A.U./mg of protein). EM analysis of one sham and one leptin placenta indicated an increase in mitophagy in the leptin-infused placenta slices as well as decreased mitochondrial circularity, suggesting that leptin increases mitochondrial death. In summary, these data indicate that leptin induces mitochondrial dysfunction which in turn may contribute to placental dysfunction and reduced uteroplacental fetal growth in PE. 1R01HL169576, 4R00HL146948. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Predicting the outcome of the buckwheat oral challenge test: a first evaluation assuming a single serving of boiled buckwheat noodles.

Background. Global increase in buckwheat consumption has led to a surge in buckwheat allergy reports. However, studies scrutinizing the predictive accuracy of buckwheat-specific immunoglobulin E (IgE) antibody levels in correlation with symptom manifestation remain limited. A critical concern is the discrepancy between the total buckwheat amount featured in prior studies and the quantity consumed per occasion. We aimed to determine open Oral Food Challenge (OFC) positivity rates with buckwheat, using a single serving of boiled buckwheat noodles, and assess the predictability of positive responses using buckwheat-specific IgE levels. Methods. Patients aged 20 years or younger, suspected of buckwheat allergy, were subjected to an OFC involving consumption of 100 g (4,800 mg of protein) of boiled buckwheat noodles for those under six years, and 200 g (9,600 mg of protein) for those six years or older. The predictive accuracy of the OFC, corresponding with buckwheat-specific IgE antibody levels, was evaluated using Receiver Operating Characteristic (ROC) analysis. Results. Our study involved 80 patients who undertook a buckwheat OFC. Among these, 14 (17.5%) tested positive for a buckwheat allergy, with 3 (3.8%) developing anaphylaxis. The comparative analysis of buckwheat-specific IgE antibody levels did not offer a reliable predictive measure for OFC outcomes. However, a past history of symptom manifestation following buckwheat consumption was significantly correlated with a positive OFC. Conclusions. Forecasting OFC outcomes based on buckwheat-specific IgE antibody levels poses a challenge, even when taking into account the total quantity of buckwheat that can be consumed in a single occasion.

Improved performance of Pseudomonas fluorescens lipase immobilized on a magnetic mesoporous solvothermal hybrid-nanocarrier and its application in ultrasonication assisted estolide synthesis

The exponential growth of the biotechnological market necessitates the exploring novel, economically viable materials for enzyme immobilization to enhance the biocatalyst performance. This study involves preparing a hybrid carrier with a magnetic mesoporous nature, using milled rice husk (MRH) and multi-walled carbon nanotubes (MWCNTs) via a one-pot solvothermal process at 200 °C for 6 h. The carrier, rich in-NH2 groups, was chemically bonded to Pseudomonas fluorescens lipase (PFL) using EDC/NHS cross-linkers. The synthesized support achieved a maximum PFL loading of 94.92 mg of protein per gram of support. Moreover, the maximum activity recovery was achieved at 1196 %, under the optimal conditions: immobilization temperature 25℃, pH 9.0, immobilization time 2 h and PFL mass concentration 4 mg/mL. Subsequently, the derived enzyme was used to catalyze ricinoleic acid esterification to synthesize estolide under ultrasound-assisted conditions. The optimized principal parameters for reaction temperature, reaction time, and immobilized lipase loading were respectively determined to be 48.99 °C, 1.60 h, and 3.09 % via response surface methodology. Ricinoleic acid conversion reached 65.23 % under the optimal conditions with the assistance of 40 % power and 40 kHz ultrasonic frequency. Furthermore, remarkable operational stability was also observed, with a retention rate of 49.28 %, even after 10 consecutive batches. This study demonstrates promising prospects for industrial-scale application using ultrasound as a fast and ecologically friendly procedure.

SYNERGISTIC ANTI-TUMOUR EFFECT OF DOXORUBICIN-HYDROCHLORIDE WITH CRM197, AN INHIBITOR OF HB-EGF, IN SQUAMOUS-CELL CARCINOMA

Recent studies have shown that cross reacting material (CRM197), a nontoxic variant of diphtheria toxin, may play an important role in treating cancers with poor prognoses. Doxorubicin-hydrochloride (DOX) is an antineoplastic prescription medicine for the treatment of certain types of cancer. Considering that CRM197 is a known carrier in targeted delivery, CRM197-DOX complexes might be a step towards targeted therapy and reduced overall toxicity. Aim. This study aims to explore the potential to inhibit the growth of tumour cells. To achieve that goal, we evaluated the usage of CRM197-DOX complexes in squamous carcinoma cell line A431 and compared it with the effect on other immortalised cell lines. The methods used in this research include derivation and purification of recombinant CRM197 through immobilized-metal affinity chromatography and electrophoresis in a denaturing polyacrylamide gel. DOX-loaded CRM197 complexes were formed by the addition of a 5 μM solution of doxorubicin to 0.5 μg of protein with further dialysis in PBS. The cell viability assay was conducted using free CRM197 at different concentrations, DOX, CRM197 with DOX, as well as DOX-loaded CRM197. DOX-loaded CRM197 along with CRM197 with the addition of DOX showed significant differences in cell viability compared to control wells. CRM197- Results. DOX complexes have an evident inhibitory effect on epidermoid carcinoma cell growth and can be used as treatment against epithelial tumours, with CRM197 as a promising carrier for targeted drug delivery. Conclusions. CRM197-DOX complexes show evident inhibition of epidermoid carcinoma cell growth and can be used as treatment against epithelial tumours, especially those overexpressing the proHB-EGF and its receptors, EGFR1 and EGFR4, with CRM197 as a promising carrier for targeted drug delivery.

The tumour-derived extracellular vesicle proteome varies by endometrial cancer histology and is confounded by an obesogenic environment.

Endometrial cancer, the most common gynaecological cancer worldwide, is closely linked to obesity and metabolic diseases, particularly in younger women. New circulating biomarkers have the potential to improve diagnosis and treatment selections, which could significantly improve outcomes. Our approach focuses on extracellular vesicle (EV) biomarker discovery by directly profiling the proteome of EVs enriched from frozen biobanked endometrial tumours. We analysed nine tissue samples to compare three clinical subgroups-low BMI (Body Mass Index) Endometrioid, high BMI Endometrioid, and Serous (any BMI)-identifying proteins related to histological subtype, BMI, and shared secreted proteins. Using collagenase digestion and size exclusion chromatography, we successfully enriched generous quantities of EVs (range 204.8-1291.0µg protein: 1.38×1011-1.10×1012 particles), characterised by their size (∼150nm), expression of EV markers (CD63/81), and proposed endometrial cancer markers (L1CAM, ANXA2). Mass spectrometry-based proteomic profiling identified 2075 proteins present in at least one of the 18 samples. Compared to cell lysates, EVs were successfully depleted for mitochondrial and blood proteins and enriched for common EV markers and large secreted proteins. Further analysis highlighted significant differences in EV protein profiles between the high BMI subgroup and others, underlining the impact of comorbidities on the EV secretome. Interestingly, proteins differentially abundant in tissue subgroups were largely not also differential in matched EVs. This research identified secreted proteins known to be involved in endometrial cancer pathophysiology and proposed novel diagnostic biomarkers (EIF6, MUC16, PROM1, SLC26A2).