High Protein Research Articles

High Protein Milk Affected the Increase of the Hb Levels of Third-trimester of Pregnancy with Anemia

Anemia is a risk factor that contributes to 50% of all maternal deaths. The main factors causing anemia are low iron intake and infection. Protein intake, especially animal protein intake, helps increase iron absorption. This study aims to determine the effect of providing high protein milk intake on increasing hemoglobin levels in pregnant women in the third trimester with iron deficiency. This type of research is Pre-Experimental with One Group Pretest-Posttest Design. The population in this study was all third-trimester of pregnant women with pale faces in the of the Gondanglegi Public Health Center Kabupaten Malang. The sample was 33 people taken by using the total sampling technique. The measuring test was the Wilcoxon Marked Rank Test. The examination results showed that the average hemoglobin level before treatment was 10.1 gr/dl, and after treatment was 11.1 gr/dl. The results showed that there was an effect of giving high-protein milk on increasing hemoglobin levels in third-trimester pregnant women with pale skin in Gondanglegi Health Center (p-value = 0.000). This study concludes that milk protein consumption basically affects the increase in hemoglobin levels in pregnant women. It is recommended for pregnant women who get anemia to focus on their food intake, especially protein consumption.

Looking back at the TEDDY study: lessons and future directions.

The goal of theTEDDY (The Environmental Determinants of Diabetes in the Young) study is to elucidate factors leading to the initiation of islet autoimmunity (first primary outcome) and those related to progression to type 1 diabetes mellitus (T1DM; second primary outcome). This Review outlines the key findings so far, particularly related to the first primary outcome. The background, history and organization of the study are discussed. Recruitment and follow-up (from age 4 months to 15 years) of 8,667 children showed high retention and compliance. End points of the presence of autoantibodies against insulin, GAD65, IA-2 and ZnT8 revealed the HLA-associated early appearance of insulin autoantibodies (1-3 years of age) and the later appearance of GAD65 autoantibodies. Competing autoantibodies against tissue transglutaminase (marking coeliac disease autoimmunity) also appeared early (2-4 years). Genetic and environmental factors, including enterovirus infection and gastroenteritis, support mechanistic differences underlying one phenotype of autoimmunity against insulin and another against GAD65. Infant growth and both probiotics and high protein intake affect the two phenotypes differently, as do serious life events during pregnancy. As the end of the TEDDY sampling phase is approaching, major omics approaches are in progress to further dissect the mechanisms that might explain the two possible endotypes of T1DM.

Lipid Priming of Adipose Mesenchymal Stromal Cells with Docosahexaenoic Acid: Impact on Cell Differentiation, Senescence and the Secretome Neuroregulatory Profile.

Priming strategies that improve the functionality of MSCs may be required to address issues limiting successful clinical translation of MSC therapies. For conditions requiring high trophic support such as brain and spinal cord injuries, priming MSCs to produce higher levels of trophic factors may be instrumental to facilitate translation of current MSC therapies. We developed and tested a novel molecular priming paradigm using docosahexaenoic acid (DHA) to prime adipose tissue-derived mesenchymal stromal cells (ASCs) to enhance the secretome neuroregulatory potential. Comprehensive dose-response and time-course assays were carried to determine an optimal priming protocol. Secretome total protein measurements were taken in association with cell viability, density and morphometric assessments. Cell identity and differentiation capacity were studied by flow cytometry and lineage-specific markers. Cell growth was assessed by trypan-blue exclusion and senescence was probed over time using SA-β-gal, morphometry and gene expression. Secretomes were tested for their ability to support differentiation and neurite outgrowth of human neural progenitor cells (hNPCs). Neuroregulatory proteins in the secretome were identified using multiplex membrane arrays. Priming with 40µM DHA for 72h significantly enhanced the biosynthetic capacity of ASCs, producing a secretome with higher protein levels and increased metabolic viability. DHA priming enhanced ASCs adipogenic differentiation and adapted their responses to replicative senescence induction. Furthermore, priming increased concentrations of neurotrophic factors in the secretome promoting neurite outgrowth and modulating the differentiation of hNPCs. These results provide proof-of-concept evidence that DHA priming is a viable strategy to improve the neuroregulatory profile of ASCs.

Augmenting Protein Degradation Capacity of PROTAC through Energy Metabolism Regulation and Targeted Drug Delivery.

The ubiquitin-proteasome system (UPS) is responsible for degrading over 70-80% of cellular proteins. Consequently, proteolysis-targeting chimeras (PROTACs) are developed to induce the ubiquitination and subsequent degradation of proteins of interest (POIs) by the UPS. To amplify the therapeutic efficacy of PROTACs, energy metabolism regulation is first harnessed to boost UPS function in tumor cells. Proteomic and ubiquitinome analyzes reveal that total ubiquitinated proteins and proteasome activity are significantly increased in 143B and MDA-MB-231 tumor cells following fasting-mimicking diet (FMD) treatment. As a result, the degradation efficiency of PROTACs targeting focal adhesion kinase (FAK-P) or bromodomain-containing protein 4 (BRD4-P) is significantly enhanced in FMD-treated 143B and MDA-MB-231 tumor cells. Then, silica-coated iron oxide nanoparticles are developed modified with tumor cell membranes for targeted delivery of PROTACs. Magnetic resonance imaging (MRI) and fluorescence imaging confirm that nanocarriers significantly improve the delivery efficiency of PROTACs in FMD-treated 143B or MDA-MB-231 tumors. In vivo studies demonstrate that the antitumor efficacy of FAK-P and BRD4-P is greatly augmented when combined with targeted delivery and FMD treatment. Overall, this study presents a strategy to enhance the efficacy of PROTACs in cancer therapy.

From Waste to Protein: Optimizing Rice Husk Utilization for Sustainable Single Cell Protein Production

The increasing global demand for protein has prompted a search for sustainable and cost-effective alternatives to traditional animal and dairy sources. Single-cell protein (SCP) produced from microorganism’s offers a promising solution due to its high protein content and rapid growth rates. This study addresses the challenge of utilizing rice husk, an abundant yet underutilized agricultural waste in Nigeria, as a substrate for SCP production. Through optimization of the production of SCP using Bacillus sp. AT-b3 and Bacillus sp. CMF 12 as model organisms. Molecular identification through 16S rRNA sequencing confirmed Bacillus sp. AT-b3 and Bacillus sp. CMF 12 as the most potent isolates for SCP production. Optimization studies were conducted using the One-Factor-at-a-Time (OFAT) method to determine the ideal fermentation conditions. The results showed that the optimal temperature for SCP production was 40 °C, with Bacillus sp. AT-b3 demonstrating superior production efficiency. pH optimization revealed that neutral pH (pH 7) was ideal for maximizing SCP production, with Bacillus spp. AT-b3 outperforming Bacillus sp. CMF 12 at this pH. Substrate concentration studies indicated that 2.0% was optimal for SCP production, and incubation time optimization indicated 48 hours as the optimal period for maximum yield. Amino acid profiling of the SCP produced showed significant variations between the two isolates. Bacillus sp. CMF 12 was richer in essential amino acids like Arginine and Methionine, while Bacillus sp. AT-b3 had a higher Glycine content at (p<0.05). The findings of this study suggest that both strains have potential applications in nutritional supplements, with Bacillus sp. AT-b3 being particularly suited for industrial-scale SCP production. This study concludes that Bacillus sp. AT-b3 is an efficient SCP producer under optimal conditions of neutral pH, moderate temperature, and appropriate substrate concentration. Further research is recommended to explore pilot-scale production, alternative substrates, and comprehensive safety assessments.

Risk Classification of Patients With Advanced Urothelial Carcinoma Treated With Enfortumab Vedotin.

Enfortumab Vedotin (EV) is a widely used antibody-drug conjugate for patients with advanced urothelial carcinoma (UC) who have previously been treated with platinum-based chemotherapy and immune checkpoint inhibitors. However, limited information is currently available on prognostic factors and risk classification. Therefore, the present study attempted to identify clinical factors that predict outcomes in patients with advanced UC treated with EV and to develop a novel risk classification model. We conducted a multicenter retrospective study including patients with advanced UC treated with EV. Oncological outcomes were assessed using progression-free survival (PFS) and overall survival (OS), and prognostic factors for PFS and OS were investigated. We then examined the usefulness of risk classification based on the prognostic factors identified. Median PFS and OS were 7.1 and 16.3 months, respectively. High C-reactive protein levels (CRP level ≥0.5 mg/dl) and hypercalcemia (corrected calcium level >10.2 mg/dl) were identified as prognostic factors for PFS (p=0.012 and p=0.003, respectively) and OS (p=0.035 and p<0.001, respectively). We then divided patients into three risk groups: no prognostic factors group, one prognostic factor group, and two prognostic factors group. Significant differences were observed in PFS and OS among the three groups (p<0.001 and p<0.001, respectively) and c-indices were 0.766 for PFS and 0.800 for OS. The risk classification using CRP and hypercalcemia is useful for predicting the outcomes of patients with advanced UC treated with EV.

Thrombocytopenia With High C-reactive Protein in Myeloma Patients Treated With Proteasome Inhibitor and/or Immunomodulatory Drugs.

Background/Aim: Thrombocytopenia is a poor prognostic factor in patients with myeloma; however, the factors associated with thrombocytopenia have not been extensively discussed. This study aimed to investigate the clinical significance of thrombocytopenia, defined as 130×103/μl or less, in patients with newly diagnosed multiple myeloma (NDMM) treated with proteasome inhibitors and/or immunomodulatory drugs. This is a retrospective review of medical records of myeloma patients treated between 2000 and 2021. A total of 241 patients were included in this study, with a median age of 72 years. Overall survival (OS) and time to next treatment (TTNT) were assessed using Kaplan-Meier analysis and Cox regression analysis. Prognostic factors were evaluated by univariate and multivariate analyses. The incidence of thrombocytopenia was 17.8%. In the median follow-up period of 46.6 months, OS and TTNT in the thrombocytopenia group were significantly shorter than those in the non-thrombocytopenia group using multivariate analysis (p<0.001 and p<0.001). C-reactive protein (CRP) level was not associated with thrombocytopenia, and high CRP predicted short OS and TTNT independently from thrombocytopenia. When the low (neither thrombocytopenia nor high CRP), intermediate (either thrombocytopenia or high CRP), and high (thrombocytopenia and high CRP) risk groups were defined, the OS and TTNT among these groups showed significant differences; the hazard ratios for survival in the high and intermediate risk groups were 7.022 and 2.598, and for TTNT, they were 4.216 and 1.887, respectively, compared to the low-risk group. Thrombocytopenia was associated with the activity of NDMM and predicted prognosis in NDMM. When combined with high CRP levels, thrombocytopenia serves as a new indicator of poor prognosis in these patients.

Unpalatable solutions: Consumer resistance towards Insect‐Based foods is moderated by uncertainty avoidance

AbstractTraditional livestock meat poses sustainability challenges, while insect proteins offer a low‐impact alternative. Cultural barriers, such as uncertainty avoidance, hinder widespread acceptance of insect‐based foods, despite a growing market. Research on consumer attitudes and purchase intentions towards insect‐based foods yields mixed results, prompting a meta‐analysis to explore cultural influences and contextual factors. This meta‐analysis investigates consumers’ willingness to try insect‐based food products. Analysis of 24 publications (97 studies, with 13,609 observations; participant Mage = 34.6), supports the notion that consumers around the world are not readily willing to adopt insect‐based food products. Moderating this effect, at the country level, high uncertainty avoidance negatively affects consumers’ intentions to try insect‐based products. Two follow‐up studies replicate the findings of the meta‐analysis, incorporating an individual‐level assessment of the moderating effect of uncertainty avoidance, and testing the mediating effect of food neophobia on consumers’ (un)willingness to adopt insect‐based food products. A third study provides evidence as how to buffer the negative reactions of consumers towards insect‐based foods: after sensory evaluation of a cricket‐flour nacho in a laboratory setting, participants were more willing to try it and generally evaluated the product and the experience more favorably, compared to their previously stated expectations. Theoretical contributions and practical implications for marketing strategy and communication, to overcome the negative perceptions related to insect‐based food, along with limitations and future research directions, are discussed.

Dextran-enriched pea-based ingredient from a combined enzymatic and fermentative bioprocessing. Design of an innovative plant-based spread.

In this study a plant-based spread was developed using dextran-enriched ingredients derived from pea flours, supplemented with defatted durum wheat germ and almond flour. Optimization of fermentation with Leuconostoc pseudomesenteroides DSM 20193, both with and without enzymatic hydrolysis, aimed to enhance exopolysaccharide production and the nutritional value of pea flours. Best results were achieved through enzymatic hydrolysis with Veron PS protease followed by fermentation at 25°C, resulting in elevated dextran levels and increased peptides and total free amino acid concentration in green and yellow pea-based ingredients. The yellow pea-based ingredient was selected for the final plant-based spread formulation, blended at 35% w/w, with 45% w/w defatted durum wheat germ, and 20% w/w almond flour. The resultant spread exhibited elastic and solid-like characteristics like milk-based spreadable cheese and yogurt, boasting 'high protein' (12.49 g/100g) and 'high fiber' (11.01 g/100g) designations. It maintained chemical, biochemical, and microbiological stability over a 10-day shelf-life under refrigerated conditions. Sensory evaluation confirmed the acceptability of the plant-based spread (PBS), highlighting a well-balanced aroma and a grainy, adhesive texture. This research underscores the potential of an integrated approach utilizing food-grade enzymes and fermentation for the in-situ production of dextran to create innovative, clean label, and plant-based foods.

The Commercial Application of Insect Protein in Food Products: A Product Audit Based on Online Resources.

Insect protein has received considerable attention as an alternative to conventional animal proteins with its high nutritional contents and eco-friendly credentials. Exploring commercially available insect-protein-enhanced foods, this study aims to profile and compare such products in the ultra-processed category with products protein-enhanced with dairy (e.g., milk and whey) and plants (e.g., pea and rice). A global product audit was conducted drawing from English-language online retail portals to determine the product formats and statistically compare their nutritional contents with products fortified with non-insect proteins. The results show that four categories-flour/powder, pasta/noodle, starch-based snacks (e.g., chips, crackers, and cookies), and energy bars-are involved with food enhanced with insect protein. Flour/powder and pasta/noodles with insects demonstrated comparable protein contents to non-insect equivalents, highlighting insects' potential as effective protein sources. However, insect protein's performances in snacks and energy bars were less favourable, with significantly lower protein contents compared to products enhanced with non-insect sources. This may be attributed to the high fat content of insects, which may also contribute to undesirable flavours in complex foods, limiting their usage. The study highlights the need for industry innovation and scientific collaboration to overcome the challenges to widely applying insects as food ingredients, offering benefits for both the industry and consumers.

Structural duality enables a single protein to act as a toxin–antidote pair for meiotic drive

In sexual reproduction, selfish genetic elements known as killer meiotic drivers (KMDs) bias inheritance by eliminating gametes that do not carry them. The selective killing behavior of most KMDs can be explained by a toxin-antidote model, where a toxin harms all gametes while an antidote provides resistance to the toxin in carriers. This study investigates whether and how the KMD element tdk1 in the fission yeast Schizosaccharomyces pombe deploys this strategy. Intriguingly, tdk1 relies on a single protein product, Tdk1, for both killing and resistance. We show that Tdk1 exists in a nontoxic tetrameric form during vegetative growth and meiosis but transforms into a distinct toxic form in spores. This toxic form acquires the ability to interact with the histone reader Bdf1 and assembles into supramolecular foci that disrupt mitosis in noncarriers after spore germination. In contrast, Tdk1 synthesized during germination of carrier spores is nontoxic and acts as an antidote, dismantling the preformed toxic Tdk1 assemblies. Replacement of the N-terminal region of Tdk1 with a tetramer-forming peptide reveals its dual roles in imposing an autoinhibited tetrameric conformation and facilitating the assembly of supramolecular foci when autoinhibition is released. Moreover, we successfully reconstituted a functional KMD element by combining a construct that exclusively expresses Tdk1 during meiosis ("toxin-only") with another construct that expresses Tdk1 specifically during germination ("antidote-only"). This work uncovers a remarkable example of a single protein employing structural duality to form a toxin-antidote pair, expanding our understanding of the mechanisms underlying toxin-antidote systems.

Effect of dietary protein on serum hepcidin and iron in adults with obesity and insulin resistance: A randomized single blind clinical trial.

Background and AimsBoth obesity and iron deficiency are public health problems. The association between the two problems could be explained by chronic low-grade inflammation in obesity, which could stimulate hepcidin expression and modify iron concentration that the consumption of high-protein diets could prevent. Thus, this study aimed to compare the effects of high-protein diets with a predominance of animal or vegetable protein on serum hepcidin and iron concentrations in adults with obesity. Methods and ResultsThis randomized clinical trial involved adults with obesity and insulin resistance, who were assigned to either a high animal protein (AP) group or a high vegetable protein (VP) group for a one-month intervention. Both groups followed a calorie-restricted diet, reducing energy intake by 750 kcal/day. Baseline and final measurements included serum concentrations of hepcidin and iron, biochemical parameters, anthropometric data, and body composition. A total of 33 participants (63% female) were included in the study. Significant weight loss was observed in both groups after the intervention. Adjusted for weight loss percentage, the AP group showed a significant increase in hepcidin concentration (from 22.3 ± 14.7 to 27.5 ± 19.5 ng/mL) compared to the VP group (from 17.9 ± 15.1 to 17.2 ± 10.1 ng/mL) (p < 0.01), with no changes in serum iron concentration. Additionally, the VP diet significantly reduced serum adiponectin (p = 0.04) and C-reactive protein (p = 0.03) levels. ConclusionsIn adults with obesity following the AP diet for one month, hepcidin levels increased without affecting serum iron concentrations. Trial registrationNCT03627104

Chinese expert consensus on allergen component resolved diagnosis.

Allergen component resolved diagnosis (CRD) is a method for identifying specific protein molecules that cause hypersensitivity. Unlike traditional methods that use crude allergen extracts containing multiple component species, CRD focuses on individual allergen protein molecules for more precise diagnosis. The World Allergy Organization (WAO) recommends CRD as a supplement to clinical history and allergen extract testing, and in some cases, it can replace crude extract tests. CRD involves the use of natural or recombinant proteins to detect specific IgE antibodies directed at individual allergenic components. This method allows for a more detailed analysis of a patient's allergic response compared to the use of whole allergen extracts. The Allergy Prevention and Control Specialty Committee of the Chinese Preventive Medicine Association, in collaboration with multidisciplinary experts, developed an expert consensus that incorporates the consensus of the European Academy of Allergy and Clinical Immunology (EAACI), WAO, and important domestic literature on CRD in recent years. The consensus aims to standardize the algorithm of allergen diagnosis and provides a reference for clinical practice. It also offers guidance for clinicians on the common protein families identified by CRD, the scenarios where CRD is applicable, and the significance of detecting common allergen components. Despite its potential, CRD is not widely used in clinical practice in China due to the lack of allergen component reagents and a general unawareness among clinicians about CRD's application and interpretation of test results. The expert consensus developed by the Chinese Preventive Medicine Association aims to address this gap and enhance the clinical application of CRD in China.

RUNX2 is stabilised by TAZ and drives pulmonary artery calcification and lung vascular remodelling in pulmonary hypertension due to left heart disease.

Calcification is common in chronic vascular disease, yet its role in pulmonary hypertension due to left heart disease is unknown. Here, we probed for the role of runt-related transcription factor-2 (RUNX2), a master transcription factor in osteogenesis, and its regulation by the HIPPO pathway transcriptional coactivator with PDZ-binding motif (TAZ) in the osteogenic reprogramming of pulmonary artery smooth muscle cells and vascular calcification in patients with pulmonary hypertension due to left heart disease. We similarly examined its role using an established rat model of pulmonary hypertension due to left heart disease induced by supracoronary aortic banding. Pulmonary artery samples were collected from patients and rats with pulmonary hypertension due to left heart disease. Genome-wide RNA sequencing was performed, and pulmonary artery calcification assessed. Osteogenic signalling via TAZ and RUNX2 was delineated by protein biochemistry. In vivo, the therapeutic potential of RUNX2 or TAZ inhibition by CADD522 or verteporfin was tested in the rat model. Gene ontology term analysis identified significant enrichment in ossification and osteoblast differentiation genes, including RUNX2, in pulmonary arteries of patients and lungs of rats with pulmonary hypertension due to left heart disease. Pulmonary artery calcification was evident in both patients and rats. Both TAZ and RUNX2 were upregulated and activated in pulmonary artery smooth muscle cells of patients and rats. Co-immunoprecipitation revealed a direct interaction of RUNX2 with TAZ in pulmonary artery smooth muscle cells. TAZ inhibition or knockdown decreased RUNX2 abundance due to accelerated RUNX2 protein degradation rather than reduced de novo synthesis. Inhibition of either TAZ or RUNX2 attenuated pulmonary artery calcification, distal lung vascular remodelling and pulmonary hypertension development in the rat model. Pulmonary hypertension due to left heart disease is associated with pulmonary artery calcification that is driven by TAZ-dependent stabilisation of RUNX2, causing osteogenic reprogramming of pulmonary artery smooth muscle cells. The TAZ-RUNX2 axis may present a therapeutic target in pulmonary hypertension due to left heart disease.

Characteristics and biological mechanism of protein degradation by the black solider fly (Hermetia illucens L.) larvae gut strain Bacillus subtilis S4

The black solider fly larvae (BSFL) can efficiently convert nitrogen in organic waste into insect protein. Bacillus subtilis S4, an efficient protein-degrading bacterium from the BSFL gut, was isolated and identified to explore the mechanism of nutrient metabolism underlying BSFL nitrogen utilization. Results showed that B. subtilis S4 could effectively increase larval biomass in a bean stem bioconversion system. In vitro high-performance liquid chromatography analysis showed that B. subtilis S4 completely degraded casein into Val, Ile, Phe, Leu, Tyr, Lys, Gly, and Met. Various protease genes with secretion expression ability were annotated in B. subtilis S4. They included peptidoglycan DL - endopeptidase, aminopeptidase, extracellular metalloprotease, peptidoglycan endopeptidase, and peptidase M15. Therefore, the BSFL intestinal microbe B. subtilis S4 could effectively degrade protein and promote larval biomass accumulation, which could provide novel insights into the combined conversion of organic waste into proteins by microbes and insects.

Analysis on microbial communities and characteristic flavor metabolic of PXDB-meju by partially substituting wheat flour with soybean flour and gluten flour

Pixian Douban (PXDB) is one of the most popular condiments in China due to its unique flavor. Wheat flour that contains abundant nutrients is an important raw material in producing flavors during PXDB fermentation. In this study, wheat flour was substituted with soybean flour and gluten flour that have higher proteins in portions of 10.4% and 4.2% (F1), 8.9% and 7.2% (F2), 9.6% and 5.8% (F3). The results indicated that the substitutions increased the amino acid nitrogen content and improved flavor quality compared with traditional group (CT). Especially, the key amino acids including spartate, glutamic, arginine and lysine, and the phenylacetaldehyde as one of the most important volatile compounds exhibited preferable higher contents in F2 group than those in CT group. Metagenomic analysis showed that the abundances of predominant bacteria, including Kosakonia_cowanii, unclassified_f__Enterobacteriaceae and unclassified_g__Enterobacter, were higher in the F2 compared to the CT. Lupinus_albus and Plutella_xylostella were the top two fungi in relative abundance, with higher growth rates in F2 than in CT. Furthermore, metabolism pathway analysis revealed higher relative abundance of enzymes producing key amino acids and phenethylaldehyde in the F2 compared to the CT. Meanwhile, these enzymes were exclusively annotated to the Kosakonia_cowanii, Bacillus_velezensis and Escherichia_coli in F2. This study provided a theoretical foundation for improving PXDB flavor quality in industry production.

Efficient enzyme-free isolation of brain-derived extracellular vesicles.

Extracellular vesicles (EVs) have gained significant attention as pathology mediators and potential diagnostic tools for neurodegenerative diseases. However, isolation of brain-derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise the integrity of EV proteins and overall functionality. Here, we describe that collagenase digestion, commonly used for BDEV isolation, produces undesired protein cleavage of EV-associated proteins in brain tissue homogenates and cell-derived EVs. In order to avoid this effect, we studied the possibility of isolating BDEVs with a reduced amount of collagenase or without any protease. Characterization of the isolated BDEVs from mouse and human samples (both female and male) revealed their characteristic morphology and size distribution with both approaches. However, we show that even minor enzymatic digestion induces 'artificial' proteolytic processing in key BDEV markers, such as Flotillin-1, CD81, and the cellular prion protein (PrPC), whereas avoiding enzymatic treatment completely preserves their integrity. We found no major differences in mRNA and protein content between non-enzymatically and enzymatically isolated BDEVs, suggesting that the same BDEV populations are purified with both approaches. Intriguingly, the lack of Golgi marker GM130 signal, often referred to as contamination indicator (or negative marker) in EV preparations, seems to result from enzymatic digestion rather than from its actual absence in BDEV samples. Overall, we show that non-enzymatic isolation of EVs from brain tissue is possible and avoids artificial pruning of proteins while achieving an overall high BDEV yield and purity. This protocol will help to understand the functions of BDEV and their associated proteins in a near-physiological setting, thus opening new research approaches.

The water channels aquaporin-1 and aquaporin-3 interact with and affect the cell polarity protein Scribble in 3D in vitro models of breast cancer.

Cellular changes in carcinomas include alterations in cell proliferation, cell migration, cell-cell adhesion, and cellular polarity. In vitro studies have revealed that the water channels, aquaporin-1 (AQP1) and AQP3, can influence cell migration and cell-cell adhesion. Of note, we previously showed that AQP1 overexpression reduced levels of cell-cell adhesion proteins, whereas AQP3 increased levels when overexpressed in normal epithelial cells. Expression of AQP1 and AQP3 in breast carcinoma is associated with lymph node metastasis, recurrence, and poor survival of patients with breast cancer. In this study, we investigated if AQP1 and AQP3 affected cell polarity in breast cancer by studying the relationship between the major polarity protein Scribble and AQP1 and AQP3. In breast cancer tissue samples, the protein expression of AQP1, AQP3, and Scribble did not show an obvious correlation. However, in a GST pull-down assay, AQP1 and AQP3 interacted with Scribble. AQP1 overexpression reduced the size of 3D spheroids as well as reduced Scribble levels at cell-cell contacts, whereas AQP3 overexpression showed no significant change in spheroid size compared with control, AQP3 overexpression also reduced Scribble levels at cell-cell contacts. Of note, AQP1 overexpression increased cell migration and induced cell detachment and dissemination from migrating breast cancer cell sheets, whereas AQP3 overexpression did not. Thus, AQP1 and AQP3 differentially affect 3D-grown breast cancer spheroids, and especially AQP1 may contribute to cancer development and spread via negatively affecting cellular junctions and cell polarity proteins as well as increasing cell migration and cell detachment.NEW & NOTEWORTHY Overexpression of the water channels aquaporin-1 and aquaporin-3 reduced levels of the key polarity protein Scribble at cell-cell junctions, suggesting potential implications in breast cancer progression and metastasis.

Endochondral ossification: insights into the cartilage mineralization processes achieved by an anhydrous freeze substitution protocol

Growth plate cartilage (GP) serves as a dynamic site of active mineralization and offers a unique opportunity to investigate the cell-regulated matrix mineralization process. Transmission electron microscopy (TEM) provides a means for the direct observation of these mechanisms, offering the necessary resolution and chemical analysis capabilities. However, as mineral crystallinity is prone to artifacts using aqueous fixation protocols, sample preparation techniques are critical to preserve the mineralized tissue in its native form. We optimized cryofixation by high-pressure freezing followed by freeze substitution in anhydrous acetone containing 0.5% uranyl acetate to prepare murine GP for TEM analysis. This sample preparation workflow maintains cellular and extracellular protein structural integrity with sufficient contrast for observation and without compromising mineral crystallinity. By employing appropriate sample preparation techniques, we were able to observe two parallel mineralization processes driven by chondrocytes: 1) intracellular- and 2) extracellular-originating mineralized vesicles. Both mechanisms are based on sequestering calcium phosphate (CaP) within a membrane-limited structure, albeit originating from different compartments of the chondrocytes. In the intracellular originating pathway, CaP accumulates within mitochondria as globular CaP granules, which are incorporated into intracellular vesicles (500-1000 nm) and transported as granules to the extracellular matrix (ECM). In contrast, membrane budding vesicles with a size of approximately 100-200nm, filled with needle-shaped minerals were observed only in the ECM. Both processes transport CaP to the collagenous matrix via vesicles, they can be differentiated based on the vesicle size and mineral morphologies. Their individual importance to the cartilage mineralization process is yet to be determined. Statement of SignificanceWe do not fully understand the process by which epiphyseal cartilage mineralizes - a vital step in endochondral bone formation. Previous work has proposed that mitochondria and intracellular vesicles are storage sites for the delivery of mineral to collagen fibrils. However, these concepts are founded on results from in vitro models of mineralization; no prior work has observed mineral-containing intracellular vesicles or mitochondria in developing epiphyseal cartilage. Here we developed a new cryofixation preparation route for transmission electron microscopy (TEM) imaging that has disclosed a cell-regulated process of mineralization in epiphyseal cartilage. High resolution TEM images revealed an involvement of mitochondria and intracellular and extracellular vesicles in delivering transient mineral phases to the collagen fibrils to promote cartilage mineralization.

The effect of clinically relevant antibiotics on bacterial extracellular vesicle release from E. coli.

Sepsis, a leading cause of death in hospitals, can be defined as a dysregulated host inflammatory response to infection, which can lead to tissue damage, organ failure, and cardiovascular complications. Although there is no cure for sepsis, the condition is typically managed with broad-spectrum antibiotics to eliminate any potential bacterial source of infection. However, a potential side-effect of antibiotic treatment is the enhanced release of bacterial extracellular vesicles (BEVs), membrane-bound nanoparticles containing proteins and other biological molecules from their parent bacterium. Some of the Gram-negative EV cargo, including Peptidoglycan associated lipoprotein (Pal) and Outer membrane protein A (OmpA), have been shown to induce both acute and chronic inflammation in host tissue. We hypothesize that antibiotic concentration and its mechanism of action can have an effect on the amount of released BEVs, which could potentially exacerbate the host inflammatory response. In this study, we evaluated nine clinically relevant antibiotics for their effect on EV release from Escherichia coli. Several beta-lactam antibiotics caused significantly more EV release, while quinolone and aminoglycosides caused relatively less vesiculation. Further study is warranted to corroborate the correlation between an antibiotic's mechanism of action and its effect on EV release, but these results underline the importance of antibiotic choice when treating sepsis patients.