Protein Loading Research Articles

Recovery of Cleaning Solutions from Dairy Manufacturing Effluents Using Membrane Technology

The recovery concept of cleaning solutions, based on single-phase detergents from cleaning-in-place (CIP) effluents from the dairy industry, is presented. The first step consists of ultrafiltration (UF) (with a cut-off of 5 or 10 kDa) to reduce the high load of milk proteins, followed by nanofiltration (NF) (with a cut-off of 200 Da) to separate low molecular weight lactose. Membrane steps were performed in the concentration mode, achieving a recovery of 75% of the solutions. UF modules reduced 70–85% of chemical oxygen demand (COD), 99% of milk proteins, and 45–70% of lactose, limiting the susceptibility of NF modules to fouling. Combined with nanofiltration, the efficiency of the purification system is 100% for proteins and more than 99% for lactose. The solutions recovered in the proposed purification variants are recognized as sodium hydroxide solutions with a surfactant admixture, and they can be successfully re-used for cleaning processes in the production plant.

Determination of stability and activity of immobilized lipase for transesterification reaction in fluorous solvent and deducing the reaction mechanism by molecular docking study

The objective of the present study was to immobilize lipase and use it for transesterification reaction in a fluorous solvent. Polymeric substance tylose (Tyl) was chosen as immobilization support. Tylose immobilized lipase from Candida rugosa (CRL) was subjected to fluorous solvents treatment and was found most stable in perfluorooctane (PFO). The immobilized Tyl-CRL was characterized for protein loading, solvent stability, surface morphology, restoration of secondary structure, and thermal stability. Immobilized CRL was further applied for the synthesis of phenethyl propionate in a fluorous solvent. All reaction parameters were optimized using the response surface method (RSM). Optimized reaction parameters were substrate ratio 1:3, immobilized lipase quantity 30 mg, and temperature 45 °C. Prepared biocatalyst was evaluated for recyclability, and it was found that catalytic activity decreased by 77 % up to the fifth recycle run. The optimized synthesis protocol showed a wide substrate scope. The docking study showed very good interaction of substrates with lipase supporting the experimental findings. The developed methodology offers a safer, more efficient approach to synthesizing various esters used as nature-identical flavors.

A Comparative Study on the Interaction Between Protein and PET Micro/Nanoplastics: Structural and Surface Characteristics of Particles and Impacts on Lung Carcinoma Cells (A549) and Staphylococcus aureus.

The interaction between particles and proteins is a key factor determining the toxicity responses of particles. Therefore, this study aimed to examine the interaction between the emerging pollutant polyethylene terephthalate micro/nanoplastics from water bottles with bovine serum albumin. The physicochemical characteristics of micro/nanoplastics were investigated using nuclear magnetic resonance, x-ray diffraction, Fourier transform infrared, dynamic light scattering, and x-ray energy dispersive spectroscopy after exposure to various concentrations and durations of protein. Furthermore, the impact of protein-treated micro/nanoplastics on biological activities was examined using the mitochondrial activity and membrane integrity of A549 cells and the activity and biofilm production of Staphylococcus aureus. The structural characteristics of micro/nanoplastics revealed an interaction with protein. For instance, the assignment of protein-related new proton signals (e.g., CH2, methylene protons of CH2O), changes in available protons s (e.g., CH and CH3), crystallinity, functional groups, elemental ratios, zeta potentials (-11.3 ± 1.3 to -12.4 ± 1.7 to 25.5 ± 2.3 mV), and particle size (395 ± 76 to 496 ± 60 to 866 ± 82 nm) of micro/nanoplastics were significantly observed after protein treatment. In addition, the loading (0.012-0.027 mM) and releasing (0.008-0.013 mM) of protein also showed similar responses with structural characteristics. Moreover, the cell-based responses were changed regarding the structural and surface characteristics of micro/nanoplastics and the loading efficiencies of protein. For example, insignificant mitochondrial activity (2%-10%) and significant membrane integrity (12%-28%) of A549 cells increased compared with control, and reductions in bacterial activity (5%-40%) in many cases and biofilm production specifically at low dose of all treatment stages (13%-46% reduction) were observed.

Dietary protein load affects the energy and nitrogen balance requiring liver glutamate dehydrogenase to maintain physical activity

Provision of amino acids to the liver is instrumental for gluconeogenesis while it requires safe disposal of the amino group. The mitochondrial enzyme glutamate dehydrogenase (GDH) is central for hepatic ammonia detoxification by deaminating excessive amino acids towards ureagenesis and preventing hyperammonemia. The present study investigated the early adaptive responses to changes in dietary protein intake in control mice and liver-specific GDH knockout mice (Hep-Glud1-/-). Mice were fed chow diets with a wide coverage of protein contents; i.e. suboptimal 10%, standard 20%, over optimal 30%, and high 45% protein diets; switched every 4 days. Metabolic adaptations of the mice were assessed in calorimetric chambers before tissue collection and analyses. Hep-Glud1-/- mice exhibited impaired alanine induced gluconeogenesis and constitutive hyperammonemia. The expression and activity of GDH in liver lysates were not significantly changed by the different diets. However, applying an in situ redox-sensitive assay on cryopreserved tissue sections revealed higher hepatic GDH activity in mice fed the high-protein diets. On the same section series, immunohistochemistry provided corresponding mapping of the GDH expression. Cosinor analysis from calorimetric chambers showed that the circadian rhythm of food intake and energy expenditure was altered in Hep-Glud1-/- mice. In control mice, energy expenditure shifted from carbohydrate to amino acid oxidation when diet was switched to high protein content. This shift was impaired in Hep-Glud1-/- mice and consequently the spontaneous physical activity was markedly reduced in GDH knockout mice. These data highlight the central role of liver GDH in the energy balance adaptation to dietary proteins.

Less is better: various means to reduce protein load in the endoplasmic reticulum.

The endoplasmic reticulum (ER) is an important organelle that controls the intracellular and extracellular environments. The ER is responsible for folding almost one-third of the total protein population in the eukaryotic cell. Disruption of ER-protein folding is associated with numerous human diseases, including metabolic disorders, neurodegenerative diseases, and cancer. During ER perturbations, the cells deploy various mechanisms to increase the ER-folding capacity and reduce ER-protein load by minimizing the number of substrates entering the ER to regain homeostasis. These mechanisms include signaling pathways, degradation mechanisms, and other processes that mediate the reflux of ER content to the cytosol. In this review, we will discuss the recent discoveries of five different ER quality control mechanisms, including the unfolded protein response (UPR), ER-associated-degradation (ERAD), pre-emptive quality control, ER-phagy and ER to cytosol signaling (ERCYS). We will discuss the roles of these processes in decreasing ER-protein load and inter-mechanism crosstalk.

<i>In vitro</i> study of the bactericidal activity for new derivatives of quaternary ammonium salts based on pyridoxine against gram-positive / gram-negative bacteria

BACKGROUND: Preventive care and successful treatment of infectious diseases (including hospital-acquired infections) are among the most important tasks of modern healthcare. Not many effective antiseptics and disinfectants available can solve this problem. Quaternary ammonium salts are currently one of the promising classes of disinfectants; however, their use is limited by the ability of microorganisms to develop resistance to the compounds. Modification of quaternary ammonium salts with substituents of various profiles is a possible way to solve this problem. AIM: To conduct a comparative analysis of the bactericidal activity of two original quaternary ammonium compounds based on pyridoxine in comparison with benzalkonium chloride. MATERIALS AND METHODS: The bactericidal profiles of 0.1 and 0.2% aqueous solutions of original quaternary ammonium salts based on pyridoxine were studied against two widespread pathogens: Staphylococcus aureus and Escherichia coli. Antibacterial activity data were obtained using a standard in vitro test (bactericidal activity test on a metal surface; exposure time of 1, 5, and 15 min). In addition to standard conditions, antibacterial efficacy was studied under protein loading conditions, in which BSA solution was added to the surface. RESULTS: For the studied compounds, the reduction factor (RF, reduction lgCFU of microorganisms) was on average ≥3. Protein loading led to a decrease in efficiency by 1–3 times. An interesting effect of exposure time-dependent specificity reversal to the S. aureus and E. coli was observed. CONCLUSIONS: The results showed the high activity of the studied compounds, which corresponds to the effectiveness criterion of RF ≥3 for commercial compounds used in this field.

Impaired cathepsin D in retinal pigment epithelium cells mediates Stargardt disease pathogenesis.

Recessive Stargardt disease (STGD1) is an inherited juvenile maculopathy caused by mutations in the ABCA4 gene, for which there is no suitable treatment. Loss of functional ABCA4 in the retinal pigment epithelium (RPE) alone, without contribution from photoreceptor cells, was shown to induce STGD1 pathology. Here, we identified cathepsin D (CatD), the primary RPE lysosomal protease, as a key molecular player contributing to endo-lysosomal dysfunction in STGD1 using a newly developed "disease-in-a-dish" RPE model from confirmed STGD1 patients. Induced pluripotent stem cell (iPSC)-derived RPE originating from three STGD1 patients exhibited elevated lysosomal pH, as previously reported in Abca4-/- mice. CatD protein maturation and activity were impaired in RPE from STGD1 patients and Abca4-/- mice. Consequently, STGD1 RPE cells have reduced photoreceptor outer segment degradation and abnormal accumulation of α-synuclein, the natural substrate of CatD. Furthermore, dysfunctional ABCA4 in STGD1 RPE cells results in intracellular accumulation of autofluorescent material and phosphatidylethanolamine (PE). The altered distribution of PE associated with the internal membranes of STGD1 RPE cells presumably compromises LC3-associated phagocytosis, contributing to delayed endo-lysosomal degradation activity. Drug-mediated re-acidification of lysosomes in the RPE of STGD1 restores CatD functional activity and reduces the accumulation of immature CatD protein loads. This preclinical study validates the contribution of CatD deficiencies to STGD1 pathology and provides evidence for an efficacious therapeutic approach targeting RPE cells. Our findings support a cell-autonomous RPE-driven pathology, informing future research aimed at targeting RPE cells to treat ABCA4-mediated retinopathies.

#2919 Renal functional reserve assessment in patients with coronary microvascular dysfunction and preserved renal function—experience of a center

Abstract Background and Aims Renal Functional Reserve (RFR) is a field of scientific debate and may be a valuable diagnostic tool for the early detection of subclinical renal disease. According to literature, chronic kidney disease is related to cardiovascular disease (CVD), however the available data on the association of early renal dysfunction and CVD are sparse. The aim of the present study is to evaluate RFR in patients with coronary microvascular dysfunction (CMD) and preserved renal function [eGFR≥ 60 ml/min/1.73 m2 (CKD-EPI) and proteinuria< 400 mg/24 h]. Method This is a single-center, prospective study enrolling patients with INOCA. We are presenting preliminary results. In the absence of significant coronary artery stenosis, functional coronary circulation assessment was performed for all patients and we estimated coronary flow reserve (CFR) and index of microvascular resistance (IMR). In all participants, RFR was estimated by endogenous creatinine clearance after oral protein load (cooked meal, 1.2 gr/kg). Normal RFR was defined as ≥30 ml/min/1.73 m2. Also, patients with INOCA were offered 24-hour Ambulatory Blood Pressure Monitoring (ABPM). Results A total of 22 participants have been enrolled so far in study: 10 without CMD—control group [7 female, mean age: 54.4 ± 8.1 years) and 12 with INOCA—CMD group (9 female, 75%, mean age: 53.5 ± 10.3 years). CMD patients were classified into 2 groups, structural and functional endotype (CFR<2.5 and an IMR≥25 were considered abnormal). The RFR value for CMD group is 7.4 ± 6.3 ml/min/1.73 m2, while for non-CMD group 36.3 ± 5.8 ml/min/1.73 m2 (p < 0.005), relationship that remained statistically significant after controlling for confounding factors. Regarding the two endotypes of MVD, it was found that RFR for the patients with the functional endotype is 3.5 ± 2.4 ml/min/1.73 m2, while for those with the structural endotype 9.3 ± 6.9 ml/min/1.73 m2 (p-value = 0.061). Furthermore, no statistically significant relationship emerged between RFR and IMR, CFR indices (p = 0.347, p = 0.263). According to data from 24 hr ABPM, there were no significant differences in ambulatory blood pressure (systolic and diastolic) between the two endotypes of CMD (p = 0.594, p = 0.243), as well as no relationship with RFR (p = 0.553, p = 0.594). However, the proportion of non-dippers (for both SBP and DBP) was significantly higher in functional CMD endotype (p < 0.05). It was not found correlation between RFR and non-dipping phenotype, both for SBP and DBP (p = 0.561, p = 0.238). Conclusion Abnormal RFR has been observed in all patients with microvascular dysfunction. In addition, patients with functional CMD show a lower RFR value compared to those with structural. RFR is not related to blood pressure or the non-dipping pattern. Given the hypothesis that the assessment of renal functional reserve is an early diagnostic tool for subclinical renal disease, the early identification of patients with specific phenotypes—such as INOCA and pathological RFR—could contribute to the individualization of therapeutic interventions.

Renal functional reserve assessment in patients with coronary microvascular dysfuntion and preserved renal function

Abstract Funding Acknowledgements None. Introduction Renal Functional Reserve (RFR) is a field of scientific debate and may be a valuable diagnostic tool for the early detection of subclinical renal disease. According to literature, chronic kidney disease is related to cardiovascular disease (CVD), however the available data on the association of early renal dysfunction and CVD are sparse. Aim The aim of the present study is to evaluate RFR in patients with coronary microvascular dysfunction (CMD) and preserved renal function [eGFR≥ 60 ml/min/1.73m2 (CKD-EPI) and proteinuria< 400mg/24h]. Methods This is a single-center, prospective study enrolling patients with CMD. We are presenting preliminary resuts. In the absence of significant coronary artery stenosis, functional coronary circulation assessment was performed for all patients. Coronary flow reserve (CFR) and index of microvascular resistance (IMR) were measured invasively. In all participants, RFR was estimated by endogenous creatinine clearance after oral protein load (cooked meal, 1.2gr/kg). Normal RFR was defined as ≥30 ml/min/1.73m2. Also, patients with CMD were offered 24-hour Ambulatory Blood Pressure Monitoring (ABPM). Results A total of 25 participants have been enrolled so far in study: 11 without CMD - non-CMD group [7 female, 64%, mean age: 52.9±8.8 years) and 14 with INOCA – CMD group (11 female, 79%, mean age : 53.5±10.3 years). CMD patients were classified into 2 groups, structural and functional endotype (CFR<2.5 and IMR≥25 were considered abnormal). The RFR value for CMD group is 7.4±6.3 ml/min/1.73m2, while for control group 36.3±5.8 ml/min/1.73m2 (p<0.005), This difference between groups was found to be statistically significant after controlling for confounding factors. It was found that RFR value for functional CMD endotype was 3.8±2.6 ml/min/1.73m2, while for patients with structural endotype 9.6±7,2 ml/min/1.73m2 (p=0.06). Furthermore, no statistical significance was found between RFR and IMR, CFR indices (p =0.35, p =0.23 respectively). According to data from 24hr ABPM , there were no significant differences in ambulatory blood pressure (systolic and diastolic) between the two endotypes of CMD (p=NS for all), as well as no relationship with RFR (p=0.6, p=0.6 respectively) was observed. However, the proportion of non-dippers (for both SBP and DBP) was significantly higher in functional CMD endotype (p<0.05). It was not found correlation between RFR and non-dipping phenotype, for both SBP and DBP (p=NS). Conclusion Abnormal RFR has been observed in all patients with microvascular dysfunction. In addition, patients with functional CMD show a lower RFR value compared to those with structural. RFR is not related to levels of BP or the non-dipping pattern. Given the hypothesis that the assessment of renal functional reserve is an early diagnostic tool for subclinical renal disease, the early identification of patients with specific phenotypes could contribute to the individualization of therapeutic interventions.

Hygienic characteristics of ethanol for preventive use in skin antiseptics

Aim – to study the ethyl alcohol 70% of the Lux brand in respect to its chemical and analytical quality, toxicological safety, microbial effectiveness and to assess its use in skin antiseptics. Material and methods. The studies were performed using organoleptic, physical, chemical, toxicological and microbial analysis in accordance with standard modern methods. Results. Ethyl alcohol 70% has chemical, analytical and microbial quality indicators that meet the requirements of the State Pharmacopeia of the Republic of Belarus. According to toxicological indicators, it belongs to class IV – lowhazard substances, with no irritating effect, which corresponds to the regulatory toxicological indicators of the safety of skin antiseptics. During the primary epicutaneous application on volunteers, it does not cause subjective and objective symptoms of sensitization; it belongs to substances that do not have an irritating effect and sensitizing ability. Standard test cultures and clinical strains of microorganisms are highly sensitive to ethyl alcohol 70% in a qualitative suspension test tube and micromethod without protein load and moderately sensitive in the diskdiffusion method with an exposure of 1 min. In relation to these microorganisms, the antimicrobial activity of ethyl alcohol 70% is above 99.999% and a reduction factor above 5 lg at exposure of 1 min in a quantitative suspension method without a protein load. Ethyl alcohol 70% is also characterized by a high efficiency of normal skin disinfection and E.coli contaminated skin of volunteers over 99% and a reduction factor above 2 lg and 4 lg, respectively. Conclusion. Ethyl alcohol 70% of the brand "Lux" is a highquality, toxicologically safe and microbial effective drug. It can be recommended for skin antisepsis of the injection field (class A), as well as hygienic hand antisepsis (class B) and antiseptic sanitization of the skin in the Republic of Belarus and Russian Federation.

Biodynamer Nano-Complexes and -Emulsions for Peptide and Protein Drug Delivery.

Therapeutic proteins and peptides offer great advantages compared to traditional synthetic molecular drugs. However, stable protein loading and precise control of protein release pose significant challenges due to the extensive range of physicochemical properties inherent to proteins. The development of a comprehensive protein delivery strategy becomes imperative accounting for the diverse nature of therapeutic proteins. Biodynamers are amphiphilic proteoid dynamic polymers consisting of amino acid derivatives connected through pH-responsive dynamic covalent chemistry. Taking advantage of the amphiphilic nature of the biodynamers, PNCs and DEs were possible to be prepared and investigated to compare the delivery efficiency in drug loading, stability, and cell uptake. As a result, the optimized PNCs showed 3-fold encapsulation (<90%) and 5-fold loading capacity (30%) compared to DE-NPs. PNCs enhanced the delivery efficiency into the cells but aggregated easily on the cell membrane due to the limited stability. Although DE-NPs were limited in loading capacity compared to PNCs, they exhibit superior adaptability in stability and capacity for delivering a wider range of proteins compared to PNCs. Our study highlights the potential of formulating both PNCs and DE-NPs using the same biodynamers, providing a comparative view on protein delivery efficacy using formulation methods.

Valorization of black carrot pomace and pea powder for co-production of polygalacturonase and pectin lyase

AbstractPectic enzyme groups, particularly polygalacturonase and pectin lyase, are vital components of a high-value microbial enzyme category widely employed in applications within the fruit juice and wine industries. The exploration of alternative carbon and nitrogen sources remains crucial for enhancing enzyme production while reducing costs. This study evaluates the impact of carbon (black carrot pomace) and nitrogen (pea protein) loading on fermentable sugar content, protein content, and enzyme activities during both batch and fed-batch cultivation. Additionally, three distinct valorization techniques—thermal (steam), thermochemical (steam assisted with dilute acid), and microwave-assisted with dilute acid pretreatments—were assessed for their effectiveness in hydrolyzing black carrot pomace as a carbon source. The findings indicate that microwave-assisted dilute acid, coupled with enzymatic saccharification, resulted in the highest fermentable sugar production (0.493 g/g), achieving an 87.3% conversion yield. Pea protein demonstrated more favorable outcomes with the highest polygalacturonase activity (20.50 ± 0.52 U/L) and pectin lyase activity (46.44 ± 3.45 U/L) compared to whey protein and yeast extract used as nitrogen sources. Meanwhile, the highest polygalacturonase and pectin lyase activity, along with the highest total protein content (52.25 ± 0.06 mg/L), was recorded under the same culture conditions, reaching 164.34 ± 2.26 and 188.22 ± 1.72 U/L, respectively, after 72 h, representing approximately 1.18- and 1.34-fold increases from the batch system. Consequently, these results prove that fed-batch cultivation, utilizing black carrot pomace hydrolyzate as a feeding substrate and pea protein as a nitrogen source, significantly increases polygalacturonase and pectin lyase activity compared to batch cultivation.

Complex hydrogel for cartilage regeneration and anti-inflammation

With the increasing incidence of osteoarthritis (OA) in elderly patients and the challenges posed by its multifaceted pathogenesis in developing effective treatments, this study explores the effective protein loading mode of Progranulin (PGRN) and its therapeutic potential for treating OA. GelMA hydrogels prepared with different concentrations of PGRN, employing AC-PEG-NHS as a crosslinking agent for drug encapsulation, were used to test the sustained release effect of the composite hydrogel drug delivery system. Additionally, we conducted in vitro assays to investigate the regulatory effects on rat bone marrow mesenchymal atromal cells (BMSCs), chondrocytes, and mouse macrophages - focusing on cell proliferation, and differentiation. For in vivo studies, histological staining techniques were utilized to assess inflammatory cell polarization and cartilage repair in a rat knee injury model. The results demonstrated that PGRN-loaded hydrogels significantly accelerated cell proliferation, enhance differentiation of BMSCs into chondrocytes, maintained a healthy chondrocyte phenotype, and induce M2-type polarization while inhibiting M1-type polarization in macrophages. Histological analyses also showed markedly improved tissue repair in the group treated with GelMA–NHS–PGRN hydrogels. As a result, these GelMA–NHS–PGRN hydrogels present a promising therapeutic approach for OA, potentially mitigating chronic inflammation and facilitating cartilage repair through controlled release of PGRN. Key worldPGRN, Osteoarthritis, Anti-inflammatory hydrogels, Cartilage regeneration.

Magnetic biocatalytic nanoreactors based on graphene oxide with graded reduction degrees for the enzymatic synthesis of phytosterol esters

As we know, the carriers of enzymatic nanoreactors with different amphipathy have significant influence on the interfacial enzyme activities and its performance. In this study, the magnetic graphene oxide (GO) enzyme carriers were constructed by assembling graphene oxide with different reduction degrees (rGO(x)) and polyethyleneimine (PEI) modified nano-flowery Fe3O4 into composite materials through electrostatic interactions, and x here represents the reduction reaction hours of GO. The SEM, TEM, contact angles and BET tests qualified the ideal enzyme carrier, PEI-Fe3O4-rGO(12), by adsorption-crosslinking method, the Candida rugosa lipase (CRL) was immobilized to obtain the lipase nanoreactor, PEI-Fe3O4-rGO(12)@CRL. The nano-sheet and nano-flower structures provided a larger specific surface area and suitable pore size, and the CRL had higher protein load on the carrier material (163.11 mg/g). In addition, using hydrolyzed p-nitrophenol palmitate as the model, the PEI-Fe3O4-rGO(12)@CRL can still show 76.5 % residual activity after 8 repeated uses. At the same time, the immobilized lipase showed good catalytic performance in the biosynthesis of phytosterol esters. The optimal esterification conditions were determined by single factor experiment (isooctane, sterol concentration 150 μmol/mL, alkyd molar ratio 1:2, enzyme dosage 120 mg, 26 h, 55 °C, the esterification rate was 76.24 %). Therefore, the optimal biocatalyst PEI-Fe3O4-rGO(12)@CRL may has good performance in the biosynthesis of other novel foods.

Impairment of the Glial Phagolysosomal System Drives Prion-Like Propagation in a Drosophila Model of Huntington's Disease.

Protein misfolding, aggregation, and spread through the brain are primary drivers of neurodegenerative disease pathogenesis. Phagocytic glia are responsible for regulating the load of pathological proteins in the brain, but emerging evidence suggests that glia may also act as vectors for aggregate spread. Accumulation of protein aggregates could compromise the ability of glia to eliminate toxic materials from the brain by disrupting efficient degradation in the phagolysosomal system. A better understanding of phagocytic glial cell deficiencies in the disease state could help to identify novel therapeutic targets for multiple neurological disorders. Here, we report that mutant huntingtin (mHTT) aggregates impair glial responsiveness to injury and capacity to degrade neuronal debris in male and female adult Drosophila expressing the gene that causes Huntington's disease (HD). mHTT aggregate formation in neurons impairs engulfment and clearance of injured axons and causes accumulation of phagolysosomes in glia. Neuronal mHTT expression induces upregulation of key innate immunity and phagocytic genes, some of which were found to regulate mHTT aggregate burden in the brain. A forward genetic screen revealed Rab10 as a novel component of Draper-dependent phagocytosis that regulates mHTT aggregate transmission from neurons to glia. These data suggest that glial phagocytic defects enable engulfed mHTT aggregates to evade lysosomal degradation and acquire prion-like characteristics. Together, our findings uncover new mechanisms that enhance our understanding of the beneficial and harmful effects of phagocytic glia in HD and other neurodegenerative diseases.

Assessment of subclinical renal dysfunction in post COVID-19 patients

ABSTRACT Introduction Kidney damage in COVID-19 is common and may lead to CKD. Early recognition of progression is mandatory, so there is a need for sensitive markers of kidney damage. Renal functional reserve (RFR) is described as the ability of the kidneys to augment their function in special situations. Aim of Work Was to assess subclinical renal dysfunction post-COVID-19. Methods This cross-sectional study investigates 31 patients previously infected with COVID-19 within 2–3 months and 31 healthy subjects with matched age and sex. Assessment of RFR was done by calculating the estimated GFR using the CKD-EPI equation before and 2 hours after 80 g protein load by intravenous infusion. Results RFR was significantly lower in post-COVID-19 patients (mean of 11.19 ± 4.76) than the control (mean of 25.81 ± 4.42, p < 0.001). Conclusion Despite preserved eGFR, there is a reduction in RFR, indicating progressive loss of nephron mass post-COVID.

Widespread nuclear lamina injuries defeat proteostatic purposes of α-synuclein amyloid inclusions.

Biogenesis of inclusion bodies (IBs) facilitates protein quality control (PQC). Canonical aggresomes execute degradation of misfolded proteins while non-degradable amyloids sequester into insoluble protein deposits. Lewy bodies (LBs) are filamentous amyloid inclusions of α-synuclein, but PQC benefits and drawbacks associated with LB-like IBs remain underexplored. Here, we report that crosstalk between filamentous LB-like IBs and aggresome-like IBs of α-synuclein (Syn-aggresomes) buffer the load, aggregation state, and turnover of the amyloidogenic protein in mouse primary neurons and HEK293T cells. Filamentous LB-like IBs possess unorthodox PQC capacities of self-quarantining α-synuclein amyloids and being degradable upon receding fresh amyloidogenesis. Syn-aggresomes equilibrate biogenesis of filamentous LB-like IBs by facilitating spontaneous degradation of α-synuclein and conditional turnover of disintegrated α-synuclein amyloids. Thus, both types of IB primarily contribute to PQC. Incidentally, the overgrown perinuclear LB-like IBs become degenerative once these are misidentified by BICD2, a cargo-adapter for the cytosolic motor-protein dynein. Microscopy indicates that microtubules surrounding the perinuclear filamentous inclusions are also distorted, misbalancing the cytoskeleton-nucleoskeleton tension leading to widespread lamina injuries. Together, nucleocytoplasmic mixing, DNA damage, and deregulated transcription of stress chaperones defeat the proteostatic purposes of the filamentous amyloids of α-synuclein.

Renal vascular control during normothermia and passive heat stress does not differ between healthy younger men and women.

Men are likely at greater risk for heat-induced acute kidney injury compared with women, possibly due to differences in vascular control. We tested the hypothesis that the renal vasoconstrictor and vasodilator responses will be greater in younger women compared with men during passive heat stress. Twenty-five healthy adults [12 women (early follicular phase) and 13 men] completed two experimental visits, heat stress or normothermic time-control, assigned in a block-randomized crossover design. During heat stress, participants wore a water-perfused suit perfused with 50°C water. Core temperature was increased by ∼0.8°C in the first hour before commencing a 2-min cold pressor test (CPT). Core temperature remained clamped and at 1-h post-CPT, subjects ingested a whey protein shake (1.2 g of protein/kg body wt), and measurements were taken pre-, 75 min, and 150 min post-protein. Beat-to-beat blood pressure (Penaz method) was measured and segmental artery vascular resistance (VR, Doppler ultrasound) was calculated as segmental artery blood velocity ÷ mean arterial pressure. CPT-induced increases in segmental artery VR did not differ between trials (trial effect: P = 0.142) nor between men (heat stress: 1.5 ± 1.0 mmHg/cm/s, normothermia: 1.4 ± 1.0 mmHg/cm/s) and women (heat stress: 1.4 ± 1.2 mmHg/cm/s, normothermia: 2.1 ± 1.1 mmHg/cm/s) (group effect: P = 0.429). Reductions in segmental artery VR following oral protein loading did not differ between trials (trial effect: P = 0.080) nor between men (heat stress: -0.6 ± 0.8 mmHg/cm/s, normothermia: -0.6 ± 0.6 mmHg/cm/s) and women (heat stress: -0.5 ± 0.5 mmHg/cm/s, normothermia: -1.1 ± 0.6 mmHg/cm/s) (group effect: P = 0.204). Renal vasoconstrictor responses to the cold pressor test and vasodilator responses following an oral protein load during heat stress or normothermia do not differ between younger men and younger women in the early follicular phase of the menstrual cycle.NEW & NOTEWORTHY The mechanisms underlying greater heat-induced acute kidney injury risk in men versus women remain unknown. This study examined renal vascular control, including both vasodilatory (oral protein load) and vasoconstrictor (cold presser test) responses, during normothermia and heat stress and compared these responses between men and women. The results indicated that in both conditions neither renal vasodilatory nor vasoconstrictor responses differ between younger men and younger women.

Inter-coat protein loading of active ingredients into Tobacco mild green mosaic virus through partial dissociation and reassembly of the virion

Chemical pesticide delivery is a fundamental aspect of agriculture. However, the extensive use of pesticides severely endangers the ecosystem because they accumulate on crops, in soil, as well as in drinking and groundwater. New frontiers in nano-engineering have opened the door for precision agriculture. We introduced Tobacco mild green mosaic virus (TMGMV) as a viable delivery platform with a high aspect ratio and favorable soil mobility. In this work, we assess the use of TMGMV as a chemical nanocarrier for agriculturally relevant cargo. While plant viruses are usually portrayed as rigid/solid structures, these are “dynamic materials,” and they “breathe” in solution in response to careful adjustment of pH or bathing media [e.g., addition of solvent such as dimethyl sulfoxide (DMSO)]. Through this process, coat proteins (CPs) partially dissociate leading to swelling of the nucleoprotein complexes—allowing for the infusion of active ingredients (AI), such as pesticides [e.g., fluopyram (FLP), clothianidin (CTD), rifampicin (RIF), and ivermectin (IVM)] into the macromolecular structure. We developed a “breathing” method that facilitates inter-coat protein cargo loading, resulting in up to ~ 1000 AIs per virion. This is of significance since in the agricultural setting, there is a need to develop nanoparticle delivery strategies where the AI is not chemically altered, consequently avoiding the need for regulatory and registration processes of new compounds. This work highlights the potential of TMGMV as a pesticide nanocarrier in precision farming applications; the developed methods likely would be applicable to other protein-based nanoparticle systems.

Abstract 1588: Identification of new signaling pathways between fibroblasts &amp; tumor cells in pancreatic cancer

Abstract We explore the unstudied role of nanoparticles (NPs), recently discovered secreted non-vesicular nanoparticles, in signaling between pre-cancer associated fibroblasts (preCAF), CAFs, and tumor cells. We generated two novel primary lines preCAF PFUM1 from a patient with high-grade intraductal papillary mucinous neoplasms and CAF FUM2 from a patient with pancreatic ductal adenocarcinoma, all tests performed between passages 2 and 3, fibroblast purity validated by RT-PCR and ACTA2 immunofluorescence. Small extracellular vesicles (sEVs), exomeres, and supermeres (the two main forms of NPs) were isolated by the ultracentrifugation method from PF-UM1 and F-UM2 with n=2-3 experiments. To validate the isolation, we used nanoparticle tracking, electron, and fluid-phase atomic force microscopy. Proteins were quantified by BCA assay. Liquid chromatography-mass spectrometry characterized the cargo from each fraction. Human cancer lines MIAPACA2 and SU8686 were treated with 10 μg of each fraction and cell proliferation was tested by phase-contrast images (Cytation 5), analyzed by Ilastik-based 1.4.0 machine-learning. We performed fluorescent Ki-67 and vimentin staining and quantified in QuPath 0.4.3. Non-parametric statistics were performed in Prism GraphPad. NPs constitute the major fraction of small particles released by fibroblasts, containing 80.5% (PFUM1) and 77.5% (FUM2) of the protein load by quantification. Mass spectrometry reveals enrichment of growth factors, epithelial-to-mesenchymal (EMT) transition, and metabolism-associated proteins in the NPs compared to sEVs. At 72 hours, cell proliferation for Su8686 reveals a proliferative effect for all fractions from both PFUM1 and FUM2 (19-63% increase compared to controls, p=0.049 to &amp;lt;0.0001). For MIAPACA2, only supermeres from PFUM1 and FUM2 consistently increased proliferation (9.5-35% increase, p=0.0051 to &amp;lt;0.0001). Ki-67 expression in MIAPACA2, increased by 9.6-23.0% following treatment by NPs (all p&amp;lt;0.0001) and by 2.9-9.2% with sEVs (p=0.011 to &amp;lt;0.0001). EMT was estimated by vimentin in MIAPACA2, as all preCAF fractions from PFUM1 reduced vimentin expression (p&amp;lt;0.001), while CAF FUM2 NPs increased vimentin expression (p&amp;lt;0.0001) while sEVs had no effect (p=0.075). In conclusion, the majority of pancreatic tumor-associated fibroblast small secretome is composed of NPs, exhibiting significant functional pro-tumoral effects. Citation Format: Ayman Al Shoukari, Maelle Batardiere, Nooshin Movahed, Camille beaussier, Jumanah Baig, Melissa gonzalez, Patricia Moraille, Eric Bonneil, Louise Rousseau, Simon turcotte, Kathleen Delgiorno, Marcus Tan, Anna Means, Elham Dianati Ajibisheh, Quoc-huy Trinh. Identification of new signaling pathways between fibroblasts &amp; tumor cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1588.