Polyacrylamide Gel Research Articles

Accelerated Screening of Alternative DNA Base‐Organic Molecule‐Base Architectures via Integrated Theory and Experiment

Organic molecule‐mediated noncanonical DNA self‐assembly expands the standard DNA base‐pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base‐pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub‐stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule‐incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA‐molecule architectures beyond Watson‐Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA‐based delivery system and probe design for sensitive detection of certain molecules.

Accelerated Screening of Alternative DNA Base-Organic Molecule-Base Architectures via Integrated Theory and Experiment.

Organic molecule-mediated noncanonical DNA self-assembly expands the standard DNA base-pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base-pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub-stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule-incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA-molecule architectures beyond Watson-Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA-based delivery system and probe design for sensitive detection of certain molecules.

Novel high entropy alloy/NiAl2O4 photocatalysts for the degradation of tetracycline hydrochloride: Heterojunction construction, performance evaluation and mechanistic insights

Novel NiAl2O4 (NAO)/high entropy alloy (HEA) (Ti), NAO/HEA (Mn) and NAO/HEA (Mo) heterojunction photocatalysts with high photocatalytic activity for the degradation of tetracycline hydrochloride (TC) under simulated sunlight irradiation were synthesized by the polyacrylamide gel method combined with a low temperature sintering technique. The effects of environmental parameters, including drug concentration, catalyst content and pH value, on the photocatalytic activity of NAO/HEA photocatalysts were investigated in detail on the basis of the response surface methodology (RSM) and experimental results. The optimal initial TC concentration, catalyst content, and pH were 100 mg/L, 1 g/L, and 5, respectively. The photocatalytic activity of NAO/HEA photocatalyst (84.6 %) was significantly improved compared to the degradation percentage of HEA (Ti) photocatalyst (36.5 %). The roles of active free radicals including h+, •O2−, •OH and oxygen vacancy in photocatalysis were verified using multiple characterization methods. In-depth analysis was conducted on the degradation pathway of TC, the safety of degradation intermediates, and the photocatalytic mechanism of NAO/HEA (Ti) photocatalyst. The Z - scheme heterojunction can significantly improve the transport efficiency of photogenerated carriers and inhibit the recombination rate of electron hole pairs in the system, which provides a new idea for exploring the application of new HEA - based heterojunction photocatalysts in the field of photocatalysis.

Development of a contrast-enhanced ultrasound guided high intensity focused ultrasound system for coagulation of liver parenchyma.

The liver is the most common organ injured in blunt abdominal trauma and makes up roughly 5% of all trauma admissions. Current treatments are invasive and resource-intensive, which may delay care. We aim to develop and validate a contrast-enhanced ultrasound (CEUS)guided noninvasive tool to treat liver lacerations at the bedside. Two 1.8 MHz high-intensity focused ultrasound (HIFU) elements were coupled to a C1-6 diagnostic ultrasound probe and a Logiq E10 scanner (GE HealthCare) utilizing a custom enclosure for co-registered imaging and ablation. A phantom was created from polyacrylamide gel combined with thermochromic ink whose color changes above biological ablative temperatures (60 °C). The HIFU wave was focused approximately 0.5 cm below the surface utilizing a 50% duty cycle generating 11.9 MPa for 20, 30, 40, 50, and 60s. Experiments were repeated on ex vivo chicken livers in a water bath. Finally, the livers of 4 live swine underwent up to 6 CEUS-guided treatments using parameters optimized from in vitro work. Treatment of the phantom between 20-60s, produced ablation sizes from 0.016 to 0.4 cm 3 . The relationship between time and size was exponential (R 2 = 0.992). Ablation areas were also well visualized on with ultrasound imaging. The ex vivo liver ablation size at 20s was 0.37 cm 3 , at 30s was 0.66 cm 3 , and at 100 s was 5.0 cm 3 . For the in-vivo swine experiments, the average ablation area measured 2.0x0.75 cm with a maximum of 3.5x1.5 cm. CEUS was utilized with the contrast agent Definity (Lantheus) for identification of lacerations as well as immediate post operative evaluation of therapy. These experiments demonstrate the feasibility of CEUS guided transdermal HIFU ablation and the time-dependent size of ablation. This work warrants future investigations into using ultrasound to detect active bleeding and HIFU to coagulate grade III and IV liver laceration. Therapeutic/care management.

Effects of phytosterol supplementation on lipoprotein subfractions and LDL particle quality

Phytosterols are natural components of plant-based foods used as supplements because of their known cholesterol-lowering effect. However, their effects on lipoprotein subfractions and the quality of the LDL particle have not been studied in greater detail. We aimed to evaluate the effects of phytosterols supplements on lipids, lipoproteins subfractions, and on the quality of LDL. A prospective, pilot-type, open label, cross-over study, randomized 23 males in primary prevention of hypercholesterolemia to receive diet or diet plus phytosterol (2.6 g in 2 doses, with meals) for 12 weeks, when treatments were switched for another 12 weeks. Lipoprotein subfractions were analyzed by electrophoresis in polyacrylamide gel (Lipoprint System®). The Sampson equation estimated the small and dense (sd) and large and buoyant (lb) LDL subfractions from the lipid profile. Quality of LDL particle was analyzed by Z-scan and UV–vis spectroscopy. Primary outcome was the comparison of diet vs. diet plus phytosterols. Secondary outcomes assessed differences between baseline, diet and diet plus phytosterol. Non-parametric statistics were performed with p < 0.05. There was a trend to reduction on HDL-7 (p = 0.05) in diet plus phytosterol arm, with no effects on the quality of LDL particles. Heatmap showed strong correlations (ρ > 0.7) between particle size by different methods with both interventions. Diet plus phytosterol reduced TC, increased HDL-c, and reduced IDL-B, whereas diet increased HDL7, and reduced IDL-B vs. baseline (p < 0.05, for all). Phytosterol supplementation demonstrated small beneficial effects on HDL-7 subfraction, compared with diet alone, without effects on the quality of LDL particles.This trial is registered in Clinical Trials (NCT06127732) and can be accessed at https://clinicaltrials.gov.

Development of Drying Methodology for Intact Whole Buffalo Liver, Its Characterization, Shelf Life, and Evaluation of Palatability as Pet Treat

Globally, large quantity of animal byproducts is generated from the slaughter of food animals, but there is lack of research articles related to drying of these byproducts and its use as pet food. Therefore, this study was conducted with the aim of utilization of intact whole buffalo liver by drying for pet treat, evaluating its shelf life and palatability. The intact liver surface was superficially sliced, and the surface was pierced. Thereafter, the livers were pretreated in 3% sugar and 4% salt solution (1 : 3 w/v) for 3 h followed by microwaving for 4 min and hot air drying at 60°C for 40 h (designated as T2L). The livers which were dried the same as T2L except surface piercing were referred as T1L, whereas the livers dried only using hot air oven were referred as control (CL). The moisture and protein contents of the dried CL and T2L were found to be 28.46% and 14.29% and 43.85% and 52.76%, respectively. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) image of T2L revealed the presence of few low as well as high molecular weight protein bands which were absent in CL and T1L indicating a comparatively lower level of protein degradation in T2L. The shelf life of T1L and T2L samples based on microbiological and lipid oxidation analyses was found to be more than 60 days at 25 and 4°C. Palatability studies using dogs showed that all dried samples were highly palatable. Thus, it could be concluded that intact buffalo liver could be dried using surface slicing, with/without piercing followed by salt and sugar pretreatment, 4 min microwaving, and hot air drying at 60°C for 40 h. Future study should focus on the sensory properties such as aroma, texture, and flavor and sensory analysis of the dried liver by human.

Development of PCR-based markers for identification of wheat HMW glutenin Glu-1Bx and Glu-1By alleles

BackgroundIn common wheat (Triticum aestivum L.), allelic variations in the high-molecular-weight glutenin subunits Glu-B1 locus have important effects on grain end-use quality. The Glu-B1 locus consists of two tightly linked genes encoding x- and y-type subunits that exhibit highly variable frequencies. However, studies on the discriminating markers of the alleles that have been reported are limited. Here, we developed 11 agarose gel-based PCR markers for detecting Glu-1Bx and Glu-1By alleles.ResultsBy integrating the newly developed markers with previously published PCR markers, nine Glu-1Bx locus alleles (Glu-1Bx6, Glu-1Bx7, Glu-1Bx7*, Glu-1Bx7OE, Glu-1Bx13, Glu-1Bx14(−), Glu-1Bx14(+)/Bx20, and Glu-1Bx17) and seven Glu-1By locus alleles (Glu-1By8, Glu-1By8*, Glu-1By9, Glu-1By15/By20, Glu-1By16, and Glu-1By18) were distinguished in 25 wheat cultivars. Glu-1Bx6, Glu-1Bx13, Glu-1Bx14(+)/Bx20, Glu-1By16, and Glu-1By18 were distinguished using the newly developed PCR markers. Additionally, the Glu-1Bx13 and Glu-1Bx14(+)/Bx20 were distinguished by insertions and deletions in their promoter regions. The Glu-1Bx6, Glu-1Bx7, Glu-1By9, Glu-1Bx14(−), and Glu-1By15/By20 alleles were distinguished by using insertions and deletions in the gene-coding region. Glu-1By13, Glu-1By16, and Glu-1By18 were dominantly identified in the gene-coding region. We also developed a marker to distinguish between the two Glu-1Bx14 alleles. However, the Glu-1Bx14(+) + Glu-1By15 and Glu-1Bx20 + Glu-1By20 allele combinations could not be distinguished using PCR markers. The high-molecular-weight glutenin subunits of wheat varieties were analyzed by ultra-performance liquid chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the findings were compared with the results of PCR analysis.ConclusionsSeven Glu-1Bx and four Glu-1By allele detection markers were developed to detect nine Glu-1Bx and seven Glu-1By locus alleles, respectively. Integrating previously reported markers and 11 newly developed PCR markers improves allelic identification of the Glu-B1 locus and facilitates more effective analysis of Glu-B1 alleles molecular variations, which may improve the end-use quality of wheat.

Ultra-sensitive Tb3+/G-quadruplex fluorescence detection of oxidative DNA damage coupled with DNA glycosylase

Development of simple, sensitive and rapid sensing strategy for DNA damage induced by chemical reagents is urgent demand. In this assay, a Tb3+/G-quadruplex fluorescence system was proposed for the sensitive detection of DNA damage induced by Fenton reaction. The specific guanine-rich oligonucleotides can form G-quadruplex induced by Tb3+, leading to an enhancement of Tb3+ emission due to energy transfer from guanine to Tb3+. After a DNA damage reaction with Fenton reagents, an obvious fluorescence decrease was observed due to the structure change of Tb3+/G-quadruplex. which was also confirmed by polyacrylamide gel electrophoresis and circular dichroism. The Fe2+-mediated Fenton reaction with as low as 0.01 μM Fe2+ and 0.04 μM H2O2 induced oxidative DNA damage can be detected. Additionally, the human 8-oxoguanine DNA glycosylase (hOGG 1) was introduced to amplify DNA oxidation damage, by converting DNA oxidative nucleobases to strand break. Over 30-fold lower detection limit of 0.3 nM Fe2+ and 1.2 nM H2O2 was achieved. Alternative specific DNA damage or other DNA repair enzymes produced much less fluorescence decrease, demonstrating its good selectivity. It shows the potential to become a universal screening tool for the rapid and sensitive assessment of the genotoxicity of the overwhelming number of chemicals in environment.

Effect of limited proteolysis and CaCl2 on the rheology, microstructure and in vitro digestibility of pea protein-carboxymethyl cellulose mixed gel

Limited proteolysis, CaCl2 and carboxymethyl cellulose (CMC) have individually demonstrated ability to increase the gel strength of laboratory-extracted plant proteins. However, the syneresis effects of their combination on the gelling capacity of commercial plant protein remains unclear. This was investigated by measuring the rheological property, microstructure and protein–protein interactions of gels formed from Alcalase hydrolyzed or intact pea proteins in the presence of 0.1 % CMC and 0–25 mM CaCl2. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed the molecular weight of pea protein in the mixture were < 15 kDa after hydrolysis. The hydrolysates showed higher intrinsic fluorescence intensity and lower surface hydrophobicity than the intact proteins. Rheology showed that the storage modulus (G') of hydrolyzed pea protein (PPH)-based gels sightly decreased compared to those of native proteins. 5–15 mM CaCl2 increased the G’ for both PP and PPH-based gels and decreased the strain in the creep-recovery test. Scanning electron microscopy (SEM) showed the presence of smaller protein aggregates in the PPH-based gels compared to PP gels and the gel network became denser, and more compact and heterogenous in the presence of 15 and 25 mM CaCl2. The gel dissociation assay revealed that hydrophobic interactions and hydrogen bonds were the dominant forces to maintain the gel structure. In vitro digestion showed that the soluble protein content in PPH-based gels was 10 ∼ 30 % higher compared to those of the PP counterpart. CaCl2 addition reduced protein digestibility with a concentration dependent behavior. The results obtained show contrasting effects of limited proteolysis and CaCl2 on the gelling capacity and digestibility of commercial pea proteins. These findings offer practical guidelines for developing pea protein-based food products with a balanced texture and protein nutrition through formulation and enzymatic pre-treatment.

Natural lipopeptide surfactin inhibits insulin aggregation and prevents amyloid-induced cytotoxicity and inflammation

Despite the centennial of the pioneering discovery of insulin, challenges regarding insulin aggregation at the site of repeated injection, as well as during industrial production, storage, and transportation, have cast a gloom over this remarkable success story. Aggregation substantially impacts the quality, potency, and effectiveness of insulin formulation, decreasing bioavailability, intensifying the risk of immunogenicity, and endangering patient health. Herein, the suitability of Bacillus-derived amphiphilic biosurfactant surfactin was demonstrated to evade temperature-induced fibrillation of insulin. Fluorometric assessment employing thioflavin-T and intrinsic tyrosine fluorescence indicated the dose-dependent reduction in insulin unfolding and subsequent formation of fibrils. Decreased thioflavin fluorescence at the plateau stage and about a 79% increase in fibrillation lag time was observed for samples containing an equimolar ratio of insulin to surfactin. Circular dichroism analysis further indicated about a 30% increase in α-helix content in samples containing an equimolar ratio of insulin and surfactin, as compared to controls. The findings were further bolstered by dynamic light scattering, electron microscopic analysis, and native polyacrylamide gel electrophoresis. Molecular docking and simulation studies did not indicate any structural destabilization of insulin with surfactin, having major interactions at the hydrophobic and potential amyloidogenic regions of insulin B-chain: B12-B19 (VEALYLVC) and B23-B27 (GFFYT) through hydrophobic and hydrogen bonds. Notably, an almost 22% increase in cell viability was observed for cells incubated with aggregated insulin containing equimolar concentrations of SFN. Surfactin also subdued insulin amyloid-induced upregulation of the pro-inflammatory genes. Thus, surfactin may be contemplated as an additive in biopharmaceutical formulations to imbue stability and may also be evaluated as anti-amyloid therapeutics for other pathological proteinopathies.

Enzymatic and Algebraic Methodology to Determine the Contents of Kunitz and Bowman-Birk Inhibitors and Their Contributions to Total Trypsin or Chymotrypsin Inhibition in Soybeans.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

Isolation and characterization of rabies monoclonal antibody charge variants.

Current postexposure prophylaxis of rabies includes vaccines, human rabies immunoglobulin (RIG), equine RIG, and recombinant monoclonal antibodies (mAb). In the manufacturing of rabies recombinant mAb, charge variants are the most common source of heterogeneity. Charge variants of rabies mAb were isolated by salt gradient cation exchange chromatography (CEX) to separate acidic and basic and main charge variants. Separated variants were further extensively characterized using orthogonal analytical techniques, which include secondary and tertiary structure determination by far and near ultraviolet circular dichroism spectroscopy. Charge and size heterogeneity were evaluated using CEX, isoelectric focusing (IEF), capillary-IEF, size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and western blotting. Antigen binding affinity was assessed by enzyme linked immuno-sorbent assay and rapid florescence foci inhibition test. Results from structural and physicochemical characterizations concluded that charge variants are formed due to posttranslational modification demonstrating that the charge heterogeneity, these charge variants did neither show any considerable physicochemical change nor affect its biological function. This study shows that charge variants are effective components of mAb and there is no need of deliberate removal, until biological functions of rabies mAb will get affected.

Composition of high-molecular-weight glutenin subunits and gluten quality in wheat lines with alien genetic material

Background. High-molecular-weight glutenin subunits (HMW-GSs) make the greatest contribution to the formation of baking properties in bread wheat (Triticum aestivum L.). Glutenin polymorphism of bread wheat relatives is significantly richer than in cultivated varieties. The objective of this work was to identify the HMW-GS composition and assess the gluten quality of bread wheat lines with introgressions of alien genetic material.Materials and methods. We studied the parental varieties of spring bread wheat, accessions of the tetraploid and hexaploid Triticum L. species, and 19 introgressive lines produced with their participation. Glutenins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and HMW-GSs were identified using the Payne nomenclature system. Gluten quality was determined in accordance with GOST 13586.1-68 (the 2017–2019 and 2021–2022 growing seasons). Data processing was performed using the Statistica 10.0 and MS Excel software packages.Results and conclusion. HMW-GSs uncharacteristic of T. aestivum cultivars were identified in the studied accessions of T. dicoccoides (Körn. ex Aschers. et Graebn.) Schweinf., T. spelta L., and T. kiharae Dorof. et Migusch. These HMW-GSs are of interest for enriching the wheat gene pool. HMW-GSs of related species were found in 10 out of 19 introgressive lines. A five-year observation period revealed that wheat relatives on average significantly exceeded common wheat cultivars in gluten quality, and introgressive lines did not differ significantly from the parental genotypes. Lines with high rheological properties of gluten had, as a rule, HMW-GSs of related species in their composition.

Genotyping and phenotyping of Haptoglobin gene polymorphisms and their correlation with cardiovascular disease and biochemical parameters in Iranian patients with type 2 diabetes mellitus

PurposeWe aimed to examine genotyping and phenotyping of Haptoglobin (Hp) gene polymorphisms and their correlation with cardiovascular disease and biochemical parameters in Iranian patients with type 2 diabetes mellitus. MethodsWe analyzed the phenotype (by polyacrylamide gel electrophoresis and peroxidase labeling), genotype (by extracting DNA), and biochemical characteristics to determine the relationship of Hp polymorphisms with premature CVD and its severity in 148 patients with T2DM, 150 patients with both CVD and T2DM, and 130 healthy controls at Afshar Hospital in Yazd city, Iran. ResultsAfter adjustment for age and gender, the Hp21 and Hp2–2 genotypes and phenotypes were related to an elevated risk of CVD in diabetic participants compared to the control group (OR = 10.35, 95% CI: 3.39–31.59, p = 0001and OR = 9.25, 95% CI: 2.29–28.59, p = 0001, respectively). Such an association was significantly observed in patients with diabetes compared to the control group (OR = 1.06, 95% CI: 0.25–4.20, p = 0.956); however, the frequency of Hp2–1and Hp2–2 genotypes in patients with both diabetes and CVD was not significantly different in comparison with diabetic individuals (OR = 1.65, 95% CI: 0.40–6.83, p = 0.484). After adjusting for age, sex, and disease, we also discovered that the Hp polymorphisms are correlated with low density lipoprotein cholesterol (LDL) and triglyceride (TG) levels. ConclusionBased on what we found, the genotypes and phenotypes of Hp2–2 and Hp2–1 are thought to be risk factors for type 2 diabetes. In addition, these polymorphisms might play an important role in increasing LDL and TG levels.

Biomaterial systems for evaluating the influence of ECM mechanics on anti-fibrotic therapeutic efficacy

Cardiac fibrosis is characterized by excessive accumulation and deposition of ECM proteins. Cardiac fibrosis is commonly implicated in a variety of cardiovascular diseases, including post-myocardial infarction (MI). We have previously developed a dual-delivery nanogel therapeutic to deliver tissue plasminogen activator (tPA) and Y-27632 (a ROCK inhibitor) to address MI-associated coronary artery occlusion and downregulate cell-contractility mediated fibrotic responses. Initial in vitro studies were conducted on glass substrates. The study presented here employs the use of polyacrylamide (PA) gels and microgel thin films to mimic healthy and fibrotic cardiac tissue mechanics. Soft and stiff polyacrylamide substrates or high and low loss tangent microgel thin films were utilized to examine the influence of cell-substrate interactions on dual-loaded nanogel therapeutic efficacy. In the presence of Y-27632 containing nanogels, a reduction of fibrotic marker expression was noted on traditional PA gels mimicking healthy and fibrotic cardiac tissue mechanics. These findings differed on more physiologically relevant microgel thin films, where early treatment with the ROCK inhibitor intensified the fibrotic related responses.

Predicting Myosin Heavy Chain Isoform Through Muscle Fiber Length in Older Adults

Myosin heavy chain (MHC) isoforms directly impact muscle cellular contractile characteristics and are closely associated with other cellular functions (substrate metabolism, calcium handling, etc.). As such, MHC isoform has come to define muscle “fiber type” (MHC I = slow/oxidative and MHC II = fast/glycolytic). Due to the heterogeneity of MHC isoforms expressed within human skeletal muscle tissue, it is essential to determine MHC isoform to allow proper interpretation of tissue level outcomes that may arise from variations within or between fiber types. A priori estimation of fiber type would be a useful tool for ensuring even distribution of fiber type when considering cellular assays in which MHC isoform can only be determined after the fact. Based on the assumption that fibers of various MHC isoform would differently resist mechanical strain, we’ve demonstrated that MHC I fibers can be distinguished from MHC II fibers based on the ratio of fiber to bundle length (degree of stretch) during mechanical dissection. However, our approach used samples from healthy young adults and has not been tested in tissue from older adults, where cellular stiffness is likely elevated. Therefore, our objective is to test our ability to predict MHC isoform in older adults. We hypothesize that muscle fiber strain following dissection will predict MHC isoform in older adults. Methods: Muscle biopsies from the vastus lateralis muscle were obtained from two older adults (one male, one female). A total of 129 muscle fibers were dissected and measured. Fibers were then stored individually and MHC isoform was subsequently identified via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-Page). Receiver Operating Characteristics (ROC) and confusion matrix were used to assess the predictive capacity for fiber stretch on activation as an index of fiber type. Data: An ROC area under curve (AUC) of 0.634 which indicates that there is a relatively low predictive capacity. Summary: This study aimed to test the feasibility of an approach for predicting muscle fiber type through changes in fiber length upon mechanical dissection. Established in young adults, this study tested samples obtained from older adults to explore the potential effects of age. Preliminary data show that muscle fiber length can predict muscle fiber type in older adults, though the accuracy may be limited when compared with the same approach in younger adults. This lack of accuracy may be attributed to the increase in stiffness that occurs with age, limiting the capacity for strain and thus fiber type variability. A new threshold may need to be determined to increase the accuracy of fiber type prediction. Conclusion: Preliminary data shows that fiber length can provide a prediction for MHC isoform in older adults. NIH National Institute on Aging (1R21AG077125-01A1) and Achievement Rewards for College Scientists (ARCS) Oregon. 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.

Heat Treatment and Homogenization Influence the Gastric Digestion of Bovine Milk Protein in Growing Pigs as an Adult Human Model

BackgroundBovine milk processing influences the structure of the curd formed during gastric digestion, which may alter gastric protein hydrolysis and impact amino acid (AA) release into the small intestine. ObjectivesThis study aimed to determine the influence of heat treatment and homogenization on the gastric protein digestion and AA emptying of bovine milk. MethodsNine-wk-old pigs (n = 144) consumed either raw, pasteurized nonhomogenized (PNH), pasteurized homogenized (PH), or ultra-high-temperature homogenized (UHT) bovine milk for 10 d. On day 11, fasted pigs received the milk treatment (500 mL) before gastric contents were collected at 0, 20, 60, 120, 180, and 300 min postprandially. The apparent degree of gastric protein hydrolysis (based on the release of free amino groups), apparent gastric disappearance of individual proteins [based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel band intensity], and the gastric emptying of digested protein and AA were determined. ResultsDuring the first 60 min, the rate of apparent gastric protein hydrolysis was fastest in pigs fed UHT milk (0.29%/min compared with on average 0.07%/min in pigs fed raw, PNH, and PH milk). Differences in the apparent degree of gastric protein hydrolysis and emptying were reflected in the rate of digested protein entering the small intestine. The AA gastric emptying half-time was generally shorter in pigs fed PH and UHT milk than in pigs fed raw and PNH milk. For example, the gastric release of total essential AA was >2-fold faster (P < 0.01) in pigs fed PH or UHT milk than that in pigs fed raw or PNH milk (i.e., homogenized compared with nonhomogenized milk). ConclusionsHeat treatment and homogenization increased the apparent gastric degree of protein hydrolysis and the release of digested protein into the small intestine. However, the rate of AA entering the small intestine was mainly increased by homogenization.

Physical and functional interaction between NHE3 and SGLT2 in the renal proximal tubule of male and female rats

Sodium-glucose cotransporter type 2 (SGLT2) and sodium/hydrogen exchanger-3 (NHE3) serve as the primary renal proximal tubule transporters responsible for the apical uptake of filtered glucose and NaHCO3/NaCl, respectively. Our previous studies demonstrated that chronic treatment with the selective SGLT2 inhibitor empagliflozin reduces NHE3 activity in the proximal tubule. Furthermore, we observed that the non-specific SGLT1/SGLT2 inhibitor, phlorizin, acutely inhibits NHE3 in the absence of luminal glucose, suggesting that the impact of SGLT inhibition on NHE3 may not be attributed to high tubular glucose concentrations. Additionally, our investigations revealed that SGLT2, but not SGLT1, colocalizes with NHE3 in the renal proximal tubule. This study aimed to gain insights into the functional and physical association of NHE3 and SGLT2 in the renal proximal tubule. In vivo microperfusion was conducted in male and female Wistar rats using glucose-free perfusates containing vehicle or empagliflozin (1 μM). Empagliflozin treatment significantly decreased the flow rate of bicarbonate reabsorption (JHCO3−) in both male (2.109±0.091 vs. 1.119±0.096 nmol/cm2∙s) and female rats (1.750±0.037 vs. 1.128±0.062 nmol/cm2∙s), indicating that SGLT2 inhibitors inhibit NHE3 activity through glucose-independent mechanisms. By employing nondenaturant detergents, we utilized blue native polyacrylamide gel electrophoresis (BN-PAGE) to preserve native protein-protein interactions. Double staining with antibodies against specific partner candidates revealed that bands attributed to NHE3, MAP17, PDZK1, and SGLT2 migrate similarly at ~480 kDa in samples from rat renal cortex, as supported by independent experiments. Rigorous controls were implemented to address concerns related to autofluorescence, antibody cross-reactivity, and signal specificity, confirming the validity of our results. Furthermore, complexes involving SGLT2 and NHE3 at an apparent ~480 kDa molecular weight were observed in male and female rats. Unlike SGLT2, SGLT1 bands in BN-PAGE did not overlap with the NHE3 signal, which is consistent with our earlier immunofluorescence experiments. Collectively, our data suggest that NHE3 and SGLT2 are functionally and physically associated with the renal proximal tubule. Future experiments will be undertaken to further characterize the physical association between NHE3 and SGLT2 and to determine whether the inhibition of NHE3 by SGLT2 inhibitors is mediated through the disruption of the NHE3-PDZK1-MAP17-SGLT2 multiprotein complex. Funding: São Paulo Research Foundation (FAPESP). 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.

Effects of high-intensity ultrasound on physicochemical and gel properties of myofibrillar proteins from the bay scallop (Argopecten irradians)

Myofibrillar proteins (MPs) have a notable impact on the firmness and flexibility of gel-based products. Therefore, enhancing the gelation and emulsification properties of scallop MPs is of paramount significance for producing high-quality scallop surimi products. In this study, we investigated the effects of high-intensity ultrasound on the physicochemical and gelation properties of MPs from bay scallops (Argopecten irradians). The carbonyl content of MPs significantly increased with an increase in ultrasound power (150, 350, and 550 W), indicating ultrasound-induced MP oxidation. Meanwhile, high-intensity ultrasound treatment (550 W) enhanced the emulsifying capacity and the short-term stability of MPs (up to 72.05 m2/g and 153.05 min, respectively). As the ultrasound power increased, the disulfide bond content and surface hydrophobicity of MPs exhibited a notable increase, indicating conformational changes in MPs. Moreover, in the secondary structure of MPs, the α-helix content significantly decreased, whereas the β-sheet content increased, thereby suggesting the ultrasound-induced stretching and flexibility of MP molecules. Sodium-dodecyl sulfate–polyacrylamide gel electrophoresis and scanning electron microscopy analysis further elucidated that high-intensity ultrasound induced MP oxidation, leading to modification of amino acid side chains, intra- and intermolecular cross-linking, and MP aggregation. Consequently, high-intensity ultrasound treatment was found to augment the viscoelasticity, gel strength, and water-holding capacity of MP gels, because ultrasound treatment facilitated the formation of a stable network structure in protein gels. Thus, this study offers theoretical insights into the functional modification of bay scallop MPs and the processing of its surimi products.

'Gel-Stacks' gently confine or reversibly immobilize arrays of single DNA molecules for manipulation and study.

Large DNA molecules (>20kb) are difficult analytes prone to breakage during serial manipulations and cannot be 'rescued' as full-length amplicons. Accordingly, to present, modify and analyze arrays of large, single DNA molecules, we created an easily realizable approach offering gentle confinement conditions or immobilization via spermidine condensation for controlled delivery of reagents that support live imaging by epifluorescence microscopy termed 'Gel-Stacks.' Molecules are locally confined between two hydrogel surfaces without covalent tethering to support time-lapse imaging and multistep workflows that accommodate large DNA molecules. With a thin polyacrylamide gel layer covalently bound to a glass surface as the base and swappable, reagent-infused, agarose slabs on top, DNA molecules are stably presented for imaging during reagent delivery by passive diffusion.