Polyacrylamide Gel Research Articles

Fractionation by Spatially Heterogeneous Diffusion: Experiments and the Two-Component Random Walk Model.

The fundamental question regarding the fractionation phenomenon is whether diffusion alone is responsible for it or whether an additional advection dynamic is involved. We studied the fractionation by diffusion of particles in spatially heterogeneous environments. By experimentally observing the time-sequential fractionation patterns of dye particles diffusing across a solid-solid interface of varying polyacrylamide gel densities, we found that the two-component diffusion model accurately captures the observed fractionation dynamics. In contrast, single-component diffusion models by Fick, Wereide, and Chapman do not. Our results indicate that diffusion alone can explain the fractionation phenomenon and that additional advection dynamics are not involved. The underlying physics in the fractionation phenomenon is discussed by using a two-component random walk model.

Maillard conjugates of whey protein and prebiotic dietary fibers: Emulsion properties, encapsulation efficiency and chemical stability after in vitro gastrointestinal digestion

The incorporation of dietary fibers into food formulations is a promising approach to developing new functional ingredients. In this study, we investigated the formation of Maillard conjugates using whey protein concentrate (WPC) combined with fructooligosaccharides (FOS) and xylooligosaccharides (XOS). These conjugates, innovatively utilized as emulsifiers for annatto seed oil, were subjected to in vitro gastrointestinal digestion to assess their chemical stability and effectiveness as encapsulating systems. Our study confirmed the occurrence of the Maillard reaction through different methods, including grafting degree (GD) assessment and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate molecular weight variations in the proteins. The key innovation of our research lies in demonstrating the conjugation process through carbohydrate mass balance analysis using High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD). The GD for the WPC-XOS and WPC-FOS was 46 ± 3 % and 56 ± 3 %, respectively. Our findings suggest that sugar quantification could serve as a novel approach to confirming the formation of Maillard conjugates. The emulsions stabilized by these conjugates exhibited larger oil droplet sizes compared to those stabilized by WPC alone yet maintained high encapsulation efficiency (≥ 90 %). Notably, the WPC-XOS emulsion demonstrated kinetic stability after 30 days of storage at 4°C, while the WPC-FOS emulsion showed signs of creaming after just 16 days. In vitro gastrointestinal digestion revealed that while the conjugates were susceptible to enzymatic degradation, the FOS and XOS sugars displayed gastrointestinal resistance of up to 82 % and 83 %, respectively. This study highlights the potential of WPC-XOS conjugates not only as effective emulsifiers but also as functional ingredients that enhance the nutritional and antioxidant properties of food products, presenting a novel approach to functional food development.

Physicochemical Characterization of the Oral Biotherapeutic Drug IMUNOR®.

IMUNOR is an oral biotherapeutic drug that had been developed, registered, and approved in 1997 in the Czech Republic and Slovakia. IMUNOR is a dialyzable leukocyte extract (DLE) prepared from swine leukocytes. It is characterized as a mixture of small peptides with molecular weights smaller than 12 kDa and a specific portion of nucleotides. The medical uses of IMUNOR include therapeutic applications within its registered range of indications, primarily for the treatment of immunodeficiencies, allergies, and certain acute or relapsing bacterial infections in adults and children. Despite the long-term clinical application of DLE, with strong evidence of positive therapeutic effects and no serious side effects, a detailed physicochemical specification of this mixture was lacking. We developed several methods for more in-depth physicochemical characterization of IMUNOR, including a spectrophotometric method for quantification of the total protein concentration and total DNA concentration in a mixture, several chromatographic methods for identification of individual components present in significant concentrations in IMUNOR, such as HPLC methods and the Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis method, and characterization of amino acid composition of this mixture. For the investigation of the variability among different batches of IMUNOR, five to nine representative batches from a standard manufacturing process on an industrial scale were utilized. Using the analytical methods, we verified and confirmed the batch-to-batch reproducibility of the biological product IMUNOR.

Nanoscale Surface Chemical Patterning of Soft Polyacrylamide with Elastic Modulus Similar to Soft Tissue.

Nanometer-scale control over surface functionalization of soft gels is important for a variety of applications including controlling interactions with cells for in vitro cell culture and for regenerative medicine. Recently, we have shown that it is possible to transfer a nanometer-thick precision functional polymer layer to the surface of relatively stiff polyacrylamide gels. Here, we develop a fundamental understanding of the way in which the precision polymer backbone participates in the polyacrylamide radical polymerization and cross-linking process, which enables us to generate high-efficiency transfer to much softer hydrogels (down to 5 kPa) with stiffness similar to that of soft tissue. This approach creates hydrogel surfaces with exposed nanostructured functional arrays that open the door to controlled ligand presentation on soft hydrogel surfaces.

Purification and characterization of α-amylase from Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae)

Acanthoscelides obtectus is one of the most notorious pests of stored kidney beans (Phaseolus vulgaris) worldwide. Kidney beans are an important source of food for these insects. α-Amylase is the main carbohydrate-digesting enzyme in animals including insects. In the current study, the biochemical analysis revealed higher α-amylase activity (U/ml) in 3rd and 4th larval instars but decreased gradually in subsequent developmental stages. However, the specific activity (U/mg) interestingly was highest in 1st instar and decreased in further developmental stages. During qualitative analysis of α-amylase using starch-agar and native PAGE, the maximum zone of starch lysis and a prominent band on the gel was observed in 3rd and 4th larval stages. The molecular mass of the native enzyme was also estimated and found to be 30.34 kDa. The crude α-amylase was further purified by ammonium sulfate precipitation, gel filtration on a Sephadex G-75, and ion exchange chromatography on the DEAE cellulose column. The purified amylase was found to be a monomer with a molecular mass of 15 kDa. The specific activity of the purified enzyme increased from 1.74 U/mg in the crude sample to 166.35 U/mg in the final purification step resulting in 95-fold purification with a yield of 11.14%. Further characterization of purified α-amylase revealed a pH optimum of 7.0 and a temperature optimum of 35 °C. Lineweaver-Burk plot analysis revealed Km and Vmax to be 0.09% and 3.3 U/mL, respectively. Oxalic acid, tannic acid, and HgCl2 significantly inhibited the enzyme, while the Na+, Ca++, and Mg++ ions acted as activators. In conclusion, the study revealed, the highest α-amylase activity in 3rd and 4th larval stages of Acanthoscelides obtectus followed by native and SDS PAGE resulting in molecular mass of 30.34 and 15 kDa respectively.

Enhancing structural strength and water retention of crosslinked polyacrylamide gel with the T-ZnOw

Crosslinked polyacrylamide gel serves as an effective plugging material, crucial for preventing water/gas channeling in reservoirs and enhancing the swept volume and oil recovery. However, its practical application encounters a significant challenge in maintaining long-term stability of strength and volume, particularly under high temperatures. This study investigates the reinforcement of polyacrylamide gel with three-dimensional (3D) nanomaterials, specifically tetra-needle-like zinc oxide whiskers (T-ZnOw), to enhance its structural stability and bound water content. The stress sweep tests showed that the addition of 0.15 wt% T-ZnOw increased the maximum elastic modulus of the gel to 28.5 Pa, representing a 51.2 % improvement compared to before the addition. After 60 days of high-temperature aging at 130 °C, the T-ZnOw strengthened gel still maintained an elastic modulus of 21.8 Pa. With increasing T-ZnOw concentration, the yield point of the gel increased from 4.74 Pa to 9.57 Pa, the anti-deformation of the gel has significantly increased. The stability test results in high salinity water indicate that at a salinity of 150,000 mg/L, the dehydration rate of the strengthened gel is less than 5 %, with an elastic modulus retention rate exceeding 88 %. Furthermore, thermodynamic testing confirms the enhanced thermal degradation resistance and water retention capacity of the gel. Under reservoir temperature conditions, the T-ZnOw strengthened gel exhibits a weight loss of less than 3 %. Additionally, the inclusion of T-ZnOw results in an increase in the bound water capacity index from 4.65 to 12.28. In conclusion, the integration of T-ZnOw into the polyacrylamide gel network not only enhances its mechanical performance and water retention under high-temperature conditions but also demonstrates superior stability in high-salinity environments. This innovative material shows significant potential for improving oil recovery rates in high-temperature reservoirs and can be applied to other high-temperature underground scenarios such as geothermal development and CO2 utilization and storage.

Unveiling the transformative influence of sonochemistry on formation of whey protein isolate and green tea extract (WPI-GTE) conjugates

This study investigated the formation of conjugates between whey protein isolate (WPI) and green tea extract (GTE) using three methods: redox-pair (R), ultrasound-assisted redox-pair (RU), and ultrasonication (UL). Ultrasonication significantly reduced the reaction time for synthesizing WPI-GTE conjugates compared to the standard R method (p < 0.05). The UL methods had the highest conjugate yield determined by polyphenol binding (p < 0.05). Fourier-transform infrared spectroscopy (FTIR) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the conjugate formation, indicating an increased molecular weight due to protein binding with polyphenols through covalent and non-covalent bonds. Conjugates produced via ultrasonication exhibited enhanced solubility, smaller particle size, better emulsifying capacity, and improved foaming ability compared to those formed using the traditional R method (p < 0.05). However, conjugates from the R method showed higher antioxidant activity, as evidenced by DPPH•and ABTS•+ scavenging activities (p < 0.05). In conclusion, WPI-GTE conjugates created through ultrasonic treatment demonstrate potential as dual-functional ingredients, serving as both antioxidant and emulsifier.

Synthesis and color-tunable emission of ZnGaOx with different cation ratios by a polyacrylamide gel method

ZnGaOx nanoparticles with varied ratios of Zn2+ to Ga3+ were synthesized by a polyacrylamide gel route. The phase purity and structure are confirmed by X-ray diffraction (XRD), Fourier transform infrared spectrometer (FTIR) and X-ray photoelectron spectrometer (XPS). High crystal quality and elemental distribution are determined by transmission electron microscopy (TEM) measurements. The band gaps of the samples ranging from 3.22 to 4.47 eV are estimated by ultraviolet–visible (UV–Vis) diffuse refectance spectroscopy (DRS). Under the excitation of 325 nm laser light, ZnGaOx nanoparticles with the Zn2+/Ga3+ molar ratios of 0.8:2 and 0.7:2 show two emission peaks at 415 and 510 nm. A marked emission band at 695 nm is observed from the samples with Zn2+/Ga3+ molar ratios of 0.6:2, 0.5:2 and 0.4:2. The luminescence mechanism is discussed based on the experimental results. The luminescence color of as-synthesized samples gradually shifts from blue-green to green-yellow region with decreasing Zn2+/Ga3+ ratio. Interestingly, the sample with Zn2+/Ga3+ ratio of 0.7:2 has the CIE chromaticity coordinates of (x = 0.26, y = 0.33) close to the coordinates (x = 0.33, y = 0.33) of pure white light. Together, ZnGaOx phosphors with different emission colors without doping with rare earths have considerable potential applications in tunable light emitting diodes (LEDs) and color display.

Characterization and Evaluation of Native Bacillus thuringiensis Isolate T121 Toxic to Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae)1

Abstract The present study focused on the molecular characterization and toxicity analysis of the Bacillus thuringiensis Berliner (Bt) isolate T121 against Henosepilachna vigintioctopunctata (F.) (Coleoptera: Coccinellidae). Colonies of T121 growing on solid media were circular, with creamy white hues and raised centers. Scanning electron microscopy revealed the presence of cuboidal and spherical crystals in liquid media. Polymerase chain reaction screening confirmed the presence of binary toxin vip1 and vip2 in addition to cry3Aa gene. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed four prominent bands at 74, 65, 48, and 31 kDa. In toxicity assays, T121 exhibited a median lethal concentration of 4.675 µg/ml against H. vigintioctopunctata. These findings demonstrate that the Bt isolate T121 offers promising prospects for development as a component of a sustainable pest management strategy against H. vigintioctopunctata and perhaps other coleopteran pests.

Mild osmotic stress offers photoprotection in Chlamydomonas reinhardtii under high light

The exposure of autotrophs to high light intensities significantly impacts their photosynthetic performance. When combined with unpredictable climate changes, the lethality of these effects is exacerbated and, often surpassing the organisms' threshold for tolerance. In this regard, our study centres on examining the mitigating effects of mild osmotic stress induced by 2% Polyethylene Glycol (PEG) in conjunction with high-light conditions, using Chlamydomonas reinhardtii as a model system. Cells were cultivated under low PEG-induced osmotic stress at various light intensities, and their responses were analyzed through biochemical and biophysical approaches. Remarkably, cells grown under lower PEG concentrations exhibited superior growth, increased biomass, and enhanced photosynthetic efficiency under high light compared to non-PEG-treated cells. Surprisingly, their non-photochemical quenching (NPQ) levels were lower, indicating the operation of a distinct photoprotective mechanism in PEG-grown samples. The PEG-grown cells demonstrated higher chlorophyll content but lower carotenoid content, supporting the NPQ data. Circular dichroism analysis suggested that the macro-organization of super-complexes was minimally disrupted in PEG-grown samples, even under high light. This was further supported by Blue native PAGE, which showed greater stability of the super-complexes in PEG-grown cells, implying heightened stability in pigment-protein interactions. Immunoblot analysis revealed minimal differences in core reaction center proteins between PEG-grown and non-PEG cells. Notably, this protective mechanism was absent in the cell wall-deficient mutant CC503. We propose that the partial photoprotection observed is attributed to the PEG shielding the cell wall. This result holds promise for enhancing algal biomass production under natural environmental conditions influenced by fluctuating light intensity.

A Novel Application of Virus Like Particles in the Hemagglutination Inhibition Assay.

The hemagglutination inhibition (HI) assay is a traditional laboratory procedure for detection and quantitation of serum antibodies of hemagglutinating viruses containing the hemagglutinin (HA) gene. The current study aimed to investigate the novel use of virus like particles (VLP) as an antigen for the HI assay. VLPs were prepared from a strain of H5N1 using a baculovirus expression system. The VLPs were characterized using the hemagglutination test, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and transmission electron microscopy. The comparative HI assay was performed using three different seed antigens: A/chicken/Mexico/232/94 (H5N2), A/chicken/Egypt/18-H/09(H5N1) and A/goose/Guangdong/1/1996(H5N1). The HI assay of serum antibody titrations using homologous antigens to these vaccinal seeds were compared to the VLP's antigens for the same serum. The HI titers were logically relevant to the similarity between VLP antigens and vaccinal seeds, indicating the VLPs behave similarly to the standard HI assay which uses inactivated whole virus as an antigen. VLPs could be considered as an alternative to the HI assay antigen as they show a relatedness between the similarity with vaccinal seed and serum antibodies. Compared to typical entire H5N1 viral antigen prepared in SPF eggs that require proper inactivation to avoid any public health risk, VLPs prepared in tissue culture, plants or insect cells are a safe, inexpensive and scalable alternative to inactivated whole virus antigen.

Electron microscopy of stable electrophoretic fractions of natural humic acids – the key to the understanding of their structural organization

Transmission electron microscopy (TEM) with contrast staining by uranyl acetate solution was used to study morphological differences between soil humic acids (HAs) and their A, B and C + D fractions obtained using coupling preparative low-pressure size exclusion chromatography and analytical polyacrylamide gel electrophoresis. The electrophoretic mobility of fractions varied in order C + D B A. The distribution of various morphological elements between fractions showed that large structures such as vesicle-like formations 70–150 nm long and 30–80 nm wide with clear edges were found exclusively in fraction A and occupied ~55% of the TEM image area. On the other hand, long fibrils with a length of 60–100 nm, a width of 4–6 nm and a thickness of 2–3 nm, as well as their bundles with a length of 150 nm and a diameter of 30–70 nm were identified only in the C + D fraction and occupied ~59 % area of TEM images. Smaller morphological elements such as point particles with a diameter of 2–3 nm, ring particles with a diameter of 4–6 nm, worm-shaped short particles with a length of 20–30 nm, and spheroids with a diameter of 10– 30 nm were observed in all samples, but in varying quantities. Significant morphological differences between the fractions can be explained by their composition, previously established by using a few physico-chemical methods. The ratio Car(165–108 ppm)/Calk(108–0 ppm), or aromaticity index, calculated from 13C-NMR, could be one of the indicators of the various morphological structures formation. The obtained TEM results clearly confirm the supramolecular organization of soil HAs.

Upstream alternative polyadenylation in SCN5A produces a short transcript isoform encoding a mitochondria-localized NaV1.5 N-terminal fragment that influences cardiomyocyte respiration.

SCN5A encodes the cardiac voltage-gated Na+ channel, NaV1.5, that initiates action potentials. SCN5A gene variants cause arrhythmias and increased heart failure risk. Mechanisms controlling NaV1.5 expression and activity are not fully understood. We recently found a well-conserved alternative polyadenylation (APA) signal downstream of the first SCN5A coding exon. This yields a SCN5A-short transcript isoform expressed in several species (e.g. human, pig, and cat), though rodents lack this upstream APA. Reanalysis of transcriptome-wide cardiac APA-seq and mRNA-seq data shows reductions in both upstream APA usage and short/full-length SCN5A mRNA ratios in failing hearts. Knock-in of the human SCN5A APA sequence into mice is sufficient to enable expression of SCN5A -short transcript, while significantly decreasing expression of full-length SCN5A mRNA. Notably, SCN5A -short transcript encodes a novel protein (NaV1.5-NT), composed of an N-terminus identical to NaV1.5 and a unique C-terminus derived from intronic sequence. AAV9 constructs were able to achieve stable NaV1.5-NT expression in mouse hearts, and western blot of human heart tissues showed bands co-migrating with NaV1.5-NT transgene-derived bands. NaV1.5-NT is predicted to contain a mitochondrial targeting sequence and localizes to mitochondria in cultured cardiomyocytes and in mouse hearts. NaV1.5-NT expression in cardiomyocytes led to elevations in basal oxygen consumption rate, ATP production, and mitochondrial ROS, while depleting NADH supply. Native PAGE analyses of mitochondria lysates revealed that NaV1.5-NT expression resulted in increased levels of disassembled complex V subunits and accumulation of complex I-containing supercomplexes. Overall, we discovered that APA-mediated regulation of SCN5A produces a short transcript encoding NaV1.5-NT. Our data support that NaV1.5-NT plays a multifaceted role in influencing mitochondrial physiology: 1) by increasing basal respiration likely through promoting complex V conformations that enhance proton leak, and 2) by increasing overall respiratory efficiency and NADH consumption by enhancing formation and/or stability of complex I-containing respiratory supercomplexes, though the specific molecular mechanisms underlying each of these remain unresolved.

Isolation of donor gamma globulin obtained from multiparous women and its effects upon expression of HLA-G and HLA-DR molecules on lymphocytes from mothers of children with septal congenital heart defects

A common pathogenetic mechanism of reproductive losses and congenital heart disease (CHD) is associated with immune inflammation in the “mother-embryo” system which affects differentiation and proliferation of cardiovascular progenitor cells. It is hypothesized that this link may be blocked by regulatory auto- and alloimmune antibodies to HLA-G and HLA-DR molecules. Moreover, these antibodies may be present at sufficient amounts in donor immunoglobulins, especially those obtained from the blood of multiparous women. Based on this suggestion, the aim of our study was to obtain enriched gamma globulin fraction from the blood of multiparous women and evaluate its functional effects towards HLA-DR and HLA-G molecules. Isolation of the gammaglobulin fraction (GGF) from the blood plasma of multiparous women was performed using affinity chromatography in several sessions. Purity grade of the resulting protein was analyzed by immunoelectrophoresis, electrophoretic separation of the protein fraction of blood serum and electrophoresis in 4.12% polyacrylamide gel with the addition of SDS (PAGE electrophoresis). PAAG electrophoresis showed that this GGF did not differ from commercial therapeutic intravenous immunoglobulin (IVIG). Assessment of the functional activity of GGF upon HLA-DR and HLA-G molecules was performed in the main group of women and their children with congenital heart disease (n = 38), and control group of women who gave birth to conditionally healthy children (n = 21). To determine the specificity of GGF with respect to HLA-G, HLA-DR molecules, as well as to compare its effect with autologous and allogeneic sera and IVIG, we developed an immunological testing protocol using flow cytometry. The protocol was arranged on the basis of the methodology of “cross-match” approach and Russian patent “Method for determining antibodies to HLA-G”. It was found that the blocking activity of female serum towards autologous (intrinsic) and allogeneic (embryo/fetus/child) HLA-G and HLA-DR molecules may determine the protective effect on development of congenital heart defects in the next generation. Donor human immunoglobulin showed a similar blocking effects to these molecules, possibly due to the presence of alloimmune antibodies to HLA classes I and II. The gammaglobulin fraction obtained from the donor blood of multiparous women has a more pronounced blocking effect on the HLA-G and HLA-DR expression. Hence, this immunobiological preparation can be considered a prototype of therapeutic and prophylactic agent blocking the genesis of congenital heart defects.

Robust peroxidase from Bacillus mojavensis TH309: Immobilization on walnut shell hydrochar and evaluation of its potential in dye decolorization

Peroxidases have received considerable attention as a cost-effective and environmentally friendly catalyst for bioremediation. Their rapid activity loss under harsh environmental conditions and inability to be used repetitively limit their exploitation in real-world wastewater treatment. First, a peroxidase was produced extracellularly by Bacillus mojavensis TH309 and purified 8.12-fold with a final yield of 47.10 % using Sephadex G-100 superfine resin. The pure peroxidase (BmPer) possessed a relatively low molecular weight of ∼21 kDa and was active against L-DOPA on acrylamide gel after electrophoresis. BmPer was immobilized by adsorption functionalized walnut shell hydrochar (WsH) with 61.99 ± 1.34 % efficiency and 37.07 ± 4.16 % activity loss. BmPer and its immobilized form (WsH-BmPer) exhibited maximum activity at 50 °C and pH 9. WsH-BmPer exhibited 3.23-, 2.37-, 1.65-, and 2.25-fold longer half-life than BmPer at 50, 60, 70, and 80 °C, respectively. Immobilization significantly enhanced the stability of the enzyme under acidic conditions. BmPer and WsH-BmPer showed maximal activity in the presence of 1 % salt and retained more than 85 % of their activity even after pre-incubation with 2.5 M salt for 60 min at 50 °C. Their catalytic efficiency was significantly stimulated by pre-incubation with Triton X-100 (1 mM), Tween20 (1 mM), and Mg2+ (1 and 10 mM). Immobilization strongly reduced the loss of activity caused by inhibitors including Ba2+, Hg2+, and Cu2+. Moreover, both forms of the enzyme were compatible with solvents. The Michaelis constant (Km) values of BmPer and WsH-BmPer were 0.88 and 2.66 mM for 2,4 DCP, respectively. WsH-BmPer peroxidase maintained about 82 % and 85 % of its activity when stored at 4 °C for 30 days and reused for up to 10 cycles, respectively. Furthermore, it decolorized Cibacron red (CR), Poly R-478 (PR), Remazol Brilliant Blue R (RBBR), and Methyl red (MR) dyes by 60.13 %, 91.34 %, 86.41 %, and 50.51 % within 60 min, respectively.

DELINEATING GENETIC DIVERSITY IN SOYBEAN (GLYCINE MAX L.) GENOTYPES: INSIGHTS FROM A-PAGE ANALYSIS OF GLOBULIN RESERVE PROTEINS

This study explores the genetic diversity of soybean (Glycine max) genotypes using polyacrylamide gel electrophoresis (A-PAGE) to analyze globulin reserve proteins. The research utilized 51 soybean genotypes from various origins, including Canada, Turkey, Uzbekistan and others. The A-PAGE method was employed to extract and analyze the globulin storage proteins in the grains of soybean samples. The proteins were categorized into ω, γ, β and α zones, revealing distinct electrophoretic patterns among the genotypes. A total of 22 spectra and 107 patterns were identified, demonstrating polymorphism based on the frequency of occurrence. The genetic diversity index, calculated using Nei’s formula, indicated varying levels of genetic diversity across the different zones.

Abstract Mo097: Inhibiting the Sarcomere Improves Diastolic Dysfunction in Patient-Derived hiPSC-CM Models of Pediatric Restrictive Cardiomyopathy

Introduction: Pediatric patients with Restrictive Cardiomyopathy (RCM) develop heart failure due to stiffening of the wall of the cardiac ventricle leading to diastolic dysfunction but preserved cardiac contractility. Treatment options are extremely limited, and the evaluation of new therapeutic strategies is hindered due to a lack of models that recapitulate this ventricular stiffening. Here, we evaluate two novel patient-derived human induced pluripotent stem cell (hiPSC) models of restrictive cardiomyopathy due to mutations in the thin filament, and evaluate separate sarcomere-targeted therapeutic strategies to improve diastolic function. Hypothesis: Directly decreasing the thin filament’s response to calcium will improve diastolic function in RCM models with less effect on systolic function than myosin inhibition. Methods: hiPSC-cardiomyocytes (hiPSC-CMs) were differentiated from hiPSC lines generated from two patients with pediatric RCM, as well as their isogenic controls. After replating on polyacrylamide gels and staining with fluorescent calcium indicators, diastolic tension, generated tension, and calcium sensitivity were measured at baseline and upon treatment with either a troponin calcium desensitizer (W7) or a myosin inhibitor (mavacamten). Results: hiPSC-CMs from both patients showed increased diastolic tension, generated tension, and calcium sensitivity compared to their isogenic controls. Treatment with Mavacamten improved diastolic function but severely compromised systolic function, while treatment with W7 improved diastolic function with less effect on systolic function. Conclusions: Directly inhibiting the sarcomere in models of RCM improved measures of diastolic dysfunction, but directly targeting the thin filament in these lines showed less effect on systolic function than myosin inhibition, suggesting cardiac troponin calcium desensitizers may be more beneficial in this severe diastolic disease.

Abstract Mo096: High-Throughput Functional Determination of TNNI3 Variant Pathogenicity

Introduction: Mutations in the thin filament protein TNNI3 (cardiac troponin I) can perturb calcium cycling and cause hypertrophic, restrictive, and dilated cardiomyopathies. However, the majority of TNNI3 variants observed in patients have unknown functional significance and uncertain contribution to disease. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful system for studying the effect of genetic variants on human cardiomyocyte function. TNNI3 has been challenging to study in these cells as they predominantly express TNNI1. In this study, we combined a novel scalable in vitro gene replacement method with simultaneous measurement of contractility and calcium cycling to determine the functional effect of TNNI3 variants. Hypothesis: Using our gene replacement platform, we expect that TNNI3 variants causing Hypertrophic Cardiomyopathy (HCM) will increase force generation and calcium sensitivity relative to wild-type, whereas TNNI3 variants causing Dilated Cardiomyopathy (DCM) will decrease force generation and calcium sensitivity relative to wild-type. Methods: We generated double knockout TNNI1 -/- TNNI3 -/- hiPSCs. Cardiomyocytes differentiated from these hiPSCs were transduced with lentivirus carrying either wild-type or mutant TNNI3 during replating onto polyacrylamide gels with fluorescent beads. After a week of incubation, the cells were stained with a fluorescent calcium probe, and contractility, diastolic tension, and calcium sensitivity were measured by our high-throughput physiologic imaging and analysis platform. Results: hiPSC-CMs expressing the HCM-associated variant R192H show increased peak force, diastolic tension, and calcium sensitivity relative to wild-type TNNI3, whereas hiPSC-CMs expressing the DCM-associated variant K36Q show decreased peak force and calcium sensitivity relative to wild-type TNNI3. Conclusion: We validated our TNNI3 in vitro gene replacement and high-throughput physiologic imaging and analysis platform by comparing reference HCM and DCM variants to wild-type TNNI3. Ongoing scaled studies will evaluate the naturally occurring variants in ClinVar to characterize their functional effect on cardiac physiology.

Abstract We035: PTMA-MBD3 axis is a core heart regenerative driver in mammals

Introduction: The adult mammalian heart has an extremely limited ability for regeneration after injury, whereas the fetal heart can steadily regenerate. Uncovering the molecular underpinnings of proliferative cardiomyocytes and manipulating the core genes activate cardiomyocyte proliferation may provide new insight into adult heart regrowth and functional repair post-injury. Methods and Results: We performed single-cell RNA-sequencing of mouse embryonic hearts from various developmental stages (E8.5-E17.5), and identified a cardiomyocyte population in proliferative phase with 120 highly expressed feature genes. Combining with our bulk RNA-seq data on isolated cardiomyocytes from different heart developmental periods (E14.5, P1-P56), we screened and demonstrated that Ptma is the core conservative factor in promoting mouse, rat and human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) proliferation. Cardiac specifically knocking out Ptma blocked cardiomyocyte proliferation and neonatal mouse heart regeneration after apical resection injury. AAV9-delivered Ptma overexpression extended neonatal heart regenerative time window and showed therapeutic benefit in driving adult cardiomyocyte proliferation and cardiac repair. As PTMA is a polypeptide, we further validated that PTMA derived conservative peptide Tα1 displayed more effective in treating adult cardiac injury compared to Decapeptide. PTMA is an unstructured, intrinsically disordered nuclear protein that functions by binding to target proteins. Using Co-IP, IP and Native PAGE, we found PTMA promoted cardiomycyte proliferaton by binding to MBD3, which decreases the formation of MBD3-HDAC1 NuRD complex . We further decreased MBD3-HDAC1 NuRD complex formation by inhibiting MBD3, boosting both rat and mouse cardiomyocyte proliferation. Conclusions: We identified the PTMA-MBD3 axis as an effective target for promoting mammalian heart regeneration and provided a new insight into cardiomyocyte proliferation through blocking MBD3-HDAC1 NuRD complex.

Oligomerization of protein arginine methyltransferase 1 and its effect on methyltransferase activity and substrate specificity.

Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.